MICROENCAPSULATED PROBIOTIC SUBSTANCE.

专利摘要:
Solid particles in dry powder form containing a probiotic microorganism and a support phase in which said probiotic microorganism is encapsulated, said support phase further comprising at least one nutrient source, said solid particles in dry powder form having a particle size distribution of between n and (n + 400) μm, wherein n is between 400 and 900 μm.
公开号:BE1019142A3
申请号:E2011/0633
申请日:2011-11-02
公开日:2012-03-06
发明作者:Johan Henri Herman Quintens；Van Lidth De Jeude Jehan Lienart；Thorsten Brandau；Holger Strohm
申请人:Vesale Pharma S A；Brace Gmbh；
IPC主号:

专利说明:

MICROENCAPSULATED PROBIOTIC SUBSTANCE
The present invention relates to a microencapsulated probiotic substance, in particular solid particles in dry powder form comprising a probiotic microorganism and a support phase in which said probiotic microorganism is encapsulated, said support phase further comprising at least one nutrient source as well as an enteric composition.
These microencapsulated probiotic substances are already known in the art. The document US 2010/0189767 describes a microencapsulated probiotic substance comprising at least one probiotic substance and a first coating, comprising, for example, wax, shellac, resistant starch, zein protein, ethylcellulose , methylcellulose, hydroxypropyl methylcellulose, amylase acetophthalate, cellulose acetophthalate, hydroxylpropyl methylcellulose phthalate, ethyl acrylate and methyl methacrylate in the form of a glassy matrix. The vitreous matrix here designates a matrix which is solid at ambient temperature and which has a rigid structure and which has a high elastic modulus and strength.
The microencapsulated probiotic substance according to the state of the art suffers from the disadvantages characterized in that the viability of the probiotic substance is limited to a few days when kept at physiological temperatures.
Studies and the subjective perception of consumers indicate that the consumption of probiotics has a number of effects that are perceived to be beneficial to health. Probiotic-based pharmaceuticals must be prepared, shipped and stored under refrigerated conditions, with a break in the cold chain not only producing a reduction in activity, but also entailing considerable transportation and surveillance costs related to cold chain. The objective of this invention is to increase viability during storage, with a special effort on long-term stability and thermal stability.
According to Füller's (1989) definition, probiotics are live microbial food supplements that produce a beneficial effect on the host by improving its intestinal microbial balance, while the World Health Organization (WHO) defines probiotics as being live micro-organisms (bacteria or yeasts) which, when ingested or locally applied in sufficient numbers, confer one or more specified and demonstrated beneficial effects on the health of the host. This well-accepted knowledge translates into a large number of probiotic products on the market, ranging from nutritional drinks containing probiotics to anti-acne formulations and other applications. However, pharmaceutical formulations have in common the problem of not being "attractive to consumers", since, unlike food products, for which the refrigerated storage is well accepted, it is badly accepted that the pharmaceutical formulations are stored in the refrigerator since it is the place where you put food.
Most formulation developments focus on the actual application, since probiotics, despite the fact that they themselves produce lactic acid, do not tolerate the strong acid in the human or animal stomach. .
In particular, the object of the invention is to overcome at least some of these disadvantages by providing a microencapsulated probiotic substance that ensures increased stability of the microencapsulated probiotic substance, resulting in an extended shelf life with high viabilities of the encapsulated probiotic substances even at high temperatures. physiological or superior.
To solve this problem, the present invention provides a microencapsulated probiotic substance as mentioned at the beginning, wherein said solid particles in dry powder form have a particle size distribution of between n and (n + 400) pm, wherein is between 400 and 900 μm.
The present invention therefore provides a solid particle composition in the form of a dry powder which is stable during production, transport, storage and application, even at room temperature. This novel encapsulation method has been shown to provide elongated shelf life with high viabilities of the encapsulated probiotic strains. It can also be observed that viability increases at high temperatures up to 55 ° C, and even that probiotic strains can withstand temperature peaks of 80 ° C when the configuration is correct.
However, probiotics are also sensitive during production because they are often kept in solution for an extended period of time, handling and storage may also affect them. The production of droplets under laminar flow conditions has been used as a very gentle production process, to avoid the high stresses associated with spray drying which reduces viability. This invention indicates that laminar flow flow drip, preferably with a vibrational carrier, produces particles having a high number of viable microorganisms and survival rates far superior to processes such as spray drying. as well as a well-defined size distribution of the resulting spheres. It has been shown that the low dispersion of the size improves the release characteristics and thus improves the overall effect of the probiotics.
This effect is achieved by the fact that the solid particles in dry powder form have a homogeneous distribution of particle size, which gives higher survival rates compared to existing microencapsulation techniques which give larger size distributions and therefore variable pearl protection and false survival test results. Surprisingly, it has been discovered that the vibrational drip-casting process provides monomodal and closely distributed particles with homogeneous properties, whose overall stability is greater than the known particles.
