WO2024063633A1 - Procédé d'obtention d'un probiotique protégé pour libération entérique - Google Patents

Procédé d'obtention d'un probiotique protégé pour libération entérique Download PDF

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WO2024063633A1
WO2024063633A1 PCT/MX2022/050080 MX2022050080W WO2024063633A1 WO 2024063633 A1 WO2024063633 A1 WO 2024063633A1 MX 2022050080 W MX2022050080 W MX 2022050080W WO 2024063633 A1 WO2024063633 A1 WO 2024063633A1
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product
obtaining
enteric release
probiotics
protected
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PCT/MX2022/050080
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English (en)
Spanish (es)
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Zacnite SÁNCHEZ PORTILLA
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Sanchez Portilla Zacnite
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/745—Polymers of hydrocarbons

Definitions

  • the present invention belongs to the technical field of biotechnology, particularly to the joint area of functional foods and the preservation or maintenance of the viability of microorganisms, even more specifically it refers to methods to preserve probiotics and maintain their viability.
  • BACKGROUND The incorporation of probiotics in foods to provide beneficial health effects is today of great interest in the food industry.
  • the stability in the process of obtaining probiotic-based products is not always optimal.
  • the main problem is the need to maintain its viability for extended storage periods, so its use is limited to its incorporation into refrigerated products.
  • the main challenge of solid products in which probiotic microorganisms are incorporated is to maintain their viability both in the stages of obtaining them and during storage, where the disadvantage is conditions such as temperature and humidity, which determine stability. of the products, which does not guarantee a specific concentration at the time of consuming the product, limiting itself to guaranteeing a specific concentration of the microorganisms determined at the time of obtaining the product, without ensuring that this is maintained during storage and until consumption.
  • the passage of probiotics through the stomach decreases their viability because not all of them are resistant to acidic conditions, so a limited number of bacteria reach the intestine.
  • the preparation method comprises the steps of granule preparation using maltodextrin and inulin as excipients and applying a wet granulation technology, a tableting technology and a low temperature alcohol coating technology; then mix with microcrystalline cellulose, probiotic powder and magnesium stearate and form tablets; and finally coating using the enteric coating material to finally acquire the enteric coated probiotic tablet.
  • Application CN 109674061 (A) provides a double-layer microencapsulation prebiotic and probiotic composition and a method of preparation thereof.
  • a microencapsulation protection technology is used; Natural polymers such as sodium alginate, glucose and prebiotics are used as outer wall materials; poly-L-arginine and prebiotics are used as inner wall materials.
  • Probiotics and skimmed milk powder are used as basic materials.
  • the specific double-layer microencapsulation structure provides double protection for unstable probiotics; the influence of the external environment and adverse factors (such as light, temperature, humidity, pH and bile) is avoided; the reduction of probiotic activity in the use and storage processes is effectively prevented; the stability, activity and effective fixed planting rate of the composition are maintained to the highest degree; The encapsulation efficiency is high, so the incorporation is good.
  • Document CN 101700253 (A) discloses a manufacturing method and a probiotic tablet with a high survival rate of live bacteria manufactured from a dry granulation technique and a tableting technique.
  • Excipients such as glucose, lactin, microcrystalline cellulose and the like are used as raw materials to obtain the particles; Then the active ingredient in probiotic powder and magnesium stearate are mixed to perform compression and finally obtain the probiotic tablet.
  • the invention has the advantages that the probiotic tablet is convenient to take and absorb and has obvious treatment effect due to the high survival rate of probiotics.
  • the dry method granulation technology provided by the invention has the advantages of low cost, fast discharge speed, stable technological conditions. While methods to protect probiotic bacteria can be used to maintain their viability during storage, it is important that protection keeps the probiotics active throughout the gastrointestinal tract and releases them at their site of action.
  • the purpose of the present invention is to provide a method that allows obtaining a product with an optimized dose that requires a smaller amount of probiotics, that ensures its arrival in the intestine and that provides a therapeutic effect, in addition to that viability can be preserved for long periods in non-refrigerated conditions.
  • the present invention proposes the formation of granules in which the probiotics are incorporated, and which in turn are protected with an acrylic polymer, conferring greater protection to the microorganisms even before obtaining the final pharmaceutical form, being able to be administered in this presentation or choose to obtain tablets/capsules.
  • Still another object of protection refers to a probiotic-based granule product enriched with a prebiotic with improved operating conditions in the production method to reduce the loss of viability during this stage, also achieving the incorporation of an enteric film that protects to probiotic bacteria from the environment and from their passage through the stomach to ensure that the greatest number of bacteria reach the specific site of action (colon) viable.
  • the present invention focuses its interest on a method to obtain a product with low microbial content, which ensures its arrival in the intestine and which provides a therapeutic effect, in addition to being able to preserve viability for long periods in non-refrigerated conditions.
  • the objectives of the present invention referred to above and still others not mentioned, will be evident from the description of the invention and the figures that accompany it with an illustrative and non-limiting nature, which are presented below.
  • BRIEF DESCRIPTION OF THE FIGURES Figures 1. Diagram of the preferential method for obtaining the enteric release product of Bifidobacterium animalis ATCC 25527 enriched with inulin as a prebiotic. Figures 2a-2d.
