WO2015019307A1 - Microcapsules contenant des probiotiques et leurs méthodes de fabrication - Google Patents

Microcapsules contenant des probiotiques et leurs méthodes de fabrication Download PDF

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Publication number
WO2015019307A1
WO2015019307A1 PCT/IB2014/063750 IB2014063750W WO2015019307A1 WO 2015019307 A1 WO2015019307 A1 WO 2015019307A1 IB 2014063750 W IB2014063750 W IB 2014063750W WO 2015019307 A1 WO2015019307 A1 WO 2015019307A1
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microcapsule
probiotic
microcapsules
protein
alginate
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PCT/IB2014/063750
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English (en)
Inventor
Michael T. NICKERSON
Nicholas H. LOW
Darren R. KORBER
Jiapei WANG
Nurul H. KHAN
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University Of Saskatchewan
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/38—Other non-alcoholic beverages
    • A23L2/382—Other non-alcoholic beverages fermented
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A23P—SHAPING OR WORKING OF FOODSTUFFS, NOT FULLY COVERED BY A SINGLE OTHER SUBCLASS
    • A23P10/00—Shaping or working of foodstuffs characterised by the products
    • A23P10/30—Encapsulation of particles, e.g. foodstuff additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/20—After-treatment of capsule walls, e.g. hardening
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/20—After-treatment of capsule walls, e.g. hardening
    • B01J13/206—Hardening; drying
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/04—Reinforcing macromolecular compounds with loose or coherent fibrous material
    • C08J5/0405—Reinforcing macromolecular compounds with loose or coherent fibrous material with inorganic fibres
    • C08J5/042—Reinforcing macromolecular compounds with loose or coherent fibrous material with inorganic fibres with carbon fibres
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C2220/00—Biochemical treatment
    • A23C2220/20—Treatment with microorganisms
    • A23C2220/204—Use of bacteria which are encapsulated, entrapped or immobilised; Fermentation with these bacteria
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines

Definitions

  • Some embodiments of the present invention pertain to microcapsules containing probiotics, probiotics and prebiotics, and/or synbiotics. Some embodiments of the present invention pertain to methods of making microcapsules containing probiotics, probiotics and prebiotics, and/or synbiotics.
  • Probiotics are 'live microorganisms' or 'live microbial feed supplements' that offer health benefits to their host, and include primarily bacteria from the Lactobacillus and
  • Beneficial effects on the host are thought to include: promoting digestion and mineral absorption; maintaining intestinal microbial balance; improving immune system response and resistance to pathogens; preventing intestinal tract infection; reducing the risk of cardiovascular disease, cancer, obesity and type-2 diabetes; regulating lipid levels and serum cholesterol; ameliorating lactose intolerance; as well as treating certain symptoms, such as inflammation, autoimmune responses, or allergies (Gibson & Roberfroid, 1995; Collins & Gibson, 1999; Shortt, 1999; Hooper et al., 2001; Pridmore et al., 2004; Sartor, 2004; Bielecka, 2007; Rastall, 2007; Sarkar, 2007).
  • Bifidobacteria are non-motile, non-spore forming and strictly anaerobic Gram-positive bacteria that grow at pH values between 4.5 and 8.5 (Scardovi, 1986; Rokka and Rantamaki, 2010). They are indigenous to the human intestine (Fuller, 1991) and play an important beneficial role in inhibiting proliferation of potentially harmful microorganisms in the gastrointestinal tract (Hoover, 1993; Gibson & Roberfroid, 1995; Yaeshima, 1996; Holzapfel, Haberer, Snel, Schillinger, & Huis in't Veld, 1998; Alander et al., 1999).
  • Bifidobacteria species include Bifidobacteria adolescentis, Bifidobactertium animalis,
  • Lactobacilli are large, non-spore forming, Gram-positive rods, that have anaerobic or microaerophilic respiration. Gram-positive bacteria have a thick peptidoglycan layer as part of their cell wall whereas Gram-negative bacteria have a thin layer. Anaerobic bacteria will not grow in the presence of oxygen and therefore an atmosphere devoid of oxygen must be present in order for their growth to occur. Microaerophilic organisms are organisms that require a lower concentration of oxygen than that found in air.
  • Lactobacillus bacteria are microaerophilic and are commonly used as a starter culture in yogurt production and are the most commonly used probiotics in foods.
  • Exemplary Lactobacillus species used as probiotics include L. delbreuckii subspecies bulgaricus, L. acidophilus, L. casei, L. germentum, L. plantarum, L. brevis, L.
  • Prebiotics are compounds that support probiotic growth. Prebiotics act to increase the number and activity of beneficial bacteria which already colonize the colon.