After production, probiotics are trapped in the matrix of encapsulation materials, but they can continue to degrade and decompose. It is therefore possible that an additional stabilization step must be performed, which is possible for example by drying, lyophilization, various preservation media, spray drying, etc. This invention shows that through the use of a suitable nutrient, the stabilization and viability of the probiotics upon lyophilization increases as opposed to unstabilized probiotics.
Advantageously, the solid particle in the form of a dry powder has a said particle size distribution d8o of between n and (n + 200) μm, in which n is between 400 and 900 μm.
In a preferred embodiment, the carrier phase comprises at least one substance selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan and agar.
The resulting particles must not only pass through the stomach, but they must also disintegrate in the intestine to be released and fulfill their health mission. It is therefore appropriate to use the correct combination of coating materials. In this invention, the materials should be selected such that the microspheres are released into the intestine after passing through the stomach and bile fluids, so that the survival rate is high enough to give a clinical effect. .
Preferably, said at least one substance is in the form of a hydrocolloid. The advantages obtained by choosing such hydrocolloids as the first coating include: nontoxicity, soft gels being used to trap sensitive materials such as probiotic substances, the viability of probiotic substances during the shelf life of the encapsulated products and the reversibility of the immobilization since the gels can be solubilized to release the encapsulated probiotic substances.
Advantageously, said nutritional source comprises at least one compound selected from the group consisting of a monosaccharide, a polysaccharide, an amino acid, a peptide, a protein, a vitamin, a a yeast extract, an alkali metal halide or an alkaline earth metal salt, an antioxidant, a glycerol, zinc acetate, zinc chloride, lactate, zinc, ascorbic acid, citric acid, vegetable oil and milk fat.
In a preferred embodiment, said nutrient source is present in an amount of 0.1 to 10% by weight, preferably 1 to 5% by weight based on the total weight of the microspheres prior to drying.
More preferably, the solid particles in dry powder form according to the invention comprise an outer coating selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, Agar, cellulose, hemicellulose, ethylcellulose, carboxymethylcellulose and mixtures thereof.
Other embodiments of solid particles in dry powder form according to the invention are described in the related claims.
The invention also relates to an enteric composition comprising said solid particles in dry powder form according to the invention in a suitable support.
As mentioned above, the fact that viability is preserved during production and storage and that probiotics are protected against temperature variations and acid attacks makes solid particles in the form of dry powder suitable for the manufacture of a highly effective enteric composition.
Advantageously, said suitable excipient is an enteric coating selected from the group consisting of ethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, Eudragit®, thereby rendering the enteric composition resistant to the conditions prevailing in the stomach. for an effect in the intestinal area.
Preferably, the enteric composition is in the form of a soft capsule or capsule, tablets, sachets and the like.
Other embodiments of the enteric composition according to the invention are mentioned in the related claims.
Further details and advantages of the microencapsulated probiotic according to the invention will become apparent from the description of the preferred embodiments of the invention by way of non-limiting examples.
Example 1
Alqinate-EC beads and probiotic paste
Microspheres of L. Rhamnosus in a matrix made of alginate and with an outer coating of ethylcellulose were prepared according to the following protocol: 150 g of L Rhamnosus paste (5 × 10 10 cfu) was dispersed in 150 g of solution sterile NaCl (0.85 wt% NaCl) to prepare a suspension of L Rhamnosus.
150 g of a sterile solution of alginate at 5% by weight was added to 300 g of the suspension of L. Rhamnosus.
Drip casting with a laminar flow drop breaker was performed to produce 800 microspheres by solidification in 4% by weight CaCl 2 solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
400 g of microspheres were stirred for 1 minute in 400 g of 1% by weight ethylcellulose solution in ethanol to produce the ethylcellulose coating (EC coating). The separation and washing of the coated microspheres was carried out in 0.85% by weight NaCl solution. 380 g of microspheres were stored in 380 g of a sterile 5% by weight aqueous glucose solution before lyophilization in said glucose preservation solution. A dry, fluid powder of microspheres 700 to 900 μm in diameter was obtained.
Example 2
Alqinate-kelatin pearls and probiotic paste
Microspheres of L. Rhamnosus in an alginate matrix and with a gelatin coating were prepared according to the protocol mentioned below: 200 g of pulp of L. Rhamnosus (5 x 1010 cfu) was dispersed in 200 g of sterile NaCl solution (0.85 wt% NaCl) to prepare a suspension of L Rhamnosus.
200 g of a sterile solution of 5% by weight alginate was added to 400 g of the L Rhamnosus suspension.
Drip casting with a laminar flow drop breaker was performed to produce 500 μm microspheres by solidification in a 5% by weight calcium lactate solution.
The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
550 g of microspheres were stirred for 1 h in 550 g of a 5 wt% gelatin solution to produce the crosslinked gelatin coating.
The microspheres were then stirred for 2 minutes in 550 g of a 10% by weight solution of glutaraldehyde. The separation and washing of the coated microspheres was carried out in 0.85% by weight NaCl solution.