  • Eudragit ⁇ FS30D aqueous-based polymer
  • Eudragit ⁇ S12.5 organic-based polymer.
  • the present invention refers to a method for obtaining a probiotic enriched with a granulated prebiotic with a porous microcrystalline cellulose (Avicel® PH 200) and with enteric coating.
  • the enteric release probiotic has a barrier made from a protective polymer that fulfills two functions, on the one hand, it prevents the probiotic bacteria from being released in the stomach since the product resists acidic pH, causing them to be released to the colon. , and on the other hand, it prevents the hydration of the product and the contact of the probiotic bacteria with the oxygen of the medium during storage, so its viability is maintained for longer times. extensive.
  • the method of obtaining the product keeps the metabolically inactive bacteria on a porous solid core, which advantageously does not need to be refrigerated and which can finally be used to formulate food or pharmaceutical supplements in tablets, hard gelatin capsules, sachets, or incorporated into milkshake powder or milk powder.
  • probiotic refers to live microorganisms that, when administered in adequate amounts, confer a health benefit to the host.
  • probiotics include decreasing potentially pathogenic gastrointestinal microorganisms, reducing gastrointestinal discomfort, strengthening the immune system, improving skin function, improving intestinal transit, strengthening resistance to cedar pollen allergens, decreased body pathogens, reduced flatulence and inflammation.
  • a more detailed definition of probiotics refers to microorganisms that beneficially affect a host animal by modifying its intestinal microbiota, ensuring better use of feed or improving its nutrition, improving the host's response to disease or improving the quality of the environment. atmosphere.
  • the probiotic used herein refers to adherent microorganisms or cells or bacteria also defined herein as adhesive microbes. Bacteria are typically found attached to and living in close association with surfaces. To achieve effective adherence to host surfaces, many bacteria produce multiple adhesion factors called adhesins.
  • Adhesins are components of the cell surface or appendages of bacteria that facilitate adhesion or adherence to other cells or surfaces.
  • the probiotic bacteria comprise one or more strains of Bacillus coagulans, Bacillus subtilis var natt, Bifidobacterium, Bifidobacterium sp, Bifidobacterium bifidum, Bifidobacterium brevebacterium, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium animalis, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus delbrueckus, Lactoco lactobacillus, Lactococcus lactis, and/or a mixture thereof.
  • Prebiotics are food ingredients that induce the growth or activity of beneficial microorganisms (for example, bacteria and fungi). Prebiotics are mostly oligosaccharides, and in the case of the present invention they can be selected from any of inulin (fructooligosaccharide or FOS) and transgalactooligosaccharides (GOS or TOS). Other suitable prebiotics include palatine oligosaccharides, soy oligosaccharides, gentiooligosaccharides, xylooligomers, non-degradable starches, lactosucrose, lactulose, lactitol, maltitol, polydextrose or combinations thereof.
  • Prebiotics can stimulate the proliferation of sugar-using bacteria (including bifidobacteria and lactic acid bacteria, etc.), thereby promoting releasing organic acids and generating an antibacterial acidic environment to inhibit the growth of intestinal pathogens.
  • Prebiotics can also improve the ecological balance of human intestinal microorganisms by increasing the activity of beneficial bacteria.
  • the prebiotic used is inulin orafti-GR, Beneo. Which is observed as a white powder soluble in water; It is a polysaccharide consisting of a linear chain of D-fructose molecules with a terminal glucose molecule.
  • Inulin-type fructans such as Orafti® inulin
  • chicory root is a particularly rich source of this element.
  • Orafti® inulin As it is extracted from the chicory root, using a hot water extraction method, it is 100% plant-based; After extraction, purification is carried out with high quality standards.
  • prebiotics can alter the composition of organisms in the intestinal microbiome.
  • prebiotics are typically nondigestible fiber compounds that pass undigested through the upper gastrointestinal tract and stimulate the growth or activity of advantageous bacteria that colonize the large intestine by acting as a substrate for them. Ingesting the right amount of probiotics can play an effective role in the health of the consumer.
  • bioactive refers to a compound that has an effect on a living organism, tissue/cell.
  • bioactive compounds are distinguished from essential nutrients. While nutrients are essential for the sustainability of a body, bioactive compounds or materials are not essential since the body can function properly without them, or because the nutrients perform the same function. Bioactive compounds can influence health.
  • the protected probiotic consists of microcrystalline cellulose granules (Avicel® PH 200) as a base excipient, in which a probiotic bacteria is incorporated by means of an incorporation suspension, preferably skim milk (Svelty) to the 30% weight/volume with at least one binding agent, selected from hydroxypropylmethyl cellulose (HPMC, Opadry® clear, Colorcon) at 2% weight/volume, at least one prebiotic, preferably inulin (Orafty® GR, Beneo) at 15% weight /volume, and at least one solvent, preferably physiological saline solution.
  • an incorporation suspension preferably skim milk (Svelty) to the 30% weight/volume with at least one binding agent, selected from hydroxypropylmethyl cellulose (HPMC, Opadry® clear, Colorcon) at 2% weight/volume, at least one prebiotic, preferably inulin (Orafty® GR, Beneo) at 15% weight /volume, and at least one solvent, preferably physiological saline solution
  • the microcrystalline cellulose used is Avicel® PH 200, which has the following characteristics: average particle size: 180 ⁇ m, moisture content: 5%, density: 0.29–0.36 g/cm 3 , specific surface area: 0.78 –1.18 m 2 /g.
