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ES2643148T3 - Composición en polvo seco estable que comprende microorganismos biológicamente activos y/o materiales bioactivos y métodos de producción - Google Patents

Composición en polvo seco estable que comprende microorganismos biológicamente activos y/o materiales bioactivos y métodos de producción Download PDF

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ES2643148T3
ES2643148T3 ES10781100.2T ES10781100T ES2643148T3 ES 2643148 T3 ES2643148 T3 ES 2643148T3 ES 10781100 T ES10781100 T ES 10781100T ES 2643148 T3 ES2643148 T3 ES 2643148T3
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formulation
temperature
dry powder
biologically active
vacuum
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Moti Harel
Roger Drewes
Brian Carpenter
Elena Artimovich
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Advanced Bionutrtion Corp
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Advanced Bionutrtion Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/174—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K40/00—Shaping or working-up of animal feeding-stuffs
    • A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A23K50/42—Dry feed
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/06—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/146—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic macromolecular compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
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Abstract

Una composición seca estable que comprende (i) un microorganismo o material bioactivo, (ii) al menos dos agentes estabilizadores y (iii) al menos dos agentes protectores, donde el microorganismo o material bioactivo es un probiótico; donde los al menos dos agentes estabilizadores comprenden alginato de sodio e inulina; donde los al menos dos agentes protectores comprenden una mezcla de un disacárido, y un hidrolizado de proteína; y donde el microorganismo o material bioactivo está encerrado dentro de una matriz vítrea amorfa.

