ES2393683A1 - Obtaining a protein extract through enzymatic hydrolysis from tobacco leaves and its application as a fertilizer. (Machine-translation by Google Translate, not legally binding) - Google Patents
Obtaining a protein extract through enzymatic hydrolysis from tobacco leaves and its application as a fertilizer. (Machine-translation by Google Translate, not legally binding) Download PDFInfo
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- ES2393683A1 ES2393683A1 ES201100606A ES201100606A ES2393683A1 ES 2393683 A1 ES2393683 A1 ES 2393683A1 ES 201100606 A ES201100606 A ES 201100606A ES 201100606 A ES201100606 A ES 201100606A ES 2393683 A1 ES2393683 A1 ES 2393683A1
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- tobacco leaves
- enzymatic hydrolysis
- protein extracts
- protein
- tobacco
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 42
- 235000002637 Nicotiana tabacum Nutrition 0.000 title claims abstract description 40
- 241000208125 Nicotiana Species 0.000 title claims abstract description 35
- 239000000284 extract Substances 0.000 title claims abstract description 20
- 230000007071 enzymatic hydrolysis Effects 0.000 title claims abstract description 19
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 title claims abstract description 19
- 239000003337 fertilizer Substances 0.000 title description 4
- 238000000034 method Methods 0.000 claims abstract description 34
- 150000001413 amino acids Chemical class 0.000 claims abstract description 10
- 108091005804 Peptidases Proteins 0.000 claims abstract description 8
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 8
- 102000035195 Peptidases Human genes 0.000 claims abstract description 6
- 238000011282 treatment Methods 0.000 claims description 22
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- 238000006243 chemical reaction Methods 0.000 claims description 10
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- 238000000605 extraction Methods 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 229940088598 enzyme Drugs 0.000 claims description 4
- 238000000265 homogenisation Methods 0.000 claims description 4
- 108090000787 Subtilisin Proteins 0.000 claims description 3
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 claims description 3
- 241001465754 Metazoa Species 0.000 claims description 2
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- 235000005911 diet Nutrition 0.000 claims 1
- 230000037213 diet Effects 0.000 claims 1
- 229940116540 protein supplement Drugs 0.000 claims 1
- 235000005974 protein supplement Nutrition 0.000 claims 1
- 230000002797 proteolythic effect Effects 0.000 claims 1
- 239000002689 soil Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 10
- 235000007688 Lycopersicon esculentum Nutrition 0.000 abstract description 8
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- 240000003768 Solanum lycopersicum Species 0.000 abstract 1
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- 235000018102 proteins Nutrition 0.000 description 32
- 230000008569 process Effects 0.000 description 22
- 241000196324 Embryophyta Species 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 208000031953 Hereditary hemorrhagic telangiectasia Diseases 0.000 description 11
- 238000006460 hydrolysis reaction Methods 0.000 description 11
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- 230000007062 hydrolysis Effects 0.000 description 9
- 241000227653 Lycopersicon Species 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 240000008886 Ceratonia siliqua Species 0.000 description 7
- 235000013912 Ceratonia siliqua Nutrition 0.000 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 244000061176 Nicotiana tabacum Species 0.000 description 5
- 235000013399 edible fruits Nutrition 0.000 description 5
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- 230000002255 enzymatic effect Effects 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 235000016709 nutrition Nutrition 0.000 description 4
- 108010009736 Protein Hydrolysates Proteins 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
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- 235000013312 flour Nutrition 0.000 description 3
- 230000003301 hydrolyzing effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 235000002918 Fraxinus excelsior Nutrition 0.000 description 2
- 108010038807 Oligopeptides Proteins 0.000 description 2
- 102000015636 Oligopeptides Human genes 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
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- 239000002253 acid Substances 0.000 description 2
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- 108010041102 azocasein Proteins 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 230000007073 chemical hydrolysis Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000001863 plant nutrition Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000000751 protein extraction Methods 0.000 description 2
- 239000003531 protein hydrolysate Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 1
- DEWLEGDTCGBNGU-UHFFFAOYSA-N 1,3-dichloropropan-2-ol Chemical compound ClCC(O)CCl DEWLEGDTCGBNGU-UHFFFAOYSA-N 0.000 description 1
- HUXDTFZDCPYTCF-UHFFFAOYSA-N 1-chloropropane-1,1-diol Chemical class CCC(O)(O)Cl HUXDTFZDCPYTCF-UHFFFAOYSA-N 0.000 description 1
- 241001456108 Castilla Species 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 235000017367 Guainella Nutrition 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 101710121996 Hexon protein p72 Proteins 0.000 description 1
- 235000002262 Lycopersicon Nutrition 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000287219 Serinus canaria Species 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000003905 agrochemical Substances 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 230000002009 allergenic effect Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
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- 230000001276 controlling effect Effects 0.000 description 1
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- 230000001086 cytosolic effect Effects 0.000 description 1
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- 238000010494 dissociation reaction Methods 0.000 description 1
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- 235000020776 essential amino acid Nutrition 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000004021 humic acid Substances 0.000 description 1
- 230000000774 hypoallergenic effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003621 irrigation water Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229960002715 nicotine Drugs 0.000 description 1
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 229930195732 phytohormone Natural products 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
- A01N65/38—Solanaceae [Potato family], e.g. nightshade, tomato, tobacco or chilli pepper
- A01N65/385—Tobacco
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Botany (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Molecular Biology (AREA)
- Agronomy & Crop Science (AREA)
- Physiology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
- Obtención de un extracto proteico mediante hidrólisis enzimática a partir de hojas Obtaining a protein extract by enzymatic hydrolysis from leaves
- de tabaco y su aplicación como fertilizante of tobacco and its application as a fertilizer
- 5 5
- Objeto de la invención Object of the invention
- El objeto de esta invención es la obtención de un concentrado proteico a partir de The object of this invention is to obtain a protein concentrate from
- hojas de tabaco (frescas y/o secas) no aprovechables para la fabricación de dicho tobacco leaves (fresh and / or dried) not usable for the manufacture of said
- tabaco mediante el proceso de hidrólisis enzimática lo que origina concentrados tobacco through the process of enzymatic hydrolysis which causes concentrates
- con un alto contenido proteico (80%). La mayoría de estos hidrolizados están with a high protein content (80%). Most of these hydrolysates are
- 1O 1O
- formados por péptidos, oligopéptidos y aminoácidos libres con alta solubilidad. formed by peptides, oligopeptides and free amino acids with high solubility.
