WO2022090474A1

WO2022090474A1 - Microencapsulated microbial composition

Info

Publication number: WO2022090474A1
Application number: PCT/EP2021/080142
Authority: WO
Inventors: Anette KOCK; Anette MÜLLERTZ; Daniel Bar-Shalom; Natashia Yde Mai JACOBSEN; Hanne Bjørn HØIBY; Eva-Marie LANGE
Original assignee: Deerland Probiotics & Enzymes A/S
Priority date: 2020-10-29
Filing date: 2021-10-29
Publication date: 2022-05-05

Abstract

The present invention relates to the field of nutrition. In particular, the present invention relates to the field of preparing an oral nutritional supplement comprising a microencapsulated microorganism.

Description

Microencapsulated microbial composition

Field of the invention

Background of the invention

Probiotics are live microorganisms or microbial mixtures administered to improve the subject’s microbial balance, particularly the environment of the gastrointestinal tract and the vaginal microbiota. The presence of e.g. Lactobacilli is important for maintenance of the intestinal microbial ecosystem. Lactobacilli have been shown to possess inhibitory activity toward growth of pathogenic bacteria such as Listeria monocytogenes, Escherichia coli, Salmonella spp and others. This inhibition could be due to production of inhibitory compounds such as organic acids, hydrogen peroxide, bacteriocins or reuterin or to competitive adhesion to the epithelium.

A variety of compositions for supplementing probiotics are currently available. The compositions are typically provided for improving the gut microbiota. Current formulation technologies include utilization of encapsulation and stabilization techniques for shielding the probiotics with a protective layer such that the composition comprising the microorganism may be delivered to Gl tract of the subject. Further, the focus of many formulation technologies has been to protect the viability of probiotics during distribution and storage.

Summary of the invention

One object of the present invention is to provide a probiotic formulation having improved tolerance to acids without compromising the viability of the microencapsulated microorganism. Taste and mouth feel are important quality attributes of oral nutritional supplements thus, one object of the present invention is to apply a gastric acid protection devoid of objectionable taste to powder forms of microorganisms.

To solve the problem, the present invention provides a nutritional composition as described in the below first aspect.

Microorganisms, including lyophilized bacteria, provide particular limitations to the encapsulation process, as they are living organisms that need to be viable after the process in order to obtain the desired biological effect. Parameters that affect the viability of the microencapsulated microorganisms include process parameters such as temperature, mechanical abrasion and humidity.

In one aspect, the present invention provides a method for preparing a microcapsule comprising a microorganism, said method comprising the steps of:

(i) providing at least one microorganism or a granulate of said microorganism(s);

(ii) introducing said microorganism in a fluidized bed reactor comprising a coating chamber and an expansion chamber;

(iii) fluidizing said microorganism using a fluidizing gas;

(iv) introducing a coating dispersion into the coating chamber, wherein said coating dispersion comprises one or more fatty alcohol(s) is in (molten) liquid form;

(v) allowing the formation of a microcapsule comprising said microorganism; wherein said fluidizing gas is having a temperature below the melting temperature of said fatty alcohol.

In a second aspect, the present invention provides a microcapsule comprising a microorganism obtainable by the method of the present invention.

In a third aspect, the present invention provides a composition comprising the microcapsule comprising a microorganism according to the present invention. In a further aspect, the present invention provides a microcapsule comprising a microorganism, wherein the surface of said microcapsule comprises one or more fatty alcohol (s).

Brief description of the drawings

Figure 1. Viability of B. longum and microencapsulations thereof (CFU/gram) as function of incubation under acid conditions (pH 1.5-1.7 at 37°C). Cetostearyl alcohol encapsulated B. longum, 203%WG (black triangle); cetostearyl alcohol encapsulated B. longum, 534%WG (white diamond); B. longum untreated (black diamond).

Figure 2. Viability of L. rhamnosus and microencapsulations thereof (CFU/gram) as function of incubation under acid conditions (pH 1.5-1.7). L. rhamnosus untreated (white square); cetostearyl alcohol encapsulated L. rhamnosus 500%WG (black square); cetostearyl alcohol encapsulated 3:7 L. rhamnosus 568%WG (white circle); L. rhamnosus R0343 BIO-SUPPORT™ (black circle).

Figure 3. Bacterial survival (L rhamnosus) as function of process time. Bacteria survival compared to start corrected for coating amount (%). The data labels on the figure refer to the applied amount of material. For the 400%WG sample the actual weight gain of the encapsulated particles was determined by GC-MS to 568%WG

Figure 4. Viability of B. longum and microencapsulations thereof (CFU/gram) as function of incubation under acid conditions (pH 1.5-1.7 at 37°C). B. longum encapsulated, 120%WG (black diamond); B. longum untreated (white square).

Figure 5. Viability of L. acidophilus and microencapsulations thereof (CFU/gram) as function of incubation under acid conditions (pH 1.5-1.7 at 37°C). L. acidophilus encapsulated, 187%WG (black triangle); L. acidophilus untreated (white circle).