550 g of microspheres were stored in 550 g of a sterile 10% by weight aqueous solution of maltodextrin before lyophilization in the maltodextrin preservation solution:
A dry, fluid powder of microspheres 400-600 μm in diameter was obtained.
Example 3
ALQININ-CMC-gelatin beads with probiotic paste
Microspheres of L. Rhamnosus in an alginate matrix and with a carboxymethylcellulose coating and a crosslinked gelatin coating were prepared as follows: 300 g of L. Rhamnosus paste (5 x 1010 cfu) was dispersed in 150 g sterile NaCl solution (0.85 wt% NaCl) to prepare a suspension of L Rhamnosus.
75 g of a sterile solution of 10% by weight alginate was added to 450 g of the L Rhamnosus suspension.
Dropwise casting with a laminar flow drop breaker was performed to produce 1000 microspheres by solidification in a 3% by weight calcium gluconate solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
500 g of microspheres were stirred for 10 minutes in 500 g of an aqueous solution of carboxymethylcellulose 2%. The coated microspheres were again separated and washed in 0.85% by weight NaCl solution.
500 g of microspheres were again stirred for 1 hour in 500 g of a 5% by weight gelatin solution to produce the cross-linked gelatin applied to the microspheres.
The microspheres were then stirred for 2 minutes in 500 g of 10% glutaraldehyde solution and separated and washed in 0.85% by weight NaCl solution.
500 g of microspheres were preserved in 500 g of a sterile aqueous solution of glycerol at 10% by weight and lyophylized in the glycerol preservation solution:
A dry, fluid powder of microspheres 800 to 1200 μm in diameter was obtained.
Example 4
Gelatin-guar-CMC gum beads with probiotic paste
Microspheres of Bifidobacterium Lactis in a gelatin matrix, coated with guar gum and carboxymethylcellulose were prepared as follows: 200 g of Bifidobacterium Lactis paste was dispersed in 100 g of sterile NaCl solution (0.85% NaCl). by weight) to prepare a suspension of Bifidobacterium Lactis.
150 g of a 30% sterile gelatin solution was added to 300 g of the Bifidobacterium Lactis suspension at 37 ° C.
Drip casting with a laminar flow drop breaker was performed to produce 1000 microspheres by solidification in caprylic / capric triglyceride at 5 ° C. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
400 g of microspheres were stirred for 10 minutes in 400 g of an aqueous solution of 5% by weight guar gum to produce the guar gum-coated microspheres. The coated microspheres were then separated and washed in 0.85% by weight NaCl solution.
400 g of microspheres were stirred for 10 minutes in 400 g of an aqueous solution of 2% carboxymethylcellulose to make the CMC coating. The microspheres were then separated and washed in 0.85% by weight NaCl solution.
400 g of microspheres were preserved in 400 g of a sterile aqueous solution of glycerol at 4% by weight before lyophilization in this glycerol preservation solution:
A dry, fluid powder of microspheres 800 to 1200 μm in diameter was obtained.
Example 5
Alqinate-chitosan-gelatin beads with probiotic paste
Microspheres of L. Rhamnosus in an alginate matrix and with a coating of chitosan with additional gelatin coating were prepared as follows: 400 g of L. Rhamnosus paste (5 x 1010 cfu) was dispersed in 200 g sterile NaCl solution (0.85 wt% NaCl) to prepare a suspension of L Rhamnosus.
100 g of a sterile solution of 10% alginate was added to 600 g of the suspension of L. Rhamnosus.
Drip casting with a laminar flow drop breaker was performed to produce 1000 microspheres by solidification in 2% by weight CaCl 2 solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
600 g of microspheres were stirred for 10 minutes in 1200 g of 1% by weight aqueous chitosan solution to make microspheres coated with chitosan. The separation and washing of the coated microspheres was carried out in 0.85% by weight NaCl solution.
600 g of microspheres were stirred for 1 hour in 1200 g of a 5% by weight gelatin solution to continue coating the microspheres with gelatin. The separation and washing of the coated microspheres was carried out in 0.85% by weight NaCl solution.
600 g of microspheres were preserved in 600 g of a sterile aqueous solution of glycerol at 4% by weight before lyophilization in the glycerol preservation solution:
A dry, fluid powder of microspheres 800 to 1200 μm in diameter was obtained.
Example 6
Alainate beads with probiotic paste
Microspheres of L. Rhamnosus in an alginate matrix were prepared as follows: 200 g of L Rhamnosus paste (5 x 1010 cfu) was dispersed in 150 g of a sterile 6.7% polysaccharide solution. weight and NaCl at 0.85% by weight to prepare a suspension of L Rhamnosus.
230 g of a sterile solution of alginate at 3% by weight was added to 350 g of the suspension of L Rhamnosus.
Drip casting with a laminar flow drop breaker was performed to produce 1000 microspheres by solidification in 2% by weight CaCl 2 solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
550 g of microspheres were stored in 550 g of sterile 5% by weight aqueous glucose solution and 3% by weight glycerol before lyophilization in the glycerol preservation solution:
A dry, fluid powder of microspheres 400 to 900 μm in diameter was obtained.
The enumeration of viable bacteria in the microspheres is performed as follows:
Two samples were prepared with the microsphere of L. Rhamnosus in alginate, one from the dry powder and one in the wet state:
Sample 1: microspheres of Lactobacillus rhamnosus, diameter of about 400 to 900 microns, dried with glucose / glycerol.
Sample 2: Microspheres of Lactobacillus rhamnosus, diameter of about 800 to 1200 microns, wet in a glucose / glycerol solution.
The microspheres must be dissolved before enumeration of viable bacteria. The dissolution procedures were adapted to the differences during the drying step of the microspheres: Sample 1 was prepared by weighing 100 mg dry microspheres under aseptic conditions in a sterile 15 ml conical tube and adding 2, 9 ml of 0.1 M Na citrate. The mixture is vortexed for 15 minutes (30x dilution).