- Esto hace que estos compuestos proteicos se absorban por la planta a una mayor This causes these protein compounds to be absorbed by the plant to a greater
- velocidad de manera más eficiente y, sobre todo, son una buena fuente de speed more efficiently and, above all, they are a good source of
- aminoácidos esenciales, muy importante para la nutrición de las plantas. Essential amino acids, very important for plant nutrition.
- 15 fifteen
- Estado de la técnica State of the art
- El tabaco, es a día de hoy uno de los principales cultivos a nivel mundial Tobacco is today one of the main crops worldwide
- cuyo fin no es alimenticio. En España se cultiva tabaco en 7 comunidades whose end is not nutritional. In Spain, tobacco is grown in 7 communities
- autónomas: Extremadura, Andalucía, Canarias, Castilla y León, Castilla la Autonomous: Extremadura, Andalusia, Canary Islands, Castilla y León, Castilla la
- 20 twenty
- Mancha, Navarra y País Vasco. Extremadura es la región autonómica donde se Manche, Navarra and the Basque Country. Extremadura is the autonomous region where
- concentra el 85% de la producción española, seguida de Andalucía. It concentrates 85% of Spanish production, followed by Andalusia.
- España es el tercer país de Europa con 40991 T de cosecha en 2005 y un Spain is the third country in Europe with 40991 T of harvest in 2005 and a
- valor de producción de 144,5 millones de euros en 2003 (ASTRA), dando empleo production value of 144.5 million euros in 2003 (ASTRA), giving employment
- a más de 20000 familias. to more than 20,000 families.
- 25 25
- La elaboración del tabaco, se hace en base a un proceso que consta de Tobacco production is done based on a process that consists of
- varias etapas, y que tiene por objeto producir en las hojas una serie de several stages, and which aims to produce in the leaves a series of
- transformaciones químicas que las hacen más aptas y aceptables para el uso. Sin chemical transformations that make them more suitable and acceptable for use. Without
- embargo, no todas las hojas de la planta del tabaco tienen la misma composición However, not all leaves of the tobacco plant have the same composition
- química, y por ello, en el cultivo del tabaco, no todas las hojas son aprovechadas chemistry, and therefore, in the cultivation of tobacco, not all leaves are used
- 30 30
- para la fabricación de tabaco. for the manufacture of tobacco.
- Generalmente, las 2-3 hojas que se desarrollan en la parte más baja del Generally, the 2-3 leaves that develop in the lower part of the
- tallo suelen quedar deterioradas por el contacto con las aguas de riego y con el stem tend to be damaged by contact with irrigation waters and with the
- suelo. Son hojas con menor contenido en nicotina, aroma, etc., y por tanto, resulta ground. They are leaves with lower nicotine content, aroma, etc., and therefore, it is
- conveniente suprimirlas en la primera parte del ciclo vegetativo que sigue al it is convenient to suppress them in the first part of the vegetative cycle that follows the
- 35 35
- transplante. transplant.
Por otro lado, en la fase de despunte se suelen suprimir varias hojas que salen justo debajo de la inflorescencia. Este despunte influye notablemente en la composición física y química del tabaco curado. Con el despunte, al reducir el número de hojas a recolectar, los nutrientes se acumularán en las hojas que quedan. On the other hand, in the phase of emergence, several leaves that leave just below the inflorescence are usually suppressed. This emergence greatly influences the physical and chemical composition of cured tobacco. With the emergence, by reducing the number of leaves to be collected, the nutrients will accumulate in the remaining leaves.
Del mismo modo, en la fase de despalillo y clasificación se manipulan las hojas seleccionadas para la fabricación de tabaco. Normalmente, a estas hojas se les retira la parte más gruesa del nervio, dos tercios en el caso de las hojas destinadas a tripa, y tres cuartas partes en el caso de los capotes. In the same way, in the phase of destemming and classification, the leaves selected for the manufacture of tobacco are handled. Normally, these leaves are removed the thickest part of the nerve, two thirds in the case of the leaves intended for gut, and three quarters in the case of the hoods.
Como consecuencia de todo esto, el cultivo de tabaco también origina un material vegetal no aprovechable para el proceso de elaboración de tabaco y que podría ser usado con otros fines, como por ejemplo, la obtención de biofertilizantes As a result of all this, the cultivation of tobacco also originates a plant material that cannot be used for the tobacco production process and could be used for other purposes, such as obtaining biofertilizers.
o bioestimulantes. or biostimulants.
En los últimos años, ha ido aumentando el consumo de proteínas vegetales a causa de una mayor utilización de productos proteicos de buena calidad, ya sean en forma de concentrados o de aislados proteicos. Sin embargo, el uso de estos productos se ve limitado por su baja solubilidad o por sus caracteres potencialmente alergénicos. Estas dos razones han conducido al estudio de procesos de hidrólisis enzimática para obtener nuevos productos proteicos con características nutricionales y funcionales definidas, minimizando o eliminando los inconvenientes. In recent years, the consumption of vegetable proteins has been increasing due to a greater use of good quality protein products, whether in the form of concentrates or protein isolates. However, the use of these products is limited by their low solubility or their potentially allergenic characteristics. These two reasons have led to the study of enzymatic hydrolysis processes to obtain new protein products with defined nutritional and functional characteristics, minimizing or eliminating the inconveniences.
Estos nuevos productos, denominados biofertilizantes o bioestimulantes están constituidos generalmente péptidos, aminoácidos, polisacáridos, ácidos húmicos, fitohormonas, etc que son absorbidos directamente por la planta, gastando la misma una menor cantidad de energía para dicho proceso de absorción, lo cual repercute positivamente no solo en su crecimiento sino también en la calidad y producción del fruto o grano recolectado. El objetivo de estos productos no es suministrar la nutrición, sino más bien para favorecer y estimular el metabolismo de la planta, reducción de estrés, etc (Parra do et al., 1991, 2008). Por esta razón, el desarrollo de nuevos biofertilizantes/bioestimulantes, utilizando materiales naturales, se ha convertido en el foco de interés en la investigación. These new products, called biofertilizers or biostimulants, are generally made up of peptides, amino acids, polysaccharides, humic acids, phytohormones, etc. that are directly absorbed by the plant, spending a lower amount of energy for said absorption process, which positively affects not only in its growth but also in the quality and production of the fruit or grain collected. The objective of these products is not to provide nutrition, but rather to promote and stimulate plant metabolism, stress reduction, etc. (Parra do et al., 1991, 2008). For this reason, the development of new biofertilizers / biostimulants, using natural materials, has become the focus of interest in research.