Figure 6. Correlation between encapsulation material weight gain and acid tolerance of B. longum (pH 1.5-1.7 at 37°C for 60 min). Figure 7. Acid tolerance (pH 1.5-1 .7 at 37°C) of microencapsulate of B. longum at manufacture and after 26 months storage. Abbreviations: T26; Measured after 26 months storage at the indicated temperature.

Figure 8. Correlation between encapsulation material weight gain and acid tolerance (pH 1.5-1 .7 at 37°C for 60 min) of L. acidophilus.

Figure 9. Long-term stability of B. longum and microencapsulate thereof (alone and in a nutritional supplement). The investigated nutritional supplement is a granulate powder in a stick pack. B. longum encapsulated, 203%WG (black diamond); B. longum encapsulated in a nutritional supplement, 101 %WG (white square); B. longum untreated in a nutritional supplement (black circle).

Figure 10. Comparison of long-term stability at three temperatures of microencapsulated B. longum in a nutritional supplement. The investigated nutritional supplement is a granulate powder in a stick pack. -10°C (black diamond); ambient temperature (white square); 25°C/60% relative humidity (black circle).

Detailed description of the invention

In describing the embodiments of the invention specific terminology will be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is understood that each specific term includes all technical equivalents, which operate in a similar manner to accomplish a similar purpose.

Microbial organism

In the context of the present invention the term “live microbial organism” refers to a microorganism that when ingested in adequate amounts by a subject (such as in the form a formulation as described herein) confers a health benefit to the subject. A probiotic microorganism is a live microorganism which, when administered in adequate amounts, confers a health benefit to the host by influencing the composition and or metabolic activity of the flora of the gastrointestinal (Gl) tract (FAO/WHO 2001). Health benefits reported include (i) improved digestion of lactose and reduced intestinal bloating, flatulence and discomfort; (ii) prevention of traveller's diarrhoea;

(iii) enhancing the immune system, improving resistance to infection and improving well-being; (iv) lowering serum cholesterol levels and reducing incidence of coronary heart disease; (v) treating intractable diarrhoea following antibiotic therapy; (vi) reducing allergic inflammation.

A first aspect of the present invention provides a method for preparing a microcapsule comprising a microorganism, said method comprising the steps of:

(iii) fluidizing said microorganism using a fluidizing gas;

(iv) introducing a coating dispersion into the coating chamber, wherein said coating dispersion comprises one or more fatty alcohol(s) in liquid form;

(v) allowing formation of a microcapsule comprising said microorganism; wherein said fluidizing gas is having a temperature below the melting temperature of said fatty alcohol.

The terms ‘a microcapsule comprising a microorganism’ and ‘a microencapsulated microorganism’ are interchangeable terms in the context of the present invention.

The inventors of the present invention have discovered that the microcapsule comprising a microorganism obtained from the method demonstrates improved tolerance to acids and improved viability compared to a commercially available product comprising a microorganism and protective matrix.

In one embodiment, the melting point of said fatty alcohol is above 18°C, such as above 20°C, for example above 25°C, preferably above 37°C.

Preferably, the fatty alcohol is a C12-C24 chain fatty alcohol, such as C12-C18.

Thus, in one embodiment, one or more fatty alcohol(s) is independently selected from the group consisting of C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, and C24.

In one embodiment, the coating dispersion comprises one or more fatty alcohol(s) selected from the group consisting of cetyl alcohol (C16), stearyl alcohol (C18), myristyl alcohol (C14), lauryl alcohol (C12) and cetostearyl alcohol or any combination thereof. In another embodiment, the coating dispersion comprises cetyl alcohol, stearyl alcohol and myristyl alcohol. In a further embodiment, the coating dispersion comprises cetyl alcohol and stearyl alcohol. In one embodiment, the coating dispersion comprises stearyl alcohol.

The coating dispersion may comprise one or more excipients. However, in some embodiment, the coating dispersion consists or consists essentially of a fatty alcohol selected from the group consisting of cetyl alcohol (C16), stearyl alcohol (C18), myristyl alcohol (C14), lauryl alcohol (C12) and cetostearyl alcohol or any combination thereof. In other embodiments, the coating dispersion consists or consists essentially of cetyl alcohol and stearyl alcohol.

In one embodiment, the coating dispersion comprises a mixture of cetyl alcohol and stearyl alcohol. In another embodiment, the coating dispersion comprises a mixture of cetyl alcohol, stearyl alcohol and myristyl alcohol.

In one embodiment, the coating dispersion comprises 0 to 40 wt% cetyl alcohol, 25 to 100 wt% stearyl alcohol and 0 to 20 wt% myristyl alcohol of the total weight of the coating dispersion. In another embodiment, the coating dispersion comprises a mixture of cetyl alcohol and stearyl alcohol in the ratio 3 to 7. In a further embodiment, the coating dispersion comprises a mixture of cetostearyl alcohol, and myristyl alcohol in the ratio 9 to 1.

In the context of the present invention, the term microcapsule refers to a particle with a diameter of 0.