  • Exemplary prebiotics include non-digestible carbohydrates such as: resistant starch (starch which is not hydrolysed in the small intestine), non-starch polysaccharides (hemicellulose, pectins, gums), and oligosaccharides (galactooligosaccharides, fructoligosaccharides, maltoligosaccharides), lactulose, inulin, and the like.
  • Synbiotics are nutritional supplements containing both probiotics and prebiotics. In some cases the combination of prebiotics and probiotics produces synergistic health benefits, hence the term “synbiotics” is used.
  • probiotic strains are sensitive to the harsh environmental conditions within foods, during processing and during transit through the gastrointestinal tract (Charteris, Kelly, Morelli, & Collins, 1998; Clark & Martin, 1994; Truelstrup Hansen, Allan- Wojtas, Jin, & Paulson, 2002).
  • GI lower gastrointestinal
  • encapsulation technology may be used to deliver therapeutic levels of both prebiotics and probiotics to the GI system, providing maximum health benefits of these agents.
  • Encapsulation technology involves encasing the sensitive core ingredients within a biopolymer shell, which can release its contents at controlled rates once triggered by an external sensor (e.g., temperature, pH, enzymes, etc.).
  • an external sensor e.g., temperature, pH, enzymes, etc.
  • alginate-based capsules seem to dominate.
  • Alginate is a linear heteropolysaccharide comprised of D-mannuronic and L-guluronic acids; the latter being highly- sensitive to divalent calcium ions resulting in the formation of strong egg box-like junction zones.
  • past alginate-probiotic capsules have been shown to be ineffective at adequately protecting probiotic bacteria subjected to simulated gastric juice.
  • small capsules ( ⁇ 100 ⁇ ) can be produced using an emulsification technique, allowing capsules to be incorporated into foods without affecting their sensory attributes (Cui, Goh, Kim, Choi, & Lee, 2000; Lee & Heo, 2000; Truelstrup Hansen et al., 2002; Chandramouli, Kailasapathy, Peiris, & Jones, 2004).
  • Plant proteins are becoming increasingly important to the food industry as a replacement for animal-derived proteins (e.g., gelatin and whey) for use as food and encapsulating ingredients.
  • Plant proteins represent an attractive alternative because of their low cost, renewability and functionality and as replacements for animal proteins based on consumer choices (e.g., vegans) and religious practices. They also are advantageous when used in combination with polysaccharides as wall materials, as they can be designed to have pH- or enzymatic triggers for controlled release purposes.
  • the application of plant proteins as encapsulating agents for probiotic delivery is currently very limited; however, this practice could be developed for use in non-dairy markets and products. Klemmer, Korber, Low, and Nickerson (2011) recently determined that pea protein isolate-alginate mixed capsules prepared by extrusion offered significant protection to B. adolescentis within simulated gastric juice (SGJ) over 2 h/37°C, and showed prolonged release within simulated intestinal fluids (SIF). However, capsule sizes were too large ( ⁇ 2 mm) for use as food supplements.
  • SGJ gastric juice
  • SIF simulated intestinal fluids
  • microcapsules having a smaller particle size acceptable for incorporation into food products that does not adversely affect the sensory attributes of the food product (i.e. less than about 100 ⁇ diameter).
  • Microcapsules having a smaller particle size have a greater surface area to volume ratio, leading to increased exposure to surface area and decreasing the survival of probiotics.
  • Polysaccharide-based microcapsules can be made, but provide poor survival rates for probiotics.
  • a microcapsule for protecting a probiotic and/or delivering a probiotic to an intestine of a subject comprises a biopolymer, a plant protein and a probiotic, and the microcapsule is prepared by crosslinking the biopolymer in an emulsion of the biopolymer, the plant protein, the probiotic, and an oil to encapsulate the probiotic.
  • a method of preparing a microcapsule includes combining a plant protein, a biopolymer and a probiotic to form an aqueous phase, adding an oil to the aqueous phase to form an emulsion, adding a crosslinker to crosslink the biopolymer, and separating the microcapsules from the oil phase of the emulsion.
  • the plant protein is chickpea protein isolate, pea protein isolate, and/or soy protein isolate.
  • the biopolymer is alginate, iota-carrageenan, and/or deacylated gellan gum.
  • the probiotic is from the Lactobacillus or Bifidobacterium genera. In some embodiments, the probiotic is encapsulated together with a prebiotic.
  • the microcapsules have a diameter of between about 20 and about 100 micrometers. In some embodiments, the microcapsules are incorporated into a food product. In some embodiments, the food product is yogurt, fruit juice, a cereal product, or a dried food product. In some embodiments, the microcapsule provides delivery of the encapsulated probiotic to the intestine of a mammalian subject.
  • Figure 4 shows the survival of B. adolescentis entrapped in alginate microcapsules incorporating different plant-based proteins (pea, soy, faba, and lentil) as a function of time within synthetic gastric juice.