Sample 2 was prepared by first separating the microspheres from the preservative solution (glucose / glycerol solution) using a sterile sieve (whatman filter paper). 100 mg of wet microspheres were added to a sterile 15 ml conical tube with 1.9 ml of 0.1 M Na citrate. The mixture was vortexed for 3 minutes (20X dilution).
The samples were dissolved in duplicate for both samples.
15 ml of MRS Agar were poured approximately into each plate and allowed to solidify at room temperature on a flat, cold surface.
In sterile tubes filled with 9 ml of sterile 0.1% peptone dilution blanks, 1 ml of the primary dilution (from the conical tube) is added to the 9 ml of diluent using a pipette of 1 ml to obtain a 10'1 dilution. This operation is repeated until the desired dilution series is obtained. Dilution tubes agitated according to the recommendations of standard dairy product review procedures. The experiments are performed in triplicate. 0.1 ml of each appropriate dilution is transferred to the surface of labeled sterile Petri plates in which about 15 ml of MRS Agar nutrient medium is poured. Plates were incubated at 35 ° C for a minimum of 72 hours up to 144 hours.
Counting of colonies on MRS Agar plates and counting of viable Lactobacillus rhamnosus cells per gram, taking into account the counting factor of the plates counted. Only plates with 25 to 250 colonies should be counted. (See Standard Methods for the Examination of Dairy Products, 16th Edition, pages 213-246).
RESULTS
Initial weight: - Sample 1: duplicate 1: 100 mg duplicate 2: 103 mg Average: 101.5 mg.
- Sample 2: duplicate 1: 102 mg duplicate 2: 99 mg Average: 100.5 mg
Initial dilution rate: - Sample 1: 101.5 mg in 2.9 ml = 29.6 X dilution.
- Sample 2: 100.5 mg in 1.9 ml = 19.9 X dilution
Counting cfu (colony-forming-unit, strain bacteria):
Dilution 10'5 Dilution 10'4 Dilution 10'3 Dilution 10 '2 Replication 1231231231 2
Sample 1____________
Duplicate 3 6 1 18 6 19 299 192 279>> 1: ____________
Duplicate 1 1 1 17 12 16 151 114 160>>
2: I i! II I II
Sample 2____________
Duplicate 8 6 8 32 88 52 - 171 203>> 1: ____________
Duplicat - 8 6 35 85 39 - 123>> 2: I I i l 11 11
The results of the count are as follows:
Sample 1: 192.103 x 29.6 = 5.68 1 06 cfu / g of microspheres (dry weight)
Sample 2: 635.103 x 19.9 = 1.26 1 07 cfu / g microspheres (wet weight)
As can be seen, by correctly choosing the diameter and the nutrient, a survival rate of 1/1000 can be obtained throughout the treatment. Although a larger diameter can preserve a higher number of living cells during the process, the yield of live microorganisms greater than 1 × 10 7 cfu is sufficient to obtain a probiotic effect.
Example 7
Alkylate-CMC-gelatin beads with freeze-dried probiotics
Microspheres of L. rhamnosus in an alginate matrix coated with carboxymethylcellulose and gelatin were made as follows: 150 g of lyophilized L. rhamnosus powder (8.8 x 1011 cfu / g) was dispersed in 300 g of sterile NaCl solution (0.85 wt% NaCl) to prepare a suspension of L. Rhamnosus. The number of L. rhamnosus bacteria obtained is therefore 1.32.1014 cfu in 150 g.
75 g of a sterile solution of alginate at 10% by weight was added to 450 g of the suspension of L. Rhamnosus.
Dropwise casting with a laminar flow drop breaker was performed to produce 1000 microspheres by solidification in a 3% by weight calcium gluconate solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
500 g of microspheres were stirred for 10 minutes in 500 g of an aqueous solution of carboxymethylcellulose at 2% by weight to obtain the coating of CMC. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
In addition, 500 g of microspheres were stirred for 1 h in 500 g of a 5 wt% gelatin solution, and the microspheres were then stirred for 2 minutes in 500 g of a 10% glutaraldehyde solution. by weight to obtain a crosslinked gelatin coating. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
500 g of microspheres were stored in 500 g of a sterile 10% by weight aqueous glycerol solution before lyophilization in a preservative solution. After double coating and crosslinking which do not absorb all of the coating material but only a small amount of 0.1 to 1%, the spheres were dried and 50 g of glycerol was added (500 g of 10% glycerol by weight), giving a dry matter of 203.93 g, in the form of a dry, fluid powder of microspheres of 800 to 1200 μm in diameter with a cell number of 2.9 × 10 11 cfu / g. This means that among the 1.32 × 10 14 cfu initially present in the freeze-dried L. rhamnosus form, there remains 0.61 × 10 14 cfu (203.93 g, 2.9 × 10 11). Therefore, the yield of live probiotics is about 50%, which is considerably higher than with the method of the prior art.
Example 8
Alginate-EC beads with freeze-dried probiotics
L Rhamnosus microspheres in an ethylcellulose-coated alginate matrix were made as follows: 67.5 g of lyophilized Lhamnosus powder (8.8 x 1011 cfu) was dispersed in 217.5 g of Sterile NaCl (0.85 wt% NaCl) to prepare a suspension of L rhamnosus.
150 g of a sterile solution of 5% alginate was added to 300 g of the suspension of L. Rhamnosus;
Drip casting with a laminar flow drop breaker was performed to produce 800 microspheres by solidification in a 4 wt% CaCb solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
In addition, 400 g of microspheres were stirred for 1 minute in 400 g of 1% by weight ethylcellulose solution in ethanol to prepare EC coated microspheres. The separation and washing of the microspheres are carried out in a solution of NaCl at 0.85% by weight.
380 g of microspheres were preserved in 380 g of a sterile aqueous glucose solution at 5% by weight before lyophilization in the glucose preservation solution: 96.32 g of a dry, fluid powder of microspheres of 700 to 900 pm diameter with a cell number of 1.9 x 1011 cfu were obtained. This means that among the 5,94,1013 cfu present in the first step, there remains 1.83.1013 cfu (8.8.1011 x 67.5) of live probiotics (96.32 X 1.9.1011) corresponding to about 31% of probiotics always living.