Una gran parte de estos nuevos biofertilizantes/bioestimulantes se han procesado a partir de métodos bioquímicos, generalmente por medio de hidrólisis enzimática. La hidrólisis enzimática se ha monitorizado mediante la técnica del pH-stat, utilizado un biorreactor con control de pH, temperatura y agitación y medida del consumo de NaOH. El parámetro usado para controlar la reacción hidrólítica es el grado de hidrólisis (GH), el cual se define como el porcentaje de uniones peptídicas rotas (Adler Nissen, 1977). El fundamento de la técnica del pH-stat radica en que bajo condiciones de hidrólisis neutra o alcalina la disociación del grupo amino resulta significativa y el continuo consumo de base necesario para mantener el pH constante durante la reacción es un medio de controlar la magnitud del proceso hidrolítico. Así pues, el grado de hidrólisis se define como la relación entre el número de uniones peptídicas rotas y el número total de uniones peptídicas.
A large part of these new biofertilizers / biostimulants have been processed from biochemical methods, generally by means of enzymatic hydrolysis. Enzymatic hydrolysis has been monitored using the pH-stat technique, using a bioreactor with control of pH, temperature and stirring and measurement of NaOH consumption. The parameter used to control the hydrolytic reaction is the degree of hydrolysis (GH), which is defined as the percentage of broken peptide bonds (Adler Nissen, 1977). The basis of the pH-stat technique is that under conditions of neutral or alkaline hydrolysis the dissociation of the amino group is significant and the continuous base consumption necessary to maintain the constant pH during the reaction is a means of controlling the magnitude of the process. hydrolytic Thus, the degree of hydrolysis is defined as the relationship between the number of broken peptide bonds and the total number of peptide bonds.
En este sentido, cabe a destacar una gran cantidad de trabajos cientificos así como de patentes realizadas sobre distintos productos vegetales (algarroba, trigo, maiz, girasol, arroz, etc.) utilizando la hidrólisis enzimatica, obteniendo productos caracterizados principalmente por su alto contenido en péptidos de bajo peso molecular y aminoácidos libres y que tienen una importante repercusión en el mundo agrícola debido a una mejora en la nutrición de la planta, afectando positivamente no solo a su crecimiento sino también a la producción del grano o fruto obtenido (Bautista, 1999; Bautista et al., 1996; Garcia-Martínez et al., 2007, 2008; Parrado et al., 2006, 2007a, 2007b; Pedroche et al., 2000; Romero et al., 2007; Villa nueva et al., 1999). Del mismo modo, existe una gran cantidad de patentes que tratan sobre la obtención de productos solubles utilizables en la agricultura y que se han elaborado mediante procesos de hidrólisis (ver referencias). Sin embargo, no hay patentes ni estudios de elaboración de biofertilizantes a partir de hojas de tabaco (frescas y/o secas) utilizando procesos enzimáticos directos. Se han encontrado patentado, procesos relacionados con tabaco y su conversión en fertilizantes compuestos por de polipéptidos y su uso como producto agroquímico (PCT/EP2007/002075), en este caso el proceso es muy diferente y más complejo que el se describe en esta patente, ya que utiliza substratos y procesos diferentes, en concreto los substratos son células BY2 de tabaco y procesos de fermentación con microorganismos, separación de la células del medio de fermentación, homogeneización, precipitación de las paredes celulares seguido de lavados para eliminar componentes citoplásmicos, hervido de las paredes celulares en disolución acida para separar los azucares unidos a las proteínas, digestión de las proteínas de las paredes en medio acido o enzimático ( usando enzimas proteolíticas), y finalmente, una separación de los péptidos y aminoácidos. In this sense, it is worth highlighting a large number of scientific works as well as patents made on different vegetable products (carob, wheat, corn, sunflower, rice, etc.) using enzymatic hydrolysis, obtaining products characterized mainly by their high content in low molecular weight peptides and free amino acids that have an important impact on the agricultural world due to an improvement in plant nutrition, positively affecting not only its growth but also the production of the grain or fruit obtained (Bautista, 1999 ; Bautista et al., 1996; Garcia-Martínez et al., 2007, 2008; Parrado et al., 2006, 2007a, 2007b; Pedroche et al., 2000; Romero et al., 2007; Villa nueva et al., 1999). Similarly, there are a large number of patents that deal with obtaining soluble products that can be used in agriculture and that have been produced through hydrolysis processes (see references). However, there are no patents or studies on the preparation of biofertilizers from tobacco leaves (fresh and / or dried) using direct enzymatic processes. They have been patented, processes related to tobacco and its conversion into fertilizers composed of polypeptides and its use as an agrochemical (PCT / EP2007 / 002075), in this case the process is very different and more complex than that described in this patent , since it uses different substrates and processes, specifically the substrates are tobacco BY2 cells and fermentation processes with microorganisms, separation of the cells from the fermentation medium, homogenization, precipitation of the cell walls followed by washing to remove cytoplasmic components, boiled of the cell walls in acid solution to separate the sugars bound to the proteins, digestion of the proteins of the walls in acidic or enzymatic medium (using proteolytic enzymes), and finally, a separation of the peptides and amino acids.
- 5 5
- La hidrólisis enzimática es pues el procedimiento óptimo para conseguir la extracción de las proteínas a partir de las plantas de tabaco y su conversión en hidrolizados proteicos, principalmente debido a que es un proceso biológico donde la calidad del producto final permanece inalterada. Enzymatic hydrolysis is therefore the optimal procedure to achieve the extraction of proteins from tobacco plants and their conversion into protein hydrolysates, mainly because it is a biological process where the quality of the final product remains unchanged.