  • Figure 5 shows the release of B. adolescentis entrapped in alginate microcapsules incorporating different plant-based proteins (pea, soy, faba, and lentil) as a function of time within simulated intestinal fluid.
  • Figure 8A shows an SEM (scanning electron microscopy) image of a microcapsule prepared from chickpea protein isolate and alginate at 625x magnification.
  • Figure 8B shows an SEM image of a microcapsule prepared from chickpea protein isolate and alginate at 2500x magnification.
  • Figure 9A shows an SEM image of a microcapsule prepared from pea protein isolate and alginate at 625x magnification.
  • Figure 9B shows an SEM image of a microcapsule prepared from pea protein isolate and alginate at 2500x magnification.
  • Figure 10A shows an SEM image of a microcapsule prepared from soy protein isolate and alginate at 625x magnification.
  • Figure 10B shows an SEM image of a microcapsule prepared from soy protein isolate and alginate at 2500x magnification.
  • Figure 11 A shows an SEM image of a microcapsule prepared from faba bean protein isolate and alginate at 625x magnification.
  • Figure 11B shows an SEM image of a microcapsule prepared from faba bean protein isolate and alginate at 2500x magnification.
  • Figure 12A shows an SEM image of a microcapsule prepared from lentil protein isolate and alginate at 625x magnification.
  • Figure 11B shows an SEM image of a microcapsule prepared from lentil protein isolate and alginate at 2500x magnification.
  • Some embodiments of the present invention pertain to micron- sized plant protein-based capsules for carrying probiotic bacteria, and/or mixtures of prebiotics and probiotic bacteria, and/or probiotic-prebiotic synbiotics.
  • the capsules can protect the probiotic bacteria against conditions of the stomach (e.g. gastric pH) and facilitate targeted delivery of the probiotic bacteria to the intestines of a mammalian subject.
  • conditions of the stomach e.g. gastric pH
  • the capsules are suitable for use as a food ingredient, being sufficiently small in size to avoid adverse effects on a tongue of a subject.
  • the capsules are used as nutritional supplements or ingredients in human food products and/or animal feed.
  • Gastric conditions are conditions similar to the conditions that would be experienced by a microcapsule in the stomach of a mammal. In some embodiments, the gastric conditions are similar to the conditions that would be experienced by a microcapsule in the stomach of a human. In some embodiments, "gastric conditions” that would be experienced in the stomach of a mammal means a pH of about 1.5-4.1 and a temperature of between about 36.5°C and 39.5°C. In some embodiments, gastric conditions comprise approximately 0.5% HC1 with large quantities of KCl and NaCl (Kararli, 1995).
  • gastric conditions that would be experienced in the stomach of a human means a pH of about 1.5 to 3.5 and a temperature of between about 36.5°C and about 37.5°C.
  • gastric conditions includes the presence of other compounds such as enzymes such as lysozyme and pepsin.
  • intestinal conditions are conditions similar to those that would be experienced by a microcapsule in the small intestine of a mammal.
  • the intestinal conditions are similar to those that would be experienced by a microcapsule in the small intestine of a human.
  • intestinal conditions that would be experienced in the small intestine of a mammal means a pH in the range of approximately 5.0-8.0 and a temperature of about 36.5°C to 39.5°C.
  • intestinal conditions that would be experienced in the small intestine of a human means a pH in the range of 5.0 to 7.0 and a temperature of about 36.5°C to 37.5°C.
  • intestinal conditions includes the presence of other compounds such as bile and enzymes such as pancreatin (a mixture of amylase, lipases and proteases).
  • microcapsules made from a biopolymer and a plant-based protein.
  • the microcapsules are used to contain a probiotic, a probiotic and a prebiotic, or a synbiotic.
  • the microcapsules can be used to deliver a probiotic to an intestine of a mammalian subject by protecting the probiotic from the conditions prevailing in the stomach of the subject and releasing the probiotic under the conditions experienced in the intestine of the subject.
  • the microcapsules can be used to deliver a probiotic and a prebiotic, or a synbiotic, to an intestine of a mammalian subject by protecting the probiotic and prebiotic, or synbiotic, from the conditions prevailing in the stomach of the subject and releasing the probiotic under the conditions experienced in the intestine of the subject.
  • the biopolymer is alginate.
  • the biopolymer is iota-carrageenan.
  • the biopolymer is deacylated gellan gum.
  • the biopolymer is present in the initial aqueous phase prior to emulsion at a concentration of between about 0.05 and 0.50% w/v or any value therebetween, e.g. 0.07, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 or 0.45% w/v in the initial aqueous phase prior to emulsion.
  • a higher concentration of biopolymer is used where there is a higher concentration of protein present.
  • the plant-based proteins are chick pea protein, pea protein and/or soy protein.