Example 9
Alqinate-gelatin beads with freeze-dried probiotics
Microspheres of L. rhamnosus in a gelatin-coated alginate matrix were made as follows: 100 g of lyophilized L. rhamnosus powder (8.8 x 1011 cfu) was dispersed in 300 g of sterile NaCl solution ( 0.85% by weight NaCl) to prepare a suspension of L Rhamnosus. The probiotics initially present to prepare the alginate-gelatin beads represent 8.8.1013 cfu.
200 g of a sterile solution of alginate at 5% by weight was added to 400 g of the suspension of L. Rhamnosus.
Drip casting with a laminar flow drop breaker was performed to produce 500 μm microspheres by solidification in a 5% by weight calcium lactate solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
550 g of microspheres were stirred for 1 h in 550 g of a 5 wt% gelatin solution to obtain a crosslinked gelatin coating.
The microspheres were then stirred for 2 minutes in 550 g of a 10% by weight solution of glutaraldehyde. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
550 g of the microspheres were stored in 550 g of a sterile aqueous solution of 10% maltodextrin before lyophilization in the maltodextrin preservation solution: 168.25 g of a dry, fluid powder of microspheres of 400 to 600 diameter with a cell number of 8.5 x 1010cfu were obtained, corresponding to 1.43.1013 cfu in the 168.25 g.
In conclusion, an alginate-gelatin microcapsule having a cross-linked coating has a substantially high number of surviving microorganisms, essentially for a viable commercial process since the ratio of the still-alive probiotics is 16.25, resulting in a powder containing 8, 9.1010 cfu (considerably higher than the 107 cfu required).
Example 10
CMC quar-gelatin-gum beads - freeze-dried probiotic
Microspheres of Bifido bacterium lactis in a gelatin matrix coated with guar gum and carboxymethylcellulose were prepared as follows: 100 g of freeze-dried Bifidobacterium lactis powder was dispersed in 200 g of sterile NaCl solution (0.85 NaCl). % by weight) to prepare a suspension of Bifidobacterium lactis.
150 g of 30% by weight sterile gelatin solution was added to 300 g of Bifidobacterium lactis suspension at 37 ° C.
Drip casting with a laminar flow drop breaker was performed to produce 1000 microspheres by solidification in caprylic / capric triglyceride at 5 ° C. The separation and washing of the microspheres was carried out in a 0.85% NaCl solution.
400 g of microspheres were stirred for 10 minutes in 400 g of an aqueous solution of guar gum at 5% by weight to coat the microspheres of guar gum and the separation and washing of the microspheres were carried out in a solution of NaCl at 0.85% by weight.
400 g of microspheres were stirred for 10 minutes in 400 g of an aqueous solution of carboxymethylcellulose at 2% by weight to coat the microspheres of CMC and the separation and washing of the microspheres were carried out in a solution of NaCl at 0, 85% by weight.
400 g of microspheres were stored in 400 g of a sterile aqueous solution of glycerol at 4% by weight before lyophilization in the glycerol preservation solution:
A dry, fluid powder of microspheres 800 to 1200 microns in diameter with a cell count of 2.9 x 10 11 cfu was obtained, which is greater than the value of 107 required for such an application.
In conclusion, it has been shown that the coating of carboxymethylcellulose with glycerol as a nutrient source during lyophilization gives very high survival rates in an enteric microsphere.
Example 11
Alginate-chitosan-gelatin beads with freeze-dried probiotics
L rhamnosus microspheres in an alginate matrix coated with chitosan and gelatin were made as follows: 200 g lyophilized Lhamnosus powder (8.8 x 10 11 cfu) was dispersed in 400 g sterile NaCl solution. (NaCl at 0.85% by weight) to prepare a suspension of L. Rhamnosus (1.76.1014 cfu of L rhamnosus present at the start).
100 g of a sterile solution of 10% alginate was added to 600 g of the L Rhamnosus suspension.
Drip casting with a laminar flow drop breaker was performed to produce 1000 microspheres by solidification in 2% by weight CaCl 2 solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
600 g of microspheres were stirred for 10 minutes in 1200 g of 1% by weight aqueous chitosan solution and the separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
600 g of microspheres were again stirred for 1 hour in 1200 g of a 5% by weight gelatin solution and the separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution. .
600 g of microspheres were preserved in 600 g of a sterile aqueous solution of glycerol at 4% by weight before freeze-drying in the glycerol preservation solution: 238.8 g of a dry and fluid powder of microspheres of 800 to 1 200 μm in diameter with a cell number of 2.9 × 10 11 cfu were obtained, corresponding to a total of 0.69 × 10 14 cfu (yield of live probiotics = 39.3%).
In conclusion, it has been shown that the coating of chitosan with glycerol as a nutrient source during lyophilization gives very high survival rates in an enteric microsphere.
Example 12
Alqinate beads with freeze-dried probiotics 75 g lyophilized Lhamnosus powder were dispersed in 250 g sterile 3.6 wt% polysaccharide solution and 0.85 wt% NaCl to prepare a suspension. from L Rhamnosus.
175 g of a sterile solution of alginate at 5% by weight was added to 325 g of the suspension of L Rhamnosus.
Drip casting with a laminar flow drop breaker was performed to produce 1100 microspheres by solidification in 2% by weight CaCl 2 solution. The separation and washing of the microspheres was carried out in 0.85% by weight NaCl solution.
450 g of microspheres were stored in 450 g of sterile aqueous glucose solution at 5% by weight.
The enumeration performed as previously described indicates 8.1 × 10 9 cfu / g wet weight of microspheres. A content of 1.87.1011 cfu / g was present in the freeze-dried powder, instead of the 45.1011 declared. Since the freeze-dried starting powder represents 15% of the total weight of the wet microspheres, this content corresponds to (8.1 × 10 9 X 100)% = 5.4 × 10 10 cfu / g of powder equivalent.