  • the incorporation process is carried out using a fluid bed at a process temperature of around 32 to 35 oC, at a chamber pressure of between 4.5 to 5 Bar, dosing speed of between 3.5 to 4.2 g/min and a pressure of atomization of between 1 to 1.2 Bar.
  • the product is subsequently protected with an external coating layer with an acid-resistant acrylic polymer, preferably Eudragit® S12.5 (Evonik) at a 10% increase in weight with respect to the product.
  • the polymer dispersion is prepared using isopropyl alcohol at a concentration of 5.6% total solids; the process using a fluid bed, with a temperature of 30 to 32 oC, a chamber pressure of 4.5 to 5 Bar, dosing speed of 4.5 to 5.8 g/min and an atomization pressure of 1 to 1.2 Bar.
  • External coating layers can be used to provide greater resistance to the dehydrated microorganism, particularly to protect against inactivation by gastric and/or oxidative environments and/or unfavorable pH conditions. These layers may also provide better mechanical strength, better resistance to steam treatment, or improved handling properties (e.g., improved solids flow) to the coated dehydrated microorganism.
  • the coating confers greater resistance to inactivation during secondary processes used for the preparation of food, feed, consumer health products or agricultural products and/or provides the long-term shelf life stability required in food, feed, health products consumption and agroproducts. Accordingly, the encapsulated probiotics according to the present invention can be used in functional foods, food products, consumer health products and agricultural products. This proposal is expected to be an efficient alternative to avoid cold transport chains. The inventors have advantageously found, without intending to impose any theory, that the protective film is responsible for the effect observed in the results shown below, providing a barrier to prevent the product from being hydrated, and being insoluble at acidic pH, so the microenvironment within the granule remains “stable”.
  • the invention further relates particularly to an enteric release product comprising at least one probiotic and at least one prebiotic coated with at least one protective or coating layer.
  • other ingredients can be included in the coating or inside it, such as polyhydroxy compounds, non-stick agents, compounds that have nutritional and/or health benefits (antioxidants, vitamins, minerals, prebiotics, peptides, proteins), hydrocolloids, diluents, lubricants, binders, acids, alkalis, hydrophobic species, polymers and mixtures thereof.
  • These other optional ingredients are preferably in an amount by dry weight of 0 to 70%, more preferably 10% to 60%, even more preferably 17 to 40%.
  • talc or starches are inert ingredients that could be used as a coating release agent.
  • These optional ingredients can also be used to provide an additional benefit/effect to the final product (e.g. vitamin supplements). They can also be used to adjust the pH of the coating to a value that is "compatible with the viability of the coated dehydrated microorganism.”
  • Anti-stickagents also called “anti-caking agents” that can be used in accordance with the present invention include talc, silica (precipitated or fumed silicas), starches (including native starches derived from potato, corn,).
  • Talc and corn starch are one of the most preferred agents.
  • polymers that may be comprised in the coating or protective layer according to the invention are native or modified starches derived from potatoes, corn, cellulose and cellulose derivatives (cellulose ester, cellulose ethers). ; low permeability polymers (e.g. shellac, HPMC, polyvinyl pyrrolidone, polyethylene glycol).
  • the acid-resistant acrylic polymer used in the present invention is a polymer whose solubility is pH dependent under strongly acidic conditions (approximately pH 4.5 or less) and dissolving at a pH greater than 5.5, typically methacrylic acid and methacrylate methyl (copolymers of methacrylic acid and methyl methacrylate such as Eudragit® L30 D-55, Eudragit® L100, Eudragit® L100 D-55, Eudragit® L12.5, Eudragit® S100, Eudragit® S12.5, Eudragit® FS 30-D and any combination thereof.
  • the acid resistant acrylic polymer can be selected from the group consisting of Eudragit® L30D-55, Eudragit® L100D-55, Eudragit® L12.5 and combinations thereof
  • Eudragit® L30 D-55 is almost insoluble at low pH (e.g., pH 5.5 or less), but dissolves when a pH of 5.5 or higher is reached.
  • Eudragit® L30 D-55 is also available as a 30% (m/v) aqueous dispersant of an acrylate polymer containing sodium lauryl sulfate and polysorbate 80 as emulsifiers.
  • the acid-resistant acrylic polymer used in the present invention is Eudragit® S12 .5, a copolymer of methacrylic acid and methyl methacrylate (1:2), which solubilizes at pH above 7. It is an organic solution of Eudragit® S 100 with 12.5% (w/w) of dry substance in isopropanol (Ph . Eur./USP). The organic solution contains 3% (w/w) distilled (deionized) water. The product contains 0.3% Sodium lauryl sulfate (Ph. Eur./NF).
  • Another object of the present invention is a method for the preparation of food products, feed products, consumer health products or agricultural products, in which the coated dehydrated microorganism as defined in the invention or as obtained according to the Preparation method of the present invention is subsequently added to a food product, a consumer health product or an agricultural product.