  • the plant-based protein is present at a concentration of between about 5 and 10% w/w in the initial aqueous phase prior to emulsion, or any value therebetween, e.g. about 6, 7, 8 or 9% w/w in the initial aqueous phase prior to emulsion. In some embodiments, the plant-based protein is present at a concentration of 10% w/w in the initial aqueous phase prior to emulsion. Higher concentrations of plant-based protein could potentially be used if desired, but sufficiently high concentrations of plant-based protein could lead to solubility and/or viscosity issues.
  • the biopolymer is crosslinked to provide the microcapsules.
  • suitable crosslinkers include calcium (e.g. supplied as CaCl 2 ), or magnesium (e.g. supplied as MgCl 2 ).
  • an excess of crosslinker is added to the water-in-oil emulsion to cross-link the biopolymer.
  • the crosslinker is present in a concentration in the range of about 0.1 to 0.5 M in the water-in-oil emulsion or any value therebetween, e.g. 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, or 0.45 M in the water-in-oil emulsion.
  • Some embodiments provide micron-sized plant protein-based microcapsules capable of carrying probiotic bacteria, mixtures of probiotic and prebiotics, or synbiotics through conditions of gastric pH to provide targeted delivery of the contents of the capsules within the intestines of a mammalian subject.
  • the capsules are less than approximately 100 micrometers in diameter, less than approximately 20 micrometers in diameter, or any value therebetween, e.g. less than approximately 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35 or 30 micrometers in diameter.
  • the capsules are between about 20 and about 100 micrometers in diameter.
  • the capsules are useful as feed and food supplements or ingredients.
  • the encapsulated probiotic is from the Lactobacillus or
  • the probiotic is Bifidobacteria adolescentis, Bifidobactertium animalis, Bifidobacterium bifidum, Bifidobacterium breve or Bifidobacterium longum.
  • the probiotic is Lactobacillus delbreuckii subspecies bulgaricus, L. acidophilus, L. casei, L. germentum, L. plantarum, L. brevis, L. cellobious, L. lactis or L. reuteri.
  • the encapsulated probiotic is a combination of one or more of the preceding species.
  • the probiotic is Bifidobacteria adolescentis.
  • the encapsulated prebiotic is a carbohydrate. In some embodiments, the encapsulated prebiotic is a carbohydrate. In some
  • the encapsulated prebiotic is a non-digestible carbohydrate such as: resistant starch (starch which is not hydrolysed in the small intestine); a non-starch polysaccharide such as hemicellulose, pectins, or gums; an oligosaccharide such as a galactooligosaccharide, fructoligosaccharide, maltoligosaccharide; lactulose; inulin; or the like.
  • the encapsulated prebiotic is a fructooligo saccharide, lactulose, transgalactooligosaccharide, or inulin.
  • the encapsulated probiotic is combined with a suitable prebiotic to provide a synbiotic.
  • the microcapsules protect the encapsulated probiotic from conditions experienced during the processing and storage of a feed product such as animal feed. In some embodiments, the microcapsules protect the encapsulated probiotic from conditions experienced during pelletization of the feed product. In some embodiments, the microcapsules protect the encapsulated probiotic to retain viability of the probiotic during long term storage of the feed product, for example for a period of time greater than two weeks to six months or more, or any period of time there between, e.g. four weeks, six weeks, eight weeks, ten weeks, three months, four months, five months, or the like.
  • the microcapsules protect the encapsulated probiotic from conditions experienced during preparation and/or storage of a food product, for example, low pH, extrusion, drying or the like.
  • food products in which the microcapsules according to embodiments of the present invention might be used include dairy products (e.g. yogurt having a pH in the range of about 3.5 to 4.0), cereals, dried fruit products, and/or fruit juices having moderately low pH.
  • the capsules are prepared using an emulsion technique that involves dispersing a mixture of the plant-based protein, the biopolymer and the probiotic as droplets within a continuous oil medium to create an emulsion.
  • the emulsion is formed in a vegetable oil.
  • the emulsion is formed in canola oil.
  • the emulsion is broken using a solution of cross-linker (to cross-link the biopolymer), followed by a hardening step. Microcapsules are then hardened and harvested.
  • the microcapsules are dried after being harvested. Any suitable drying method that is compatible with the encapsulated probiotic can be used to dry the microcapsules. In some embodiments, drying of the microcapsules is done by freeze-drying.
  • the microcapsules provide protection against substantial loss of viability of the probiotic contained in the microcapsules for at least two hours under simulated gastric conditions.
  • substantially loss of viability means any loss of viability greater than about 2 log CFU/g.
  • the microcapsules provide protection against a loss of viability greater than about 1 log CFU/g.
  • the microcapsules provide protection against a loss of viability greater than about 3 log CFU/g.
  • the microcapsules provide release of the probiotic contained in the microcapsules under simulated intestinal conditions.