权利要求:
Claims (10)
[1]
1. Solid particles in dry powder form containing: a) a probiotic microorganism, b) a support phase in which said probiotic microorganism is encapsulated, said support phase further comprising at least one nutrient source, c) characterized in that said solid particles in dry powder form have a particle size distribution of between n and (n + 400) pm, wherein n is between 400 and 900 μm.
[2]
Solid particles in dry powder form according to claim 1, wherein said particle size distribution d80 is between n and (n + 200) μm, wherein n is between 400 and 900 μm.
[3]
Solid particles in dry powder form according to claim 1 or claim 2, wherein said carrier phase comprises at least one substance selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar.
[4]
Solid particles in dry powder form according to claim 3, wherein said at least one substance is in the form of a hydrocolloid.
[5]
Solid particles in dry powder form according to any one of claims 1 to 4, wherein said nutritive source comprises at least one compound selected from the group consisting of a monosaccharide, a polysaccharide, an acid amino acid, a peptide, a protein, a vitamin, a yeast extract, an alkali metal halide salt or an alkaline earth metal, an antioxidant, glycerol, zinc acetate, zinc chloride, zinc lactate, ascorbic acid, citric acid, vegetable oil or milk fat.
[6]
Solid particles in dry powder form according to any one of claims 1 to 5, wherein said nutrient source is present in an amount of from 0.1 to 10% by weight, preferably from 1 to 5% by weight. relative to the total weight of solid particles in the form of dry powder.
[7]
Solid particles in dry powder form according to any one of claims 1 to 6, further comprising an outer coating selected from the group consisting of alginate, chitosan, pectin, pullulan, gelatin, carrageenan, agar, cellulose, hemicellulose, ethylcellulose, carboxycellulose, and mixtures thereof.
[8]
An enteric composition comprising said solid particles in dry powder form according to any one of claims 1 to 7, in a suitable excipient.
[9]
9. An enteric composition according to claim 8, wherein said suitable excipient is an enteric coating selected from the group consisting of ethylcellulose, hydroxypropylcellulose, carboxymethylcellulose and Eudragit®.
[10]
An enteric composition according to claim 8 or claim 9 in the form of a soft capsule or capsule, tablets, sachets and the like.