  • "food” means products suitable for human consumption.
  • the coated dehydrated microorganisms of the present invention are suitable for use in food products. Preferably, those foods that after mixing or incorporating do not require baking or treating at high temperatures.
  • the food products particularly preferred are nutritional bars, breakfast cereals, infant formulas and powdered drinks, lactic acid bacteria-based drinks, adult nutritional drinks, acidified soy/juice drinks, milk drinks.
  • feed means products suitable for animal consumption and can be selected from the group comprising "pet foods” (cakes, biscuits, chews, pet snacks), silage products and granulated feeds.
  • animal should be understood with a broad meaning. May refer to a "polygastric herbivore” that includes, but is not limited to, cattle, sheep, cervids, antilocaprids, and camelids.
  • a "polygastric ruminant” which includes, but is not limited to, cows, sheep, goats, deer, camels and giraffes. It can also refer to a “monogastric herbivore” such as horses and pigs, as well as domestic animals or pets (dogs, cats, rabbits, birds, rats, mice, guinea pigs, fish, reptiles, among others).
  • the coated probiotics of the invention can be included in pastes or other oil delivery systems to directly supplement animals. They can also be powders or other dry formulations for dressing.
  • "consumer health improvement products” include dietary supplements, nutraceuticals and over-the-counter products.
  • a consumer can be a human and/or an animal.
  • a "dietary supplement” also called a food supplement or nutritional supplement
  • a dietary supplement can be for human or animal consumption.
  • the term “nutraceutical” means a functional food that is capable of providing not only a nutritional effect and/or taste satisfaction, but is also capable of providing therapeutic (or other beneficial) effects to the consumer.
  • "over-the-counter product” means over-the-counter medications that can prevent certain diseases or reduce symptoms associated with intestinal or immune health, thereby promoting intestinal health or improving immune function.
  • agroproduct encompasses biopesticides, biofertilizers, plant care products, composts and by-products, as well as bioenergy products (bioethanol, bioester).
  • Another object of the present invention is food products, products aimed at improving health for the consumer or agricultural products comprising coated dehydrated microorganisms as defined or obtained in the present invention.
  • a high survival of bacteria is obtained after several months of storage of final products comprising the dehydrated microorganisms coated according to the invention (for example, after 1 month of storage, even after 2 months, even after 3 months, even after 5 months, even after 9 months, even after 12 months, even after 2 years, even longer, especially when stored at room temperature (e.g. 15 to 40°C, especially 20°C to 35°C, e.g. at 23 or 30 °C).
  • room temperature e.g. 15 to 40°C, especially 20°C to 35°C, e.g. at 23 or 30 °C.
  • the coated probiotic for enteric release in the present example was prepared with the Bifidobacterium animalis strain (ATCC 25527) using Avicel ⁇ PH 200 as a base material, where the probiotic is incorporated together with the prebiotic. , inulin. Eudragit ⁇ S12.5 was used as an enteric protection product.
  • the obtaining process was fluid bed coating.
  • the product obtained can be incorporated into solid pharmaceutical forms or food supplements for oral administration, maintaining its ability to provide protection to bacteria to resist passage through the gastrointestinal tract and maintain the viability of bifidobacteria during prolonged storage periods under normal conditions, avoiding the cold chain commonly used to maintain the viability of certain probiotic-based products.
  • a strain of Bifidobacterium animalis (ATCC 25527) was used, which belongs to the collection of the Microbial Ecology Laboratory (UAM-X).
  • UAM-X Microbial Ecology Laboratory
  • TPY broth enriched with inulin TPY-I
  • MRS-C Microbial Ecology Laboratory
  • Microcrystalline cellulose Avicel® PH 200, FMC Biopolymer
  • inulin Orafti®-GR, Beneo
  • skim milk Svelty
  • physiological saline solution SSF
  • Method for the preparation of the product with bifidobacteria enriched with inulin and protected with an enteric polymer Preparation of the bacterial suspension a) Inoculate Bifidobacterium animalis in TPY-I broth under anaerobic conditions at an initial OD660 of 1.0, and incubate at 37o C, with shaking at 120 rpm for 48 hours; probiotic and prebiotic; b) Centrifuge the culture for 10 min at 3500 rpm, remove the supernatant and wash with physiological saline solution (SSF); c) Resuspend the packet in 10 mL of SSF; d) Independently, prepare a suspension with skim milk (30% w/w), HPMC (1% w/w) and inulin (15% w/w) in 40mL of SSF; e) Combine the previous mixtures to create the binder suspension that is used to
  • the final product is stored in polypropylene bags at room temperature.
  • the established culture conditions allowed obtaining a concentration between 10 10 and 10 12 CFU of bifidobacteria in the incorporation suspension for the different batches obtained.
  • the parameters observed in Table 1 remained stable.
  • Table 1 Process conditions for the incorporation of bifidobacteria and their protection by granulation in the fluid bed. These conditions were established in previous experiments, which served to program the equipment and verify that the production processes of the proposed batches remained stable and under the established conditions.
  • FIG. 2a-2d shows the characterization of the morphology of Bifidobacterium animalis ATCC 25527 by scanning electron microscopy.