  • the probiotics contained in the microcapsules may provide a positive influence on the gastrointestinal system of a mammalian subject, even in cases where the probiotics are not delivered live to the small intestine of the subject.
  • microcapsules can be used to provide targeted delivery of the encapsulated probiotic or combination of probiotic and prebiotic, or synbiotic, to the intestines of a
  • mammalian subject by protecting the encapsulated material against a substantial loss of viability due to gastric conditions during transit through the stomach of a mammalian subject, and then releasing the encapsulated material upon exposure to intestinal conditions in the intestine of the mammalian subject.
  • the microcapsules are used to deliver a probiotic, probiotic and prebiotic, or synbiotic to the intestine of a mammalian subject.
  • the microcapsules are stable at gastric pH to protect the probiotic from degradation in the stomach of the mammalian subject, as can be observed by the extended survival of prebiotics under simulated gastric conditions.
  • the microcapsules release the probiotic, probiotic and prebiotic, or synbiotic in the intestine of the mammalian subject under intestinal conditions.
  • the microcapsules are prepared by forming a water-in-oil emulsion in a solution containing the plant-based protein, the biopolymer, and the material to be encapsulated. The mixture is mixed to achieve homogeneity and is then added to oil.
  • the oil is a vegetable oil such as canola oil. The resulting mixture is stirred.
  • a crosslinking agent is added.
  • the crosslinking agent is calcium chloride or magnesium chloride.
  • the aqueous phase containing the microcapsules is separated from the oil phase.
  • the microcapsules are rinsed with a suitable detergent to maintain good capsule dispersability after the microcapsules have been separated from the oil phase.
  • the microcapsules are dried in any suitable manner, for example, by freeze- drying.
  • Alginic acid sodium salt from brown algae, ⁇ -carrageenan, L-cysteine hydrochloride monohydrate, proteose peptone, bile salt, pepsin from porcine gastric mucosa, lysozyme from egg white, pancreatin from porcine pancreas, and TweenTM 80 (polysorbate 80) were all supplied from Sigma-Aldrich (Mississauga, ON, Canada); De man, Rogosa, Sharpe (Lactobacilli MRS). Broth, calcium chloride (CaCl 2 ), sodium bicarbonate (NaHC0 3 ) and sodium chloride (NaCl) were purchased from EMD Chemicals Inc.
  • Chickpea seeds kindly donated by the Crop Development Centre (Saskatoon, SK), were initially ground using a bowl grinder (Cuisinart Mini-Prep Plus) followed by a fine grind (IKA Al l basic. IKA Works Inc., Wilmington, NC) to give flour.
  • the flour was then defatted with hexane (L'Hocine, Boye, & Arcand, 2006) and then concentrated utilizing an isoelectric precipitation procedure (Can Karaca et al., 201 la).
  • the defatted flour was dispersed in MQW at a 1 to 10 (w/v) protein/MQW ratio, followed by pH adjustment to 9.0 with 1.0 M NaOH so as to facilitate protein solubility.
  • the resulting solution was stirred at 1000 rpm
  • the crude ash, lipid, moisture and protein (%N x 6.25) contents for the resulting isolate was determined according to the Association of Official Analytical Chemists (AOAC, 2003) methods: 923.03, 920.85, 925.10, and 920.87, respectively.
  • the carbohydrate content was determined on the basis of percent differential from 100%. Chemical analysis of the isolate indicated protein, moisture, lipid, ash and carbohydrate levels were 85.76%, 2.39%, 0.83%; 4.41% and 6.89%, respectively.
  • Bifidobacterium adolescentis (ATCC 15703) was stored at -70°C in a 1/1 (v/v) suspension of 15% (w/v) glycerol and 5.22% (w/v) MRS broth. Cultures were streaked onto RCM agar supplemented with 0.05% (v/v) L-cysteine HC1 (RCM-cys) plates at 37°C under anaerobic conditions (80% N 2 , 10% C0 2 and 10% H 2 ) in an anaerobic chamber (Forma
  • a total of 10.0 mL of 5.22% (w/v) MRS broth supplemented with 0.05% (w/v) L- cysteine-HCl and 1.5% (w/v) agar was inoculated with two isolated colonies of B. adolescentis for 20 hours at 37 °C under anaerobic conditions. After centrifugation for 5 minutes at 1000 x g, the resulting pellet was re-suspended in 1.00 mL of sterile 0.1% (w/v) alkaline peptone water (APW). Probiotics were then applied to encapsulation study and the final concentration (log CFU mL "1 ) of probiotics grown under anaerobic conditions at 37°C for 48 hours was determined through the spread plate count method conducted in duplicate.