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---

Raddatz et al.2020|Use of prebiotic sources to increase probiotic viability in pectin microparticles obtained by emulsification/internal gelation followed by freeze-drying

---

Nawong et al.2016|Entrapment in food-grade transglutaminase cross-linked gelatin–maltodextrin microspheres protects Lactobacillus spp. during exposure to simulated gastro-intestinal juices

---

Paramera et al.2014|Yeast cells and yeast-based materials for microencapsulation

---

JP5866361B2|2016-02-17|Method to increase the storage stability of probiotics

---

WO1999052511A1|1999-10-21|Tarch capsules containing microorganisms and/or polypeptides or proteins and a process for producing them

---

Raddatz et al.2020|Influence of the prebiotics hi-maize, inulin and rice bran on the viability of pectin microparticles containing Lactobacillus acidophilus LA-5 obtained by internal gelation/emulsification

---

Nag2011|Development of a microencapsulation technique for probiotic bacteria Lactobacillus casei 431 using a protein-polysaccharide complex: a thesis presented in partial fulfillment of the requirements of the degree of Masters of Technology in Food Technology at Massey University, Palmerston North, New Zealand

---

Oberoi et al.2021|Microencapsulation: an overview for the survival of probiotic bacteria

---

Kailasapathy2015|Biopolymers for administration and gastrointestinal delivery of functional food ingredients and probiotic bacteria

---

FR3065162B1|2019-06-28|PECTIN MICROCAPSULES, PROCESS FOR PRODUCING THE SAME AND USES THEREOF

---

Martın et al.2014|ÔØ Å ÒÙ× Ö ÔØ

---

FR3048884A1|2017-09-22|COMPOSITION FOR THE ORAL ADMINISTRATION OF AT LEAST ONE ACTIVE INGREDIENT TO A SUBJECT

---

Bekhit2015|Encapsulation of Nisin producing strain of Lactococcus lactis for active packaging development

---

Kavitake et al.0|Food Bioscience

---

WO2021234111A1|2021-11-25|Composition comprising safranal and probiotics

---

Aghajania et al.0|Microencapsulation of probiotics in yogurt: A Review

同族专利:

---

公开号 | 公开日

---

US9554590B2|2017-01-31|

---

MY161543A|2017-04-28|

---

CN103458709B|2017-10-03|

---

CA2825011A1|2012-07-26|

---

BR112013018344A2|2017-06-13|

---

US20140010918A1|2014-01-09|

---

JP5950938B2|2016-07-13|

---

JP2014502989A|2014-02-06|

---

DK2665377T3|2017-06-19|

---

WO2012098239A1|2012-07-26|

---

CA2825011C|2019-09-03|

---

RU2593327C2|2016-08-10|

---

ES2627858T3|2017-07-31|

---

EP2665377A1|2013-11-27|

---

EP2665377B1|2017-03-08|

---

CN103458709A|2013-12-18|

---

RU2013138291A|2015-02-27|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

JPS6150593B2|1984-04-18|1986-11-05|Morinaga Milk Industry Co Ltd|

---

KR100387245B1|1997-10-17|2003-08-19|일양약품주식회사|Enteric coated microgranules for stabilizing lactic acid bacteria|

---

DK1065936T3|1998-03-23|2009-11-02|Gen Mills Inc|Encapsulation of constituents in edible products|

---

EP1344458A1|2002-03-12|2003-09-17|Société des Produits Nestlé S.A.|Probiotic delivery system|

---

US20050266069A1|2002-09-06|2005-12-01|Simmons Donald L|Stable probiotic microsphere compositions and their methods of preparation|

---

US20040247580A1|2003-06-06|2004-12-09|Myung-Jun Chung|Process for preparing double-coated lactic acid bacteria powder using protein and polysaccharide and product by the same|

---

CN1613455A|2003-11-04|2005-05-11|北京东方百信生物技术有限公司|Targeting microorgan micro-capsules and their preparation|