Description

imagen1
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formulación.
En una realización, por ejemplo, una formulación que comprende un microorganismo o material bioactivo, los agentes estabilizadores y los agentes protectores se mezclan hasta homogeneidad, bajo vacío suave de
5 aproximadamente 10 a 50 Torr, en una solución. La Figura 6 muestra el efecto de diferentes densidades de la formulación sobre su expansión bajo vacío. La introducción de aire durante la mezcla de los componentes de la formulación en una solución da como resultado excesiva y descontrolable formación de espuma incluso a presión de vacío relativamente alta. La etapa de mezcla bajo vacío según la invención resuelve este problema eliminando la introducción de aire o gas en la solución de la formulación, eliminando de ese modo la excesiva y descontrolada formación de espuma de la solución.
A continuación, la solución se enfría hasta una temperatura por encima de su punto de congelación (normalmente entre -5 ºC y +5 ºC). La Figura 7 muestra el efecto de preenfriamiento de la solución de la formulación sobre su expansión bajo presión de vacío. Sorprendentemente e inesperadamente se encontró que la ebullición se puede
15 eliminar eficazmente incluso bajo una presión de vacío relativamente mayor y la expansión de la formulación se controla mejor cuando la temperatura de la solución se reduce a no más de 10 ºC por encima de su temperatura de congelación. Como se ve en la Figura 7, se puede aplicar una presión de vacío de 3 Torr sin excesiva formación de espuma siempre que se enfríe la formulación a +5 ºC y preferiblemente a -3 ºC.
Una vez enfriada, la formulación se seca a continuación bajo vacío suficiente (por ejemplo, aproximadamente 3 Torr) para mantener esa temperatura de preenfriamiento durante la etapa de secado primario. La Figura 8 muestra el efecto de la presión de vacío aplicada sobre la temperatura de la solución de la formulación. A presión de vacío relativamente alta por encima de 8 Torr, la temperatura de formulación se incrementó a más de 6 ºC y continua incrementándose rápidamente hacia la temperatura de estante o cámara. Al mismo tiempo, la solución continuará
25 formando espuma y expandiéndose más. Esta realización se distingue de la técnica anterior anteriormente discutida (véase, por ejemplo, el documento de patente americana Nº 6.534.087, en el que la presión de vacío aplicada está entre 3 a 7 Torr e incluso mayor), en la cual se aplica una presión de vacío más fuerte (<3 Torr) mientras se controla la expansión de la formulación. Este proceso da como resultado una tasa de secado significativamente más rápida (véase la Figura 9) y posibilita una alta capacidad de carga de la formulación. En esta realización, se elimina la excesiva formación de espuma y ebullición incluso bajo presiones de vacío mucho más bajas debido a que los métodos de la invención proporcionan a) una composición específica con una expansión controlada bajo vacío, b) un método que elimina la introducción de aire en la formulación durante la mezcla y c) un preenfriamiento sustancial de la formulación.
35 Los métodos típicos en la técnica anterior implican extensa formación de espuma y/o salpicado y ebullición violenta que pueden ser dañinos a productos biológicos sensibles y causar dificultades para el escalado industrial. Además, una desgasificación completa y eficaz de suspensiones viscosas es difícil y puede requerir un periodo de tiempo prolongado. Estos obstáculos se resolvieron en la presente invención llevando a cabo primero el proceso de mezcla completo bajo vacío suave para eliminar la introducción de gases incluidos en la formulación en primer lugar. Cualquier pequeña cantidad de gases solubles que puede quedar en la formulación, se elimina suavemente a continuación, permitiendo que la formulación se expanda moderadamente bajo vacío bajo. La etapa de preenfriamiento adicional de la formulación a una temperatura por encima de su temperatura de congelación proporciona un control añadido de la tasa de expansión y permite de ese modo una capacidad de carga por área de secado mucho mayor de la que se obtenía de acuerdo con la técnica anterior. Después de que se complete la fase
45 de secado primario, la formulación seca estabilizada se mantiene a temperaturas de secado secundario elevadas (hasta 70 ºC) y presiones de vacío de menos de 0,2 Torr para completar el secado de la formulación en un tiempo muy corto.
Otra realización de la invención proporciona métodos para preparar composiciones de la formulación en forma de hidrogel para la conservación de microorganismos o materiales bioactivos. Por ejemplo, una formulación que contiene una bacteria probiótica en una forma en polvo seco, un agente estabilizador y un agente protector, se mezclan en una solución, se reticula para formar un hidrogel añadiendo iones metálicos o cationes divalentes y, a continuación, se secan bajo vacío suave y baja temperatura como se describió anteriormente. La temperatura de preenfriamiento de la formulación se puede mantener mediante la conducción de calor fuera de la formulación, y/o
55 por perdida de calor latente debido a la evaporación de agua.
En una realización particular de la invención, por ejemplo, la formulación incluye un cultivo concentrado, fresco o congelado, o seco de bacterias probióticas vivas en una solución de 1 a 2,5 % de alginato de sodio (preferiblemente 1,5 % de alginato de sodio ), de 1 % a aproximadamente 5 % de inulina (preferiblemente2,5 % de inulina), de 20 % a 60 % de trehalosa (preferiblemente 40 % de trehalosa) y de 3 % a 15 % de hidrolizado de caseína (preferiblemente 8 % de hidrolizado de caseína al). La formulación se mezcla bajo vacío a una temperatura ligeramente por encima de la temperatura ambiente (generalmente entre 25 ºC a 37 ºC) hasta que todos los componentes se disuelven completamente.