- 1 O 1 o
- Una alternativa para el proceso de extracción sería una hidrólisis química, en la cual se hidrolizan/extraen las proteínas llevando a ebullición con ácido clorhídrico fuerte, seguido de enfriamiento y neutralización del hidrolizado con carbonato sódico o hidróxido sódico y separación del material sólido no hidrolizado. Las concentraciones de ácido utilizadas son altas por ejemplo: Acido Clorhídrico 35% , en la hidrólisis de proteína de maíz (ES 2 034 593), Sulfúrico 3 Molar (EP2128114A2). An alternative for the extraction process would be a chemical hydrolysis, in which the proteins are hydrolyzed / extracted by boiling with strong hydrochloric acid, followed by cooling and neutralization of the hydrolyzate with sodium carbonate or sodium hydroxide and separation of the non-hydrolyzed solid material. The acid concentrations used are high, for example: Hydrochloric acid 35%, in the hydrolysis of corn protein (ES 2 034 593), 3 Molar sulfuric acid (EP2128114A2).
- 15 fifteen
- Estudios realizados han demostrado que los hidrolizados de proteína preparados con ácido clorhídrico contienen una cantidad de compuestos indeseables cono dicloropropanoles (DCPs), especialmente 1,3 dicloropropan-2-ol y monocloropropanodioles MCPs) y ha surgido el problema de su eliminación o más bien de la prevención de su formación. Studies have shown that protein hydrolysates prepared with hydrochloric acid contain an amount of undesirable compounds known as dichloropropanoles (DCPs), especially 1,3 dichloropropan-2-ol and monochloropropanediols MCPs) and the problem of their elimination or rather of The prevention of their training.
- 20 25 20 25
- La hidrólisis enzimática es un proceso muy eficiente, que se lleva a cabo en condiciones suaves de temperatura, pH y presión, con lo que la calidad nutricional de los aminoácidos no resulta alterada, ya que de esta manera, no tiene lugar la destrucción o modificación de ciertos restos aminoacídicos, como sí ocurre con la hidrólisis química. Efectivamente para la extracción de proteínas es una técnica ampliamente utilizada, debido a su eficiencia y nulo impacto ambiental, pero no ha sido descrita en el substrato descrito en la invención, esto es, plantas de tabaco con un pretratamiento físico. Enzymatic hydrolysis is a very efficient process, which is carried out under mild conditions of temperature, pH and pressure, so that the nutritional quality of the amino acids is not altered, since in this way, destruction or modification does not take place of certain amino acid residues, as is the case with chemical hydrolysis. Indeed, protein extraction is a widely used technique, due to its efficiency and no environmental impact, but it has not been described in the substrate described in the invention, that is, tobacco plants with a physical pretreatment.
- Descripción de las figuras Description of the figures
- 30 30
- Figura 1.-Esquema del proceso de obtención de extractos proteicos Figura 2.-Aspecto de la harina de hojas de tabaco Figura 3.-Solubilidad de las HHT a distintos valores de pH Figure 1.-Scheme of the process of obtaining protein extracts Figure 2.-Appearance of the flour of tobacco leaves Figure 3.-Solubility of the HHT at different pH values
- 35 35
- Figura 4.-Aspecto de los extractos proteicos obtenidos. A. Concentrado (50 materia seca) Figure 4.-Appearance of the protein extracts obtained. A. Concentrate (50 dry matter)
B. Atomizado en forma de polvo. B. Atomized in powder form.
Figura 5.-Cromatografía de exclusión molecular, en columna Superdex 200HR Figure 5.-Molecular exclusion chromatography, in Superdex 200HR column
10/30 y distribución de los pesos moleculares de los concentrados proteicos. (A) 10/30 and distribution of molecular weights of protein concentrates. (TO)
5 BB-163, (B) H-20, (C) GR, (D) BB-5 BB-163, (B) H-20, (C) GR, (D) BB-
Figura 6.-Solubilidad de los extractos proteicos obtenidos en el proceso de Figure 6.-Solubility of the protein extracts obtained in the process of
hidrólisis enzimática enzymatic hydrolysis
Descripción de la invención La Figura 1 muestra esquemáticamente el proceso de hidrólisis enzimática propuesto para obtener concentrados proteicos a partir de hojas de tabaco. Description of the invention Figure 1 schematically shows the proposed enzymatic hydrolysis process to obtain protein concentrates from tobacco leaves.
15 Las hojas de tabaco seleccionadas correspondieron a las variedades (BB-163, B-194., H-20 y G-20). Son hojas frescas y secas que se eliminaron de la planta durante la fase de despunte y que se situaban justo debajo de la inflorescencia. Las hojas de tabaco se secan en estufa a 50 o C y se trituran posteriormente obteniendo así la harina de hojas de tabaco (HHT). La Tabla 1 15 The selected tobacco leaves corresponded to the varieties (BB-163, B-194., H-20 and G-20). They are fresh and dried leaves that were removed from the plant during the emergence phase and that were located just below the inflorescence. The tobacco leaves are dried in an oven at 50 o C and subsequently crushed, thus obtaining the flour of tobacco leaves (HHT). Table 1
20 muestra la composición química de HHT y la Figura 2 muestra una fotografía del aspecto de esta HHT. La solubilidad de dichas HHT no es alta en ningún caso (Figura 3). Sólo a pH muy alcalino logramos que la solubilidad sea algo significativa, pero en ningún caso superior al 31%. Esta baja solubilidad se debe en parte al calor aplicado en el proceso de secado para la obtención de la HHT, 20 shows the chemical composition of HHT and Figure 2 shows a photograph of the appearance of this HHT. The solubility of these HHTs is not high in any case (Figure 3). Only at very alkaline pH do we achieve that the solubility is somewhat significant, but in no case greater than 31%. This low solubility is due in part to the heat applied in the drying process to obtain the HHT,
25 aumentando así su indegradabilidad. 25 thus increasing its indegradability.