  • To observe the morphology of the bifidobacteria it is necessary to take a culture sample of Bifidobacterium animallis ATCC 25527 grown in TPY-I broth for 48 hours. This sample is washed and fixed with a dehydration technique with different concentrations of ethanol.
  • FIG 3a there are microcrystalline cellulose particles forming agglomerates (Avicel® PH 200), which have irregular edges and spaces (pores) which allow the incorporation of a greater number of bacteria, as we can see in Figure 3a.
  • Figure 3b in which remains of the incorporation material are also observed.
  • the coating process with the enteric polymer is carried out, so the surface of the granule changes its appearance, but the shape is maintained.
  • acid and alkaline conditions which is carried out under conditions of constant agitation (see resistance of the Bifidobacterium animalis culture and the enteric release product in acetate buffer pH 3 and phosphate buffer pH 7.5), cellulose residues are observed.
  • FIG. 4 shows the release profile of bifidobacteria from the enteric-coated product with the product Eudragit® S12.5 at an increase in weight of 10%. Under acidic conditions (pH 3) it shows good resistance by retaining more than 80% of the total content of incorporated bacteria; Under alkaline conditions (pH 7.5), which is the optimal pH for the solubility of the enteric polymer, a release of 42.6% is observed at 120 minutes of the experiment, reaching the total at 180 minutes.
  • each product was stored in closed polypropylene bags. This analysis makes it possible to guarantee the viability of the microorganisms contained in the product and, in turn, establish a period of expiration and in this way, ensure that when consumed, it will produce a beneficial effect.
  • the loss is 5.65. In general, this loss corresponds to approximately 50% for each product.
  • Table 2 Characteristics of the batches obtained. In general, the loss of viability in the products after one year of having been obtained and stored at 26o C and 62% relative humidity corresponds to approximately 50% for each product. Example 5.
  • the resistance of the culture at pH 3 is low, since during the first two hours of analysis a decrease in resistance is seen.
  • concentration of bifidobacteria concentration of bifidobacteria.
  • the protection with the enteric polymer protects the bacteria from the conditions of the environment in which they are found.
  • the unprotected culture lost around 3 log units in acidic pH, which represents a significant loss during passage through the stomach, especially if products whose concentration is 10 6 CFU/g of product are consumed.
  • the enteric polymer film provides physical protection to bifidobacteria and maintains viability in acidic conditions, in addition to Their release is pH dependent, so their release is specific.
  • the following examples demonstrate the application of the enteric release product based on probiotics.
  • Example 6. Characterization of the products obtained Methodology in fluid bed on the viability of Bifidobacterium animalis To determine the loss of viability of the bifidobacteria, samples were taken from the initial culture, the product obtained after the incorporation of the bifidobacteria in the avicel nucleus PH ⁇ 200, and the product obtained after the protection process with enteric polymer.
  • the samples were placed in 50 mL of medium, SSF for the culture samples and the incorporation product, and phosphate buffer pH 7.5 for the protected product, they were kept under constant agitation and samples of the medium were taken. Size of Particle size determination was carried out with a laser beam diffraction equipment (Partica, LA950, Horiba Scientific) in a powder cell. Moisture content The percentage of moisture was determined with 5 g samples on a thermobalance (OHAUS, MB45), at 100o C for 10 minutes.
  • the apparent density of a powder is the ratio of the mass of an unsettled powder sample to its volume, including the contribution of the volume of the void space between the particles. Consequently, the bulk density depends on both the density of the powder particles and the spatial distribution of the particles in the powder bed. Measurement in a graduated cylinder. 100 g of sample powder (M) were weighed and placed (without compacting) in a dry, graduated glass cylinder of 250 mL. A reading of the unsettled apparent volume (Vo) was taken with an approximation to the nearest unit on the scale. The apparent density was calculated in grams per milliliter, using the following formula, M/V0.
  • the compacted density is obtained after mechanically striking a graduated measuring container containing the same powder sample used in the apparent density test, its value being greater than the latter due to the volume reduction.
  • the volume reduction is obtained by the mechanical settlement of the powder sample, when the specimen or container that contains it is lifted and impacted from a specific height. Measurement in a graduated cylinder. The same sample used in the determination of apparent density was used without removing it from the test tube. The mouth of the test tube was covered before performing the test. The specimen was raised to a height of 10 ⁇ 5 cm and impacted 250 times on a flat, smooth surface, at a constant rate.
  • the composition of the granules, particle size and humidity are factors that influence the flow rate and is defined as the time necessary for a specific amount of powder to flow through a glass or stainless steel funnel placed at a certain height.
  • the flow rate of a powder is a manifestation of its rheological properties and is defined as the displacement of a quantity of sample per unit of time.
  • the angle of repose is a manifestation of the friction between particles and the resistance to movement; it is defined as that which corresponds to the maximum angle formed between the surface of a powder cone and the horizontal plane.
  • the angle of repose is a function of the shape, particle size distribution and surface roughness of the powder particles, for example, spherical and smooth particles have better flow properties. Both tests must be performed simultaneously.