  • Microcapsules were prepared with the chickpea protein isolates using emulsion-based technology which employed a two-phase system to make a water-in-oil emulsion (Truelstrup Hansen et al, 2002; Butler, Ng, & Pudney, 2003; Krasaekoopt, Bhandari, & Deeth, 2003; Winder et al., 2003).
  • niL of a chickpea protein (1.18 g protein power containing 1.00 g protein) solution (10% (w/w) with respect to the final volume of the aqueous phase, 10.0 mL) was prepared in MQW, adjusted to pH 7.0 with 0.5 M NaOH, and stirred at room temperature overnight (16 hours).
  • Genipin, alginate or ⁇ -carrageenan powder was added to the solution to reach a final concentration of 0.2% (w/v).
  • Genipin and alginate powder could be added directly to the solution; however, in the case of ⁇ -carrageenan, the powder was dissolved first at 60°C in advance and then cooled to room temperature before use.
  • One mL of bacterial suspension was subsequently added to the protein-crosslinker mixture to bring the final volume of the aqueous wall material solution to 10.0 mL.
  • the mixture was stirred (750 rpm) for 5 minutes to achieve homogeneity and then added into 100.00 g of canola oil, and the resulting mixture was stirred with an overhead Real Torque Digital Stirrer (Caframo, Wiarton, ON, Canada) at 1000 rpm.
  • an overhead Real Torque Digital Stirrer (Caframo, Wiarton, ON, Canada) at 1000 rpm.
  • the emulsion was stirred for 6 hours so as to afford time for crosslinking, followed by the addition of 100.00 g of MQW to break the emulsion in order to harvest the microcapsules.
  • emulsions were stirred for 0.5 hours, followed by the addition of 100.00 g of 0.1 M CaCl 2 and 0.3 M KC1, respectively, to facilitate microcapsule formation via ionic crosslinking.
  • the resulting suspension was then centrifuged for 5 minutes at 1000 x g to fully separate the oil phase from the capsule-aqueous phase.
  • the aqueous phase was rinsed with 10.0 mL of a 1.0% TweenTM 80 (polysorbate 80) solution so as to maintain good capsule dispersability.
  • the aqueous capsule phase was then transferred to a 50 mL separatory funnel and allowed to settle for 5 minutes prior to the final capsule harvest to ensure full removal of the oil phase.
  • the concentration of viable encapsulated organisms (CFU mL "1 ) was then determined by plate counting on MRS-cys agar plates.
  • Microcapsules were prepared using pea, soy, faba bean and lentil protein isolate in the same manner as described above, with the alternate protein being substituted for chickpea protein isolate.
  • Microcapsules were prepared using pea protein-iota carrageenan and pea protein-deacyl gellan gum mixtures in the same manner as described above, with the alternate biopolymer being substituted for alginate.
  • Example 1.5 Capsule Size
  • Capsule size was determined by light scattering using a Mastersizer 2000 equipped with a Hydro 2000S wet sample cell (Malvern Instruments, Westborough, MA, USA). Measurement conditions used in the Mastersizer included % obscurity (10 - 20%), a pump speed of 850 rpm, a sample absorbance default of 0.1 and refractive index values of 1.45 and 1.33 for the sample (protein) and dispersant (MQW), respectively. Experiments were performed using each capsule formulation for duplicate batches, with size analysis completed in duplicate.
  • SGJ was prepared with a total of 8.3 g of proteose peptone, 3.5 g of glucose, 2.05 g of NaCl, 0.6 g of KH 2 P0 4 , 0.11 g of CaCl 2 , 0.37 g of KC1, 0.05 g of bile, 0.1 g of lysozyme, and 13.3 mg of pepsin in 1 L of MQW with adjustment to pH 2.5 by 1 M HC1. SGJ was heated to 37°C for 30 min and sterile filtered before use. The ability of free and encapsulated B.
  • Chickpea protein-alginate capsules were produced and harvested as previously described. The free cell and the entrapped probiotics in chickpea- alginate capsules were re- suspended with 2.0 mL of 0.1% APW, and then incubated in 48.0 mL of SGJ with stirring (750 rpm) for 2 hours under anaerobic conditions at 37°C. Sampling was carried out at 10, 30, 60, and 120 minutes.
  • a 100 ⁇ ⁇ aliquot was sampled and serially diluted in 900 ⁇ ⁇ sterile 0.1% APW to relieve SGJ stress.
  • the first dilution of each sample was homogenized (Omni International Inc., GA, USA) at 13,000 rpm for 30 seconds to break up the capsule wall.
  • the number of viable surviving bacteria (log CFU mL "1 ) was determined by plate counting after 48 hours anaerobic incubation at 37°C for each time tested. Replicate plates were counted at each time interval during the survival study, and then repeated in duplicate.