HHT HHT
BB-163 H-20 GR BB-194 Humedad (g kg-1, w/w) 72 ± 10 79 ± 9 84 ± 13 80 ± 11 Proteínas (g kg-1, w/w) 239 ± 21 245 ± 26 258 ± 22 250 ± 23 Lípidos (g kg-1, w/w) 25 ± 7 21 ± 5 19 ± 6 23 ± 6 BB-163 H-20 GR BB-194 Humidity (g kg-1, w / w) 72 ± 10 79 ± 9 84 ± 13 80 ± 11 Proteins (g kg-1, w / w) 239 ± 21 245 ± 26 258 ± 22 250 ± 23 Lipids (g kg-1, w / w) 25 ± 7 21 ± 5 19 ± 6 23 ± 6
k -1k -1
Carbohidratos (g 604 ± 36 639 ± 48 680 ± 41 666 ± 42 Carbohydrates (g 604 ± 36 639 ± 48 680 ± 41 666 ± 42
g ' Cenizas (g kg-1, w/w) 95 ± 14 93 ± 11 103 ± 16 98 ± 12 lle (*) 4,1 ± 0,9 3,7±1,1 5,1±1,3 4,1±1,0 Leu (*) 12,3±1,2 7,2 ± 1,9 9,1 ± 2,1 8,3 ± 1,6 Lys (*) 2,5 ± 0,5 2,7 ± 0,8 3,3 ± 1,0 2,3 ± 0,7 Met (*) 1,8 ± 0,4 1,7 ± 0,4 0,4 ± 0,1 1,7 ± 0,2
g 'Ashes (g kg-1, w / w) 95 ± 14 93 ± 11 103 ± 16 98 ± 12 lle (*) 4.1 ± 0.9 3.7 ± 1.1 5.1 ± 1.3 4.1 ± 1.0 Leu (*) 12.3 ± 1.2 7.2 ± 1.9 9.1 ± 2.1 8.3 ± 1.6 Lys (*) 2.5 ± 0.5 2.7 ± 0.8 3.3 ± 1.0 2.3 ± 0.7 Met (*) 1.8 ± 0.4 1.7 ± 0.4 0.4 ± 0.1 1.7 ± 0.2
- Phe (*) Phe (*)
- 3,6 ± 1,0 5,2 ± 1 '1 5,7±1,1 3,1 ± 0,6 3.6 ± 1.0 5.2 ± 1 '1 5.7 ± 1.1 3.1 ± 0.6
- Thr (*) Thr (*)
- 3,6 ± 0,8 2,9 ± 0,7 5,0 ± 1,3 3,5 ± 0,8 3.6 ± 0.8 2.9 ± 0.7 5.0 ± 1.3 3.5 ± 0.8
- Trp (*) Trp (*)
- 0,8±0,1 0,8 ± 0,1 0,3 ±O, 1 0,8±0,1 0.8 ± 0.1 0.8 ± 0.1 0.3 ± O, 1 0.8 ± 0.1
- Tyr (*) Tyr (*)
- 3,4 ± 0,9 4,1 ± 1 '1 2,8 ± 0,9 1,7 ± 0,5 3.4 ± 0.9 4.1 ± 1 '1 2.8 ± 0.9 1.7 ± 0.5
- Val(*) Val (*)
- 5,5± 1,4 4,3 ± 1 '1 7,5 ± 1,9 5,2 ± 1,4 5.5 ± 1.4 4.3 ± 1 '1 7.5 ± 1.9 5.2 ± 1.4
- Tabla 1. Composición química y aminoacídica (media ± desviación standard) de Table 1. Chemical and amino acid composition (mean ± standard deviation) of
- las HHT. Los resultados son la media de tres repeticiones. the HHT. The results are the average of three repetitions.
- (*) Resultados expresados como gramos por cada 100 g de proteína (*) Results expressed as grams per 100 g of protein
- 5 5
- Una variante es la utilización de la hoja de tabaco, principalmente hojas frescas, A variant is the use of the tobacco leaf, mainly fresh leaves,
- procedentes de descartes en la recogida del material, sin secado, se procede a su coming from discards in the collection of the material, without drying, it proceeds to its
- homogenización en el agua de la hoja, la pasta resultante tendrá un 35-40% de homogenization in the leaf water, the resulting paste will have 35-40% of
- materia seca, con lo cual se ahorra agua, energía y tiempo. dry matter, which saves water, energy and time.
- 1 O 1 o
- Otra variante es la utilización de hoja de tabaco procedente de descartes del Another variant is the use of tobacco leaf from discards of
- proceso de secado/fermentación industrial. industrial drying / fermentation process.
- Los productos serán sometidos a un tratamiento físico de autoclavado, el The products will undergo a physical autoclaving treatment, the
- cual aumenta la susceptibilidad de extracción proteica por parte de las proteasas. which increases the susceptibility of protein extraction by proteases.
- El proceso se realiza a una presión interna al menos de 103 kPa, lo cual The process is carried out at an internal pressure of at least 103 kPa, which
- 15 fifteen
- provoca que el vapor alcance una temperatura de 121 grados centígrados. Siendo causes the steam to reach a temperature of 121 degrees Celsius. Being
- el tiempo del proceso entre 15-20 minutos. The process time between 15-20 minutes.
- A continuación comienza el proceso de hidrólisis enzimático (tabla 3), dicha Then the enzymatic hydrolysis process begins (table 3), said
- hidrólisis enzimática se lleva a cabo en un bioreactor con control de pH, Enzymatic hydrolysis is carried out in a bioreactor with pH control,
- temperatura y agitación, como solvente agua de la red y como medio activo temperature and stirring, as a solvent in the water network and as an active medium
- 20 twenty
- enzimas proteolíticas, preferentemente Subtilisina. A una concentración de proteolytic enzymes, preferably Subtilisin. At a concentration of
- substrato entre 15-20% peso /volumen y una concentración de proteasas entre substrate between 15-20% weight / volume and a concentration of proteases between
- 0.1-0.3% peso/volumen de reacción, a partir de una solución stock de proteasa 0.1-0.3% weight / volume of reaction, from a stock protease solution
- 70.000 Unidades de actividad/Ensayo de azocaseína)). 70,000 Activity Units / Azocasein Test)).