  • a sample from the culture and a 50 mg sample of the enteric release product were placed separately in 5 mL of pH 3 acetate buffer, keeping it under constant agitation; Samples were taken from each medium at 0 and 60 minutes, time in which the resistance in acidic pH was concluded. Each of the media was centrifuged and the supernatant was discarded. The residues of both culture and product were placed independently in phosphate buffer pH 7.5 and kept under constant agitation for 3 hours. Samples of the medium were taken at 120 and 180 minutes.
  • In vitro release of Bifidobacterium animalis from enteric release products The acid resistance test was performed using a sample of the product in pH 3 acetate buffer, keeping it agitated for 1 hour, taking a sample at time 0 and 60 min.
  • Figure 7b shows the growth of the bifidobacteria culture in TPY medium, using inulin as a carbon source (TPY-I).
  • the fresh culture sample was analyzed by scanning electron microscopy at different magnifications ( Figures 8a-8d) to observe the morphology of Bifidobacterium animalis. Products obtained from bifidobacteria protected with pH-dependent polymers.
  • the bacterial suspension obtained was incorporated into the different nuclei, avicel ⁇ PH 200, inulin and the avicel/inulin granules, subsequently protected by incorporating the pH-dependent polymer; For both processes, the established conditions were maintained.
  • Table 5 describes the characteristics of the products made using avicel ® PH 200, inulin and the core obtained with avicel ⁇ /inulin, as solid substrates for the incorporation of bifidobacteria, and their subsequent protection with a pH-dependent polymer by means of of fluid bed granulation. Table 5. Characteristics of the different batches of products obtained.
  • FIG 9 shows the concentration of bifidobacteria found in the samples analyzed to determine the impact of the product obtaining processes on the viability of these microorganisms.
  • the average initial concentration of the culture was 10.11 Log10 CFU; In the incorporation process, around 1 Log10 CFU is lost, and in the protection process with the enteric polymer, the loss is 0.1 Log10 CFU.
  • Bifidobacteria resistance test Figure 10 shows the resistance of bifidobacteria from a culture of Bifidobacterium animalis, and samples of the enteric release products using inulin (BIP-In) and avicel (BIP-Av) as nuclei.
  • the resistance of the crop to pH 3 is low, since during the first two hours of analysis there is a decrease in the concentration of bifidobacteria.
  • the initial concentration was 8.8 Log10 of CFU for the culture, 7.85 Log10 of CFU for BIP-In and 7.95 Log10 of CFU for and BIP-Av respectively.
  • the release of bifidobacteria is gradual, however, the difference lies in the amount of bacteria released, which for BIP-In is greater compared to BIP-Av, which causes a notable release not to be observed in the pH change. 3 at pH 7.5 as observed in the line corresponding to BIP-Av.
  • inulin has a spherical and smooth shape, while avicel ⁇ PH 200 (Figure 13e) occurs in irregular and porous granules, the particle size of inulin is smaller compared to avicel ⁇ PH 200. As solids are incorporated onto the surface of the nuclei, their porosity decreases, however, in inulin it is difficult to notice this difference ( Figure 13b). It is worth mentioning that during the processes carried out in a fluid bed, the shape of the particles is maintained, except when inulin is protected with Eudragit ⁇ S12.5, since there is an agglomeration of particles that form larger, round granules. Analysis of the release profiles according to the modified variables in each product obtained.
  • Figure 14 shows the release profile of the products obtained with avicel ⁇ PH 102 and PH 200, whose difference lies in the particle size. As can be seen, the product whose particle size is larger (PH 200) does not present bifidobacteria release in acidic conditions compared to the smaller avicel (PH 102).
  • the first was to use it as a nucleus for the incorporation of bifidobacteria, for which inulin (Orafti ⁇ -GR) from the Beneo company was used;
  • inulin Oleo company
  • the performance of this material during the fluid bed process was not favorable since its particle size and solubility made the process difficult in which the parameters such as dispersion speed and atomization pressure were constantly moved to obtain the product, whose behavior It is seen in Figure 15.
  • Both the solubility of inulin and the reduced particle size allow the release of bifidobacteria in a shorter time compared to the other two products.
  • the second strategy was to obtain a granulated product taking advantage of the good technological characteristics of avicel and including inulin as a prebiotic and binding solution.
  • This product was used as a core for the incorporation of bifidobacteria and protection with the pH-dependent polymer.
  • a homogeneous product was obtained and its release profile (Figure 15) shows a significant improvement compared to the product obtained with inulin as a core, however, when compared with the product in which avicel was used, there is no an important difference that justifies its use.
  • the third strategy consisted of using avicel directly, since microcrystalline cellulose is considered a material of choice for the development of solid pharmaceutical forms due to its physical characteristics that give it technological advantages over other materials, as already mentioned, there are different types of avicel and the difference lies in their size, surface area, density and porosity.
  • PH 200 was chosen because of its good flow properties, its high porosity and because its average particle size is around 180 ⁇ m.
  • the prebiotic was added within the bifidobacteria incorporation process. It should be noted that this is the formulation that was selected to obtain the product since the process conditions remain stable and the product is homogeneous. As seen in Figure 15, the best release profile is observed with the co-processing (discarded by the obtaining method), and the profile corresponding to avicel PH 200 as a core, which involves only two fluid bed granulation processes and gives rise to a product with good characteristics in terms of the release of bifidobacteria.