65 En una realización adicional de la invención, todos los ingredientes se disuelven en la solución a temperatura elevada, a continuación, se enfría la suspensión a una temperatura entre 0 ºC a -5 ºC y un polvo seco de
9
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para el ensayo en el que el secador se mantiene a 30 ºC y comparado con aquellos en los que el secador se mantiene a 50 ºC se muestran en la Tabla de a continuación.
Actividad del agua después del secado
0,23 0,26
Pérdidas durante el secado
0,6 log 0,7 log
Pérdidas durante el almacenamiento
0,8 log 0,9 log
5 Ejemplo 15
Se mezclan diez (10) gramos de Lactobacillus acidophilis seco con 10 g de trehalosa y se apartan brevemente mientras 65,3 g de trehalosa, 3 g de alginato de sodio, 5 g de inulina y 16,7 g de hidrolizado de suero de leche se mezclan juntos como un polvo seco y se añaden lentamente a 100 g de agua desionizada a 50 ºC en un mezclador 10 planetario doble cubierto, y se mezclan durante 5 minutos a 40 rpm. La suspensión se enfría a 4 ºC. A esta suspensión enfriada se añade la premezcla de Lactobacillus acidophilis y trehalosa, y el mezclado se continúa durante unos 5 minutos adicionales a 4 ºC. La suspensión homogénea se extiende uniformemente sobre una bandeja a una capacidad de carga de 100 g/pies cuadrados, y la bandeja se coloca sobre un estante en un liofilizador (Modelo 25 SRC, Virtis, Gardiner, NY). La temperatura del estante se fija a 5 ºC para mantener la 15 temperatura de la suspensión fría. Se aplica vacío para reducir la presión a 3 Torr, en dicho momento la temperatura del estante se eleva a 30 ºC. Después de 2 horas la presión se reduce más hasta 150 miliTorr con la temperatura del estante mantenida aún a 30 ºC. El secado se continúa durante unas 3 horas adicionales y en dicho momento la temperatura del producto se ha elevado a una temperatura dentro de 2 ºC de la temperatura del estante. El producto secado se saca del liofilizador. La actividad del agua de la formulación seca en este momento es de Aw=0,23
20 medida por un instrumento Hygropalm Aw1. Las pérdidas de viabilidad durante los procesos de formulación, preparación y secado ascienden a 0,6 logs.
Ejemplo 16
25 Se añaden lentamente cien (100) gramos de premezcla de soja a 100 g de agua desionizada a 35 ºC en un mezclador doble cubierto, y se mezclan durante 10 minutos a 40 rpm. Diez (10) gramos de Bifidobacterium lactis Bb12 seco se añaden lentamente mezclando a 20 rpm, y la suspensión se mezcla durante unos 5 minutos adicionales. La suspensión homogénea se extiende uniformemente sobre una bandeja a una capacidad de carga de 100 g/pies cuadrados, y la bandeja se coloca sobre un estante en un liofilizador (Modelo 25 SRC, Virtis, Gardiner, NY). La
30 temperatura del estante se fija a 5 ºC para enfriar la suspensión. Se aplica vacío para reducir la presión a 3 Torr, en dicho momento la temperatura del estante se eleva a 30 ºC. Después de 2 horas la presión se reduce más hasta 150 miliTorr con la temperatura del estante mantenida aún a 30 ºC. El secado se continúa durante unas 3 horas adicionales y en dicho momento la temperatura del producto se ha elevado a una temperatura dentro de 2 ºC de la temperatura del estante. El producto secado se saca del liofilizador y la actividad del agua de la formulación seca en
35 este momento es de Aw=0,26 medida por un instrumento Hygropalm Aw1. Las pérdidas de viabilidad durante los procesos de formulación, preparación y secado ascienden a 0,7 logs.
El ensayo de estabilidad de almacenamiento de la formulación seca bajo condiciones de almacenamiento acelerado de 32 ºC y HR al 20 % muestra una pérdida de viabilidad de las bacterias probióticas estabilizadas en la formulación
40 de la invención de solamente de 0,7 logs después de cuatro semanas.
Ejemplo 17
45 Los mismos parámetros que el Ejemplo Nº 1, excepto que la mezcla realizada en el mezclador Ross es bajo 25 pulgadas de vacío para dar una densidad de suspensión de 1,2 g/cc. La actividad del agua de la formulación seca en este punto es de Aw=0,26 medida por el instrumento Hygropalm Aw1. Las pérdidas de viabilidad durante los procesos de formulación, preparación y secado ascienden a 0,5 logs.
50 El ensayo de estabilidad de almacenamiento de la formulación seca bajo condiciones de almacenamiento acelerado de 32 ºC y HR al 20 % muestra una pérdida de viabilidad de las bacterias probióticas estabilizadas en la formulación de la invención de solamente 0,7 logs después de cuatro semanas.
Ejemplo 18
55 Se añaden cien (100) gramos de un concentrado líquido fresco de bacterias LGG (que contienen sólidos al 10 % y el resto agua) a un mezclador planetario doble cubierto y se calientan a 35 ºC. A esto se añaden lentamente 100 g de premezcla de suero de leche (Tabla 1). La suspensión resultante se mezcla 10 minutos a 40 rpm. La suspensión homogénea se extiende uniformemente sobre una bandeja a una capacidad de carga de 100 g/pies cuadrados, y la
60 bandeja se coloca sobre un estante en un liofilizador (Modelo 25 SRC, Virtis, Gardiner, NY). La temperatura del estante se fija a 5 ºC para enfriar la suspensión. Se aplica vacío para reducir la presión a 3 Torr, en dicho momento la temperatura del estante se eleva a 30 ºC. Después de 2 horas la presión se reduce más hasta 150 miliTorr con la temperatura del estante mantenida aún a 30 ºC. El secado se continúa durante unas 3 horas adicionales y en dicho
16
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CA2763074A1 (en) 2010-12-02
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US20220081471A1 (en) 2022-03-17
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