BB-163 H-20 GR BB-194 Humedad (g kg-1, w/w) 78 ± 6 72 ± 8 79 ± 6 76 ± 5 Proteínas (g kg-1, w/w) 801 ± 36 815 ± 42 791 ± 31 822 ± 40 Lípidos (g kg-1, w/w) 9 ± 1.1 9 ± 1.0 8 ± 0.9 9 ± 1.2
BB-163 H-20 GR BB-194 Humidity (g kg-1, w / w) 78 ± 6 72 ± 8 79 ± 6 76 ± 5 Proteins (g kg-1, w / w) 801 ± 36 815 ± 42 791 ± 31 822 ± 40 Lipids (g kg-1, w / w) 9 ± 1.1 9 ± 1.0 8 ± 0.9 9 ± 1.2
k -1k -1
Carbohidratos (g g ' 63 ± 11 68 ± 9 80 ± 13 74 ± 12 Cenizas (g kg-1, w/w) 52± 10 40 ± 7 45 ± 9 52± 12 Arg (*) 2.7 ± 0.5 2.8 ± 0.4 2.9 ± 0.4 2.8 ± 0.6
Carbohydrates (gg '63 ± 11 68 ± 9 80 ± 13 74 ± 12 Ashes (g kg-1, w / w) 52 ± 10 40 ± 7 45 ± 9 52 ± 12 Arg (*) 2.7 ± 0.5 2.8 ± 0.4 2.9 ± 0.4 2.8 ± 0.6
Tyr (*) 3.8 ± 0.9 4.1 ± 0.9 2.9 ± 0.5 2.9 ± 0.6 Ala(*) 5.6±1.1 5.5 ± 1.0 6.1 ± 0.9 5.9 ± 1.0 Asp (*) 5.9 ± 1.5 5.7 ± 1.1 6.0± 1.3 6.9± 1.4 Cis (*) 0.9 ± 0.1 0.9±0.1 0.9 ± 0.1 0.7 ± 0.1 Glu (*) 20.1 ± 1.2 22.4 ± 1.5 22.3 ± 2.0 21.0 ± 1.8 Gly (*) 4.0±1.2 3.1 ± 0.7 3.9 ± 0.8 4.2 ± 1.1 Pro(*) 8.5± 1.6 8.8± 1.4 8.3 ± 1.7 8.0 ± 1.9 Ser(*) 4.5 ± 1.0 4.8 ± 1.2 4.9±1.1 4.8± 1.4 Trp (*) 0.5 ± 0.1 0.6±0.1 0.5 ± 0.1 0.5 ± 0.1 His (*) 1.8 ± 0.5 1.9 ± 0.4 2.0 ± 0.7 2.9 ± 0.5 lle (*) 3.8 ± 0.9 4.1 ± 0.8 4.0 ± 1.0 5.6 ± 1.2 Leu (*) 7.8±1.1 7.5±1.0 7.8±1.3 7.4±1.6 Val(*) 4.1±1.0 4.4 ± 0.8 4.5 ± 1.3 3.8 ± 0.9 Lys (*) 2.8 ± 0.8 2.7 ± 0.6 3.0 ± 0.4 2.1 ± 0.7 Met (*) 1.1 ±0.3 2.0 ± 0.8 1.0 ± 0.2 3.0 ± 0.6 Tyr (*) 3.8 ± 0.9 4.1 ± 0.9 2.9 ± 0.5 2.9 ± 0.6 Wing (*) 5.6 ± 1.1 5.5 ± 1.0 6.1 ± 0.9 5.9 ± 1.0 Asp (*) 5.9 ± 1.5 5.7 ± 1.1 6.0 ± 1.3 6.9 ± 1.4 Cis ( *) 0.9 ± 0.1 0.9 ± 0.1 0.9 ± 0.1 0.7 ± 0.1 Glu (*) 20.1 ± 1.2 22.4 ± 1.5 22.3 ± 2.0 21.0 ± 1.8 Gly (*) 4.0 ± 1.2 3.1 ± 0.7 3.9 ± 0.8 4.2 ± 1.1 Pro (*) 8.5 ± 1.6 8.8 ± 1.4 8.3 ± 1.7 8.0 ± 1.9 Ser (*) 4.5 ± 1.0 4.8 ± 1.2 4.9 ± 1.1 4.8 ± 1.4 Trp (*) 0.5 ± 0.1 0.6 ± 0.1 0.5 ± 0.1 0.5 ± 0.1 His (*) 1.8 ± 0.5 1.9 ± 0.4 2.0 ± 0.7 2.9 ± 0.5 lle (*) 3.8 ± 0.9 4.1 ± 0.8 4.0 ± 1.0 5.6 ± 1.2 Leu (*) 7.8 ± 1.1 7.5 ± 1.0 7.8 ± 1.3 7.4 ± 1.6 Val (*) 4.1 ± 1.0 4.4 ± 0.8 4.5 ± 1.3 3.8 ± 0.9 Lys (*) 2.8 ± 0.8 2.7 ± 0.6 3.0 ± 0.4 2.1 ± 0.7 Met (*) 1.1 ± 0.3 2.0 ± 0.8 1.0 ± 0.2 3.0 ± 0.6
- Tabla 3. Composición química y aminoacídica (media ± desviación standard) de Table 3. Chemical and amino acid composition (mean ± standard deviation) of
- los extractos proteicos obtenidos en el proceso de hidrólisis enzimática. Los datos Protein extracts obtained in the enzymatic hydrolysis process. The data
- son la media de tres repeticiones. They are the average of three repetitions.
- 5 5
- (*) Resultados expresados como gramos por cada 100 g de proteína (*) Results expressed as grams per 100 g of protein
- En primer lugar es muy importante, previo al proceso de hidrólisis, la First of all it is very important, prior to the hydrolysis process, the
- homogeneización y solubilización de los sustratos. El desarrollo de la reacción homogenization and solubilization of substrates. The reaction development
- enzimática se realiza un pH=8-9,5 y P=50-60°C, lo que supone las condiciones Enzymatic pH is performed = 8-9.5 and P = 50-60 ° C, which assumes the conditions
- 1O 1O
- óptimas de máxima actividad de las enzimas empleadas. La temperatura se regula optimal maximum activity of the enzymes used. The temperature is regulated
- con baño termostatizado y el pH con NaOH 6N para no incrementar en exceso el with thermostated bath and the pH with 6N NaOH so as not to excessively increase the
- volumen de reacción. La etapa de disolución del sustrato dura hasta que el pH se reaction volume The substrate dissolution stage lasts until the pH is
- equilibra (estabiliza); después de esta etapa empieza la hidrólisis con la adición de balances (stabilizes); after this stage hydrolysis begins with the addition of
- la enzima, el pH bajará a liberarse los restos amino terminales, por lo que se the enzyme, the pH will lower to release the amino terminal residues, so
- 15 fifteen
- requiere la adición de NaOH para seguir manteniendo el pH óptimo de actuación requires the addition of NaOH to continue maintaining the optimum pH of action
- de la enzima. of the enzyme.