  • the thickness of the protective layer directly influences the rate at which bacteria are released from the granules. Theoretically, a greater increase in polymer weight would mean maintaining the release for a longer time due to the thickness of the protective layer formed around the granule; This phenomenon can be observed in Figure 17, where it is shown that at 10% Eudragit S12.5, close to 100% of the bifidobacteria content is released after 120 minutes of the experiment, while the profile corresponding to 20% polymer It releases around 20% of the content at the same sampling time, reaching 100% after 300 minutes, which is a problem since the product may not release the bifidobacteria; Although it is true that the behavior improves in the first hours of the experiment, the problem mentioned above, added to the fact that it involves an extra process and double the product, means that the formulation is discarded.
  • Example 7 Administration of the product based on enteric release bifidobacteria to rats fed a diet high in fat and fructose Materials Diets administered to the groups of The RatDiet 5012 rodent food was ground in a blade mill until it became a uniform fine powder, which served as the basis for the preparation of both the normal diet (DN) and the high-fat and fructose diet (HFF). its acronym in English).
  • DN normal diet
  • HFF high-fat and fructose diet
  • HFF high-fat and fructose
  • P érez-Ram ⁇ rez, 2017 Test 1 An experiment was designed in which the product based on enteric release bifidobacteria was administered for 2 weeks in a rat model fed with a high-fat and fructose diet, in order to observe the behavior during their adaptation to diets, and evaluate the effect of the administration of bifidobacteria protected with a pH-dependent polymer on weight gain and the formation of fat vesicles in the liver.
  • Treatment 1 Normal diet (ND)
  • Treatment 2 Normal diet plus fresh culture of bifidobacteria (ND+B)
  • Treatment 3 Normal diet plus product with protected bifidobacteria (ND+P)
  • Treatment 4 High-fat and fructose diet (HFF)
  • Treatment 5 High-fat and fructose diet plus fresh culture of bifidobacteria ( HFF+B)
  • Treatment 6 High-fat, high-fructose diet plus product with protected bifidobacteria (HFF+P) Experimental animals were maintained on 12-hour light/dark cycles.
  • Test 2 Based on the previous experiment, a three-week experiment was designed. The first week was dedicated to adaptation to ground food (DN) for all groups, the second week the administration of probiotics began (culture and product to the respective groups), which was maintained until the end of the experiment; During the third week, feeding was started with the diets corresponding to each treatment, whether it was a normal diet or a high-fat diet.
  • DN ground food
  • Treatment 1 Normal diet (DN) ⁇
  • Treatment 2 Normal diet plus fresh culture of bifidobacteria (DN+B) ⁇
  • Treatment 3 Normal diet plus product with protected bifidobacteria (DN+P) ⁇
  • Treatment 4 High-fat and fructose diet (HFF) ⁇
  • Treatment 5 High-fat and fructose diet plus fresh culture of bifidobacteria (HFF+B)
  • Treatment 6 High fat and fructose diet plus product with protected bifidobacteria (HFF+P) ⁇
  • Treatment 7 High fat and fructose diet plus placebo (HFF+Pb)
  • the fresh culture was administered as a control to observe the effect of protecting the bacteria; Both the culture and the product were administered at a concentration of 1x10 5 CFU/day.
  • mice were maintained on 12-hour light/dark cycles. Water consumption was ad libitum and food was rationed at 30 g of food/day per rat.
  • the first week of the experiment was an adaptation week to ground food, so all experimental groups were fed a normal diet ( Figure 2). Starting in the second week, the administration of bifidobacteria began every third day at a concentration of 1x10 5 CFU/day, to the groups corresponding to the culture and the bifidobacteria product. In this week, the rats were fed the normal diet.
  • each group was fed a normal diet or a high-fat and high-fructose diet depending on the experiment (DN, DN+B, DN+P, HFF, HFF+B, HFF+P and HFF+Pb).
  • the weight of the rats was monitored.
  • the rats were sacrificed to obtain blood and liver samples, which were subsequently analyzed.
  • Sacrifice and sampling Rats were anesthetized with isoflurane and blood was collected by cardiac puncture. Subsequently, the liver was removed, washed with SSF, and the weight was recorded.
  • Liver dissection was done in a standardized manner, taking two portions of the four main lobes, identified as lobe 1 (L1), lobe 2 (L2P (small) and L2G (large)), lobe 3 (L3) and lobe 4 ( L4), a portion was frozen at -80 oC and another was used to later embed it in paraffin and obtain sections for histological analysis.
  • the standardization of the samples was carried out to identify the areas of highest concentration of fat vesicles.
  • Figure 3 shows a photograph of the rat liver and the order in which the lobes were dissected. Analysis of blood samples Blood samples were centrifuged, and serum was recovered to measure glucose levels, liver profile and lipid profile.
  • liver samples A histological analysis was made of tissue sections embedded in paraffin and stained with hematoxylin and eosin. The slides obtained were analyzed under an optical microscope and photographs were taken of the four lobes of each rat (six per lobe); The photographs were analyzed with the help of the ImageJ ⁇ program, and the number of identified fat vesicles was counted with the particle analysis tool.
  • An analysis parameter was sphericity, since the vesicles appear as well-defined circumferences with smooth edges, which allows a difference to be made between them and some structures typical of the liver.