  • the free cell and entrapped probiotics in chickpea- alginate capsules were re-suspended with 2 mL of 0.1% APW, and then added into 48.0 mL SIF, pH 6.5 (adjusted with 0.1 M NaOH), with stirring (750 rpm) for 3 hours under anaerobic conditions at 37°C. Sampling was carried out at 10, 30, 60, 120, and 180 minutes. The diluted samples were directly incubated without mechanical homogenization to break the capsules. The number of viable surviving bacteria (log CFU mL "1 ) was determined by plate counting after 48 hours anaerobic incubation at 37°C for each time tested. Replicate plates were counted at each time interval during the release study, and repeated in duplicate.
  • Bifidobacterium adolescentis was entrapped within a 10.00% (w/w) chickpea protein capsule crosslinked with (0.20% w/v) genipin or in the presence of (0.20% w/v) alginate or ⁇ - carrageenan. Overall, the entrapment process did not significantly suppress the growth of B. adolescentis regardless of the capsule formulation (p>0.05). Prior to encapsulation, the number of viable B.
  • adolescentis was 8.6 + 0.1 log CFU mL "1 , whereas afterwards cell counts ranged between 8.5 + 0.1 and 8.7 + 0.1 log CFU mL "1 for the protein capsules with alginate or ⁇ - carrageenan, respectively.
  • viable cell counts were reduced to 7.8 + 0.1 log CFU mL "1 .
  • Figure 1 shows viable log CFU mL "1 over a 2 hour acid challenge (pH 2.0/25°C) for encapsulated and free cells, and free cells with genipin. Viable cell counts after 2 hours were significantly reduced in all capsule formulations relative to time zero (p ⁇ 0.01), as well as amongst themselves (p ⁇ 0.01).
  • Chickpea protein- alginate capsules offered the greatest protection to B. adolescentis (4.6 + 0.1 log CFU mL "1 ), followed by chickpea protein capsules prepared with K-carrageenan (3.5 + 0.3 log CFU mL "1 ) and genipin (1.8 + 0.1 log CFU mL "1 ) ( Figure 1).
  • the majority of free cells died within the first 30-90 minutes of exposure to pH 2.0/25°C following a similar trend as the encapsulated CPI-alginate/ ⁇ - carrageenan ( Figure 1).
  • Viable cells within the CPI-genipin capsule showed the majority of losses within the first 30-40 minutes, below that of free cells alone.
  • chickpea protein-alginate capsules appeared to offer better survival at pH 2.0/25°C than the other formulations and is of a more appropriate size ( ⁇ 100 ⁇ ) size.
  • the high surface area to volume ratio can potentially lead to reduced survival under simulated gastric conditions (Sultana et al., 2000).
  • Example 2.2 Chickpea Protein- Alginate Capsules: The Effect of Alginate Concentration on the Survival of B. adolescentis During an Acid Challenge
  • Table 1 shows changes to viable cell counts for the different formulations and loss reductions over a 2 hour period at pH 2.0/25°C. After 2 hours, all capsule formulations were found to be significantly lower than time zero (p ⁇ 0.01) and also different amongst themselves (p ⁇ 0.01). The greatest protection was found for capsules prepared with 0.10% (w/v) alginate (0.6 + 0.3 log CFU mL "1 reduction), followed by 0.05% (w/v) (1.5 + 0.2 log CFU mL "1 reduction) and then 0.20% (w/v) (2.8 + 0.4 log CFU mL "1 reduction) (Table 1).
  • Viable cells (log CFU mL 1 )
  • Alginate capsules (0.10% w/v) without chickpea protein were also prepared and subjected to the same acid challenge, to show a 3.7 + 0.7 log CFU mL "1 cell number reduction over the 2 hour period (Table 1), which is significantly greater than when chickpea protein was present (p ⁇ 0.05).
  • Table 1 Alginate capsules (0.10% w/v) without chickpea protein was also prepared and subjected to the same acid challenge, to show a 3.7 + 0.7 log CFU mL "1 cell number reduction over the 2 hour period (Table 1), which is significantly greater than when chickpea protein was present (p ⁇ 0.05).
  • an alginate-Ca network developed to maintain capsule integrity, with the globular chickpea proteins packed within interstitial spaces acting to fill pores within the capsule.
  • Alginate is a linear polysaccharide comprised of L-guluronic and D-mannuronic acid residues as homo- or co-block polymeric regions.
  • the present examples demonstrate the potential for entrapping probiotics within a chickpea protein-based capsule, either crosslinked by genipin or in the presence of alginate or ⁇ - carrageenan (plus salts).
  • a chickpea protein capsule in the presence of 0.10% (w/v) alginate offered the best protection under the tested conditions to B. adolescentis within synthetic gastric juice.
  • Capsules produced using this design were ⁇ 100 ⁇ in size, and as such, there would be no perceived adverse effects on the sensory attributes of this ingredient into foods by consumers.
  • Within simulated intestinal fluid a burst-release of B.