- La principal diferencia entre los componentes proteicos de las HHT y sus The main difference between the protein components of HHT and their
- correspondientes hidrolizados es el tamaño molecular de estos componentes. En Corresponding hydrolysates is the molecular size of these components. In
- el caso de los hidrolizados son fundamentalmente péptidos, oligopéptidos y In the case of hydrolysates, they are mainly peptides, oligopeptides and
- 20 twenty
- aminoácidos libres (Figura 5), mientras que la mayoría de los componentes de las free amino acids (Figure 5), while most of the components of
- HHT son proteínas de alto peso molecular. HHT are high molecular weight proteins.
- Al cambiar el tamaño molecular, aumenta drásticamente la solubilidad (Figura By changing the molecular size, the solubility increases dramatically (Figure
- 6), aumentando así su absorción por la planta. Además, estos hidrolizados 6), thus increasing its absorption by the plant. In addition, these hydrolysates
- 5 5
- proteicos debido a su relativamente alto grado de hidrólisis (>15%), pueden considerarse un producto potencialmente hipoalergénico para alimentación animal y como una buena fuente de nitrógeno orgánico para las plantas en caso de utilizarse como biofertilizante/bioestimulante, bien a través de la raíz o foliarmente. Proteins due to their relatively high degree of hydrolysis (> 15%), can be considered a potentially hypoallergenic product for animal feed and as a good source of organic nitrogen for plants if used as a biofertilizer / biostimulant, either through the root or foliarly.
- Modo de realización de la invención Embodiment of the invention
- EJEMPLO: EXAMPLE:
- 1 O 15 1 or 15
- A continuación se muestra un ejemplo de obtención de un extracto proteico a partir de hoja de tabaco, se ha procedido a utilizar la harina de hojas de tabaco (HHT). Una vez establecidas las condiciones óptimas del proceso hidrolítico, se ha elegido para la obtención de un extracto proteico los: temperatura 60°C, pH 8 y concentración de sustrato (15% p/v) y Subtilisina (0.2% peso/volumen, solución stock de proteasa 70.000 Unidades de actividad/Ensayo de azocaseína) este proceso se mantiene 120 minutos, alcanzándose un grado de hidrólisis final superior, en todos los casos, al 15%. El procesamiento del medio de hidrólisis continúa con diferentes etapas: centrifugación, concentración (30-60% en Materia seca) y una etapa opcional de secado y conversión en polvo (atomización). La Figura 4 muestra una fotografía del aspecto del extracto proteico que se obtiene. Below is an example of obtaining a protein extract from tobacco leaf, the use of tobacco leaf flour (HHT) has been used. Once the optimal conditions of the hydrolytic process have been established, the following are chosen to obtain a protein extract: temperature 60 ° C, pH 8 and substrate concentration (15% w / v) and Subtilisin (0.2% weight / volume, solution Protease stock 70,000 Units of activity / Azocasein test) This process is maintained for 120 minutes, reaching a final degree of hydrolysis in all cases, 15%. The processing of the hydrolysis medium continues with different stages: centrifugation, concentration (30-60% in dry matter) and an optional stage of drying and conversion into powder (atomization). Figure 4 shows a photograph of the appearance of the protein extract obtained.
- 20 25 30 20 25 30
- Para determinar los efectos de este nuevo producto, se seleccionó el cultivo de tomate (Lycopersicon escu/entum cv. Momotaro) como cultivo indicador. Del mismo modo, se utilizó un extracto hidrolizado de algarroba para comparar los efectos de este biofertilizante/bioestimulante con el obtenido. El proceso de extracción y composición química del extracto enzimático de algarrobo se encuentran detallados en un trabajo anterior (Parrado et al., 2008). Se seleccionaron semillas de tomate y se hicieron germinar en condiciones controladas de temperatura (20 °C) y humedad (60%). Las plántulas fueron transferidas a contenedores que tenían una turba comercial como sustrato. El diseño experimental fue en bloques al azar, incluyendo 9 contenedores y 8 plántulas de tomate por contenedor. Se realizaron 3 tratamientos fertilizantes, realizándose 3 repeticiones por tratamiento. Los tratamientos fueron: To determine the effects of this new product, the tomato crop (Lycopersicon escu / entum cv. Momotaro) was selected as the indicator crop. Similarly, a hydrolyzed carob extract was used to compare the effects of this biofertilizer / biostimulant with that obtained. The process of extraction and chemical composition of the enzymatic extract of carob tree are detailed in a previous work (Parrado et al., 2008). Tomato seeds were selected and germinated under controlled conditions of temperature (20 ° C) and humidity (60%). The seedlings were transferred to containers that had a commercial peat as a substrate. The experimental design was randomized blocks, including 9 containers and 8 tomato seedlings per container. 3 fertilizer treatments were performed, performing 3 repetitions per treatment. The treatments were:
- (1) tratamiento AO: contenedores fertilizados con una solución NPK (0.6 g N r1 , o.o3 g P 1-1 , o.o9 g K r1) (1) AO treatment: containers fertilized with an NPK solution (0.6 g N r1, o.o3 g P 1-1, o.o9 g K r1)
- (2) (2)
- tratamiento A 1: contenedores fertilizados con un concentrado proteico de algarroba a una dosis de 1 cm3/500 ml-1 agua treatment A 1: fertilized containers with a carob protein concentrate at a dose of 1 cm3 / 500 ml-1 water
- (3) (3)
- tratamiento A2: contenedores fertilizados con el nuevo concentrado proteico de hojas de tabaco a una dosis de 1 cm3/500 ml-1 agua A2 treatment: fertilized containers with the new protein concentrate of tobacco leaves at a dose of 1 cm3 / 500 ml-1 water
El tiempo experimental fue de 18 semanas, durante el cual se midió la altura de planta, se determinó el número de flores por planta, y el número de frutos por planta. The experimental time was 18 weeks, during which the plant height was measured, the number of flowers per plant was determined, and the number of fruits per plant.