  • HFF+P protected bifidobacteria product
  • lipid profile total cholesterol, triglycerides and high-density cholesterol (HDL)
  • liver profile alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase
  • Lipid profile Figures 21a-21f, table 11 show the results of the statistical analysis of total cholesterol, triglycerides and HDL, which seeks to find a relationship between the weight gain of the experimental animals with each of the treatments and the levels found.
  • experiment 1 there is a significant decrease in triglyceride levels and the atherogenic index in the HFF+P treatment compared to the HFF treatment;
  • the administration of bifidobacteria Protected reduces cholesterol levels.
  • Table 11 show the results of the statistical analysis of total cholesterol, triglycerides and HDL, which seeks to find a relationship between the weight gain of the experimental animals with each of the treatments and the levels found.
  • experiment 1 there is a significant decrease in triglyceride levels and the atherogenic index in the HFF+P treatment compared to the HFF treatment;
  • the administration of bifidobacteria Protected reduces cholesterol levels.
  • experiment 2 there is no clear effect since the results are not consistent. Table 11.
  • the atherogenic index is higher in the groups fed a high-fat diet, which shows a clear difference between both types of diets, although for experiment 2 it does not represent a statistically significant difference.
  • this index decreases when bifidobacteria protected with the pH-dependent polymer are administered, but this is only observed in experiment 1.
  • Liver profile When feeding experimental animals with a high-fat diet, It is expected that weight gain will be greater compared to rats fed a normal diet, as we could see in the weight gain results. To evaluate the effect of bifidobacteria administration, Liver enzyme levels were analyzed.
  • FIGS. 24a-24b show the results obtained; They show that in experiment 1, only the treatments corresponding to the high-fat diet (HFF) and the high-fat diet and administration of the bifidobacteria culture (HFF+B) are significantly different, with an average hepatic index of 4.18 and 4.06% respectively, so once again a positive effect is observed when administering protected bifidobacteria.
  • HFF high-fat diet
  • HFF+B bifidobacteria culture
  • FIG. 27a-27b shows the results of the statistical analysis, in which we can observe that, as in the results of weight gain, the administration of protected bifidobacteria has a positive effect on the accumulation of fat vesicles, since there is a statistically significant difference between this treatment and the HFF and HFF+B groups. In the group placebo, as already mentioned, there was no effect, since the difference is very significant (p ⁇ 0.00001).

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Abstract

L'invention relève du domaine technique des aliments fonctionnels et de la préservation ou du maintien de la viabilité des microorganismes. L'invention a pour objet un procédé pour l'obtention d'un probiotique enrichi d'un prébiotique granulaire avec une cellulose microcristalline poreuse et avec un enrobage entérique, permettant d'abaisser la teneur en probiotiques, assurant leur arrivée dans l'intestin en vue de l'obtention d'un effet thérapeutique, outre la possibilité de préserver la viabilité pendant des périodes prolongées dans des conditions non réfrigérées, ces ingrédients pouvant être incorporés dans des formules alimentaires ou pharmaceutiques.
PCT/MX2022/050080 2022-09-19 2022-09-19 Procédé d'obtention d'un probiotique protégé pour libération entérique WO2024063633A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
L.K. STENMAN, A. WAGET, C. GARRET, P. KLOPP, R. BURCELIN, S. LAHTINEN: "Potential probiotic Bifidobacterium animalis ssp. lactis 420 prevents weight gain and glucose intolerance in diet-induced obese mice", BENEFICIAL MICROBES, WAGENINGEN ACADEMIC PUBLISHERS, NL, vol. 5, no. 4, 1 December 2014 (2014-12-01), NL , pages 437 - 445, XP055296828, ISSN: 1876-2883, DOI: 10.3920/BM2014.0014 *
MOON HO DO: "Bifidobacterium animalis ssp. lactis MG741 Reduces Body Weight and Ameliorates Nonalcoholic Fatty Liver Disease via Improving the Gut Permeability and Amelioration of Inflammatory Cytokines", NUTRIENTS, M D P I AG, CH, vol. 14, no. 9, CH , pages 1965, XP093154714, ISSN: 2072-6643, DOI: 10.3390/nu14091965 *
NIÑO-VÁSQUEZ IVÁN A., MUÑIZ-MÁRQUEZ DIANA, ASCACIO-VALDÉS JUAN A., CONTRERAS-ESQUIVEL JUAN CARLOS, AGUILAR CRISTÓBAL N., RODRÍGUEZ: "Co-microencapsulation: a promising multi-approach technique for enhancement of functional properties", BIOENGINEERED, LANDES BIOSCIENCE, US, vol. 13, no. 3, 1 March 2022 (2022-03-01), US , pages 5168 - 5189, XP009553654, ISSN: 2165-5979, DOI: 10.1080/21655979.2022.2037363 *
SANCHEZ-PORTILLA, Z. ET AL.: "Incortporation of Bifidobacterium sp. into poder products through a fluidized bed process for enteric targeted release", J.DAIRY SCI., vol. 103, 2020, pages 11129 - 11137, [retrieved on 20230504], DOI: http://doi.org/10.3168/jds.2020- 18516 *

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