  • chickpea protein- alginate microcapsule designs could serve as a suitable probiotic carrier intended for food applications to provide targeted delivery of a probiotic to the intestines of a mammalian subject, including a human subject.
  • Figure 4 shows the protection of B. adolescentis from simulated gastric conditions by the alginate microcapsules with pea, soy, faba bean or lentil protein.
  • Figure 5 shows the release of B. adolescentis from the microcapsules under simulated intestinal conditions.
  • microcapsules made from a combination of alginate and pea protein or alginate and soy protein provide strong protection to an encapsulated probiotic under simulated gastric conditions, while allowing release of the encapsulated probiotic under simulated intestinal fluid.
  • microcapsules made from a combination of alginate and pea protein or alginate and soy protein will be able to provide targeted delivery of a probiotic through the stomach of a mammalian subject to its intestine.
  • Microcapsules were prepared using pea protein-iota carrageenan (P-I) and pea protein- deacyl gellan gum (P-D) mixtures to determine if either polysaccharide could be used instead of alginate.
  • P-I pea protein-iota carrageenan
  • P-D pea protein- deacyl gellan gum
  • microcapsules prepared using iota-carrageenan or deacyl gellan gum as the biopolymer can provide protection to probiotics under simulated gastric conditions while allowing release under simulated intestinal conditions. It can therefore be soundly predicted that iota-carrageenan and deacyl gellan gum can be used as the biopolymer in some embodiments of the present invention to provide targeted delivery of probiotics, probiotics and prebiotics, and/or synbiotics to the intestine of a mammalian subject, including a human subject.
  • SEM images were obtained using microcapsules prepared from different plant proteins (chickpea, pea, soy, faba bean or lentil protein) in conjunction with alginate polysaccharide. SEM images indicated that the microcapsules formed using chickpea or pea protein were more distinct and spherical ( Figures 8A/8B and 9A/9B). In contrast, microcapsules formed using soy, faba bean or lentil protein isolate formed using alginate appeared more as a aggregated mass rather than a distinct capsule ( Figures 10A/10B, 11 A/1 IB and 12A/12B), and was less-effective at protecting the probiotic bacteria during acid challenge experiments.
  • Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract Journal of Applied Microbiology, 84, 759-768. Clark, P. A., & Martin, J. H. (1994). Selection of bifidobacteria for use as dietary adjuncts in cultured dairy foods: III- Tolerance to simulated bile concentrations of human small intestines. Cultured Dairy Products Journal, 29, 18-21.
  • probiotic survial, adherence and antimicrobial resistence candidate selection for encapsulation in a pre proein isolate-alginate delivery system.
  • Gastrointestinal microbes increase arsenic bioaccessibility of ingested mine tailings using the simulator of the human intestinal microbial ecosystem.
  • Microencapsulation a strategy for formulation of inoculum. Biocontrol Science and

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Abstract

La présente invention concerne des microcapsules de protection de probiotiques et leurs méthodes de fabrication. Selon certains modes de réalisation, les microcapsules sont utilisées pour obtenir une délivrance de probiotiques, de combinaisons de probiotiques et de prébiotiques, et/ou de symbiotiques à travers l'estomac d'un mammifère jusqu'à son tube digestif inférieur. Selon certains modes de réalisation, les microcapsules comprennent un biopolymère et une protéine à base de plante. Selon certains modes de réalisation, les microcapsules comprennent un alginate, du carraghénane iota ou une gomme gellane désacylée, en tant que biopolymère. Selon certains modes de réalisation, les microcapsules comprennent des protéines de pois chiches, des protéines de pois ou des protéines de soja en tant que protéines à base de plante. Une émulsion contenant le biopolymère, la protéine à base de plante et la matière probiotique à encapsuler peut être soumise à une réticulation pour former la microcapsule.
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WO2017099559A1 (fr) 2015-12-10 2017-06-15 Uab "Probiosanus" Composition et procédé pour augmenter la survie et la stabilisation des bactéries probiotiques (pb) dans des compositions à base de détergent de produits d'hygiène personnelle et domestiques
US20190192439A1 (en) * 2015-12-17 2019-06-27 Universidad De Navarra Microparticles for encapsulating probiotics, obtaining said microparticles and uses thereof
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EP4093211A4 (fr) * 2020-01-24 2024-04-03 Int Flavors & Fragrances Inc Micro-encapsulation avec une protéine végétale
WO2021150456A1 (fr) * 2020-01-24 2021-07-29 International Flavors & Fragrances Inc. Micro-encapsulation avec une protéine végétale
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WO2024114679A1 (fr) * 2022-11-30 2024-06-06 内蒙古伊利实业集团股份有限公司 Microcapsule pour libération ciblée dans l'intestin et son procédé de préparation, et composition alimentaire
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