La tabla 4 muestra los parámetros analizados en el cultivo de tomate para Table 4 shows the parameters analyzed in tomato cultivation for
10 los diferentes tratamientos. El análisis estadístico mostró diferencias significativas (p <0,05) entre el tratamiento no orgánico (AO) y los dos tratamientos orgánicos (A1 y A2), mostrando los valores más altos en los parámetros estudiados para los tratamientos A 1 y A2. Parrado et al (2008) encontró la eficacia de un extracto enzimático de algarrobo en un cultivo de tomate haciendo hincapié en la 10 different treatments. The statistical analysis showed significant differences (p <0.05) between the non-organic treatment (AO) and the two organic treatments (A1 and A2), showing the highest values in the parameters studied for treatments A 1 and A2. Parrado et al (2008) found the efficacy of an enzymatic extract of carob in a tomato crop emphasizing the
15 importancia de este bioestimulantes/biofertilizante en el crecimiento de tomate y de la calidad del fruto obtenido. Los resultados encontrados para los concentrados de proteína de tabaco en hoja son ligeramente superiores a los obtenidos para los concentrados de proteínas de algarroba. Por lo tanto, creemos que al igual que los concentrados de proteínas de algarroba, las hojas de tabaco de concentrados de 15 importance of this biostimulants / biofertilizer in tomato growth and the quality of the fruit obtained. The results found for leaf tobacco protein concentrates are slightly higher than those obtained for carob protein concentrates. Therefore, we believe that like carob protein concentrates, tobacco leaves concentrate
20 proteínas puede ser utilizado como bioestimulantes 1 biofertilizante en la agricultura. 20 proteins can be used as biostimulants 1 biofertilizer in agriculture.
Tiempo Altura de planta (cm) Número de flores por planta Número de frutos por planta experimental (semanas) Time Plant height (cm) Number of flowers per plant Number of fruits per experimental plant (weeks)
Tratamiento Tratamiento Tratamiento Tratamiento Tratamiento Tratamiento Tratamiento Tratamiento Tratamiento AO A1 A2 AO A1 A2 AO A1 A2 Treatment Treatment Treatment Treatment Treatment Treatment Treatment Treatment Treatment AO A1 A2 AO A1 A2 AO A1 A2
- 8 8
- Nd Nd Nd 1at 1a 1a Nd Nd Nd 1at 1st 1st
- 10 10
- Nd Nd Nd 3ab± 1 4b ± 1 4b ± 1 Nd Nd Nd 3ab ± 1 4b ± 1 4b ± 1
- 12 12
- Nd Nd Nd ?be± 1 7bc±2 Be± 2 1at 1a 1a Nd Nd Nd ? be ± 1 7bc ± 2 Be ± 2 1at 1st 1st
- 14 14
- Nd Nd Nd 8c±2 14cd ± 2 15cd ± 2 1a 2b± 1 2b ± 1 Nd Nd Nd 8c ± 2 14cd ± 2 15cd ± 2 1st 2b ± 1 2b ± 1
- 16 16
- Nd Nd Nd 9c± 2 31d ±4 33d ±4 2b± 1 4c± 1 4c ± 1 Nd Nd Nd 9c ± 2 31d ± 4 33d ± 4 2b ± 1 4c ± 1 4c ± 1
- 18 18
- 54.1at± 1.4 82.5b ± 2.1 84.6b ± 2.5 9c±2 33d ±5 34d ±4 3bc ± 1 15d ± 3 16d ±2 54.1at ± 1.4 82.5b ± 2.1 84.6b ± 2.5 9c ± 2 33d ± 5 34d ± 4 3bc ± 1 15d ± 3 16d ± 2
Tabla 4. Parámetros obtenidos (media ± desviación standard) en el cultivo de tomate. Los datos son la media de tres repeticiones
Table 4. Parameters obtained (mean ± standard deviation) in tomato cultivation. The data is the average of three repetitions
Nd: no determinado tcolumnas (media ± desviación standard) seguidas de la misma letra no son significativamente diferentes (p<0.05) Nd: not determined tcolumns (mean ± standard deviation) followed by the same letter are not significantly different (p <0.05)
Claims (7)
- 2. 2.
- Procedimiento para la obtención de extractos proteicos a partir de hojas de tabaco mediante hidrólisis enzimática según reivindicación 1, caracterizado porque el concentrado de hojas de tabaco se obtiene: a) principalmente a partir hojas frescas, las cuales serán secadas a 50°C, trituradas y mediante la adición de agua se obtendrá el concentrado con 10-20% de materia, b) sin secado, se procede a su homogenización en el agua de la hoja, de tal manera que la pasta resultante tendrá un 35-40% de materia seca y mediante la adición de agua se obtendrá el concentrado Procedure for obtaining protein extracts from tobacco leaves by enzymatic hydrolysis according to claim 1, characterized in that the concentrate of tobacco leaves is obtained: a) mainly from fresh leaves, which will be dried at 50 ° C, crushed and by adding water, the concentrate with 10-20% of matter will be obtained, b) without drying, homogenization is carried out in the leaf water, so that the resulting paste will have 35-40% dry matter and by adding water the concentrate will be obtained
- 3. 3.
- Procedimiento para la obtención de extractos proteicos a partir de hojas de tabaco mediante hidrólisis enzimática según reivindicaciones anteriores, caracterizado porque las hojas de tabaco son pretratadas físicamente, Method for obtaining protein extracts from tobacco leaves by enzymatic hydrolysis according to previous claims, characterized in that the tobacco leaves are physically pretreated,
- 4. Four.
- Procedimiento para la obtención de extractos proteicos a partir de hojas de tabaco mediante hidrólisis enzimática según reivindicación 3 caracterizado porque el tratamiento de autoclavado se realiza a una presión interna al menos de 103 kPa, lo cual provoca que el vapor alcance una temperatura de 121 grados centígrados durante un tiempo entre 15-20 minutos. Procedure for obtaining protein extracts from tobacco leaves by enzymatic hydrolysis according to claim 3 characterized in that the autoclaving treatment is carried out at an internal pressure of at least 103 kPa, which causes the steam to reach a temperature of 121 degrees Celsius for a time between 15-20 minutes.
- 5. 5.
- Utilización de los extractos proteicos obtenidos según el procedimiento descrito en las reivindicaciones anteriores compuestos principalmente por nitrógeno orgánico en forma de péptidos y aminoácidos libres como agente con una alta potencia biofertilizante/bioestimulante en todo tipos de Use of the protein extracts obtained according to the method described in the preceding claims composed mainly of organic nitrogen in the form of free peptides and amino acids as an agent with a high biofertilizing / biostimulant power in all types of
- mediante through
- un tratamiento de autoclavado, el cual aumenta la a treatment from autoclaved, he which increases the
- susceptibilidad susceptibility
- de extracción proteica por parte de las enzimas from extraction protein by part from the enzymes
- proteolíticas. proteolytic
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