---

US20060099247A1|2004-11-10|2006-05-11|Byrd-Walsh, Llc.|Liquid, gas and/or vapor phase delivery systems|

---

CN101257804A|2005-05-18|2008-09-03|帝斯曼知识产权资产管理有限公司|Compositions for enteral application of microorganisms|

---

EP1981338A2|2006-01-13|2008-10-22|Advanced Bionutrition Corporation|Continuous spray-capture production system|

---

CN100469869C|2006-09-08|2009-03-18|哈尔滨美华生物技术股份有限公司|Embedding protection method for beneficial bacteria of intestinal tract|

---

EP2104434A1|2006-09-19|2009-09-30|Technion Research and Development Foundation Ltd.|Probiotic compositions and methods of making same|

---

CN100537759C|2006-12-04|2009-09-09|济南赛拜斯生物工程有限公司|Enteric-coated multilayer encapsulated probiotic microcapsule and preparation method thereof|

---

CN101323850B|2008-07-28|2011-05-04|天津科技大学|Lactobacillus helveticus microcapsule, preparation and use thereof|WO2014175852A1|2013-04-23|2014-10-30|Zx Pharma, Llc|Enteric coated multiparticulate controlled release peppermint oil composition and related methods|

---

CN104642539B|2013-11-25|2018-02-09|内蒙古蒙牛乳业（集团）股份有限公司|A kind of probiotic granulate, its preparation method, contain its long shelf-life acidified milk and preparation method|

---

AU2015227075A1|2014-03-06|2016-09-22|Ohio State Innovation Foundation|Probiotic formulations and methods for use|

---

US10624934B2|2014-03-06|2020-04-21|Research Institute At Nationwide Children's Hospital|Prebiotic formulations|

---

KR101640744B1|2014-05-07|2016-07-19|일동제약주식회사|Macromolecular polysaccharide binder conjugated Lactobacillus rhamnosus RHT-3201, and use thereof in prevention and treatment of atopic dermatitis|

---

JP6456377B2|2014-06-10|2019-01-23|ライオン株式会社|Tablets containing sake yeast|

---

CN108066314A|2016-11-17|2018-05-25|中国科学院大连化学物理研究所|A kind of pectin is the biological microsphere preparation method and application of wall material|

---

WO2018154593A1|2017-02-22|2018-08-30|Praj Industries Limited|Preparation of dry formulations of dairy probiotics.|

---

AU2018244082A1|2017-03-27|2019-10-03|Praj Industries Limited|Process for the preparation of powdered probiotic formulations for monograstic animals.|

---

EP3603420A4|2017-03-28|2021-04-14|Ajinomoto Co., Inc.|Food for improving intraintestinal environment|

---

FR3065162B1|2017-04-13|2019-06-28|Universite De Bourgogne|PECTIN MICROCAPSULES, PROCESS FOR PRODUCING THE SAME AND USES THEREOF|

---

US10514721B2|2017-05-08|2019-12-24|International Business Machines Corporation|Validation of clock to provide security for time locked data|

---

WO2018230939A1|2017-06-13|2018-12-20|부산대학교 산학협력단|Probiotics-delivering hydrogel formulation for protecting probiotics in acidic environment and composition for delivering probiotics comprising same|

---

AU2018364355A1|2017-11-07|2020-05-07|Purac Biochem B.V.|Acetate powder and method for the preparation thereof|

---

KR20200093608A|2017-11-27|2020-08-05|디에스엠 아이피 어셋츠 비.브이.|Freeze-dried multiparticulate solid dosage form|

---

CN111867388A|2018-02-08|2020-10-30|丹尼斯科美国公司|Heat-resistant wax matrix particles for enzyme encapsulation|

---

CN108576371A|2018-03-30|2018-09-28|大连工业大学|A kind of microcapsule embedded double protein peptide and preparation method thereof|

---

WO2021022336A1|2019-08-07|2021-02-11|Commonwealth Scientific And Industrial Research Organisation|Compositions of probiotics and biomass and methods for promoting health in a subject|

---

CN110522061B|2019-08-28|2021-09-14|青岛职业技术学院|Microcapsule and preparation method thereof|

---

WO2021061586A1|2019-09-24|2021-04-01|Gpcp Ip Holdings Llc|Microencapsulated probiotic and compositions containing the same|

---

RU2740380C1|2019-12-24|2021-01-13|Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" |Bioengineered structure based on bacterial alginate and probiotic bacteria and method for production thereof|

---

CN112375703A|2020-11-12|2021-02-19|安徽燕婉健康科技有限公司|Therapeutic milk-derived composite probiotics and preparation method and application thereof|

法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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LU91782|2011-01-21|

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LU91782A|LU91782B1|2011-01-21|2011-01-21|MICROENCAPSULATED PROBIOTIC SUBSTANCE|

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EP11151686|2011-01-21|

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EP11151686|2011-01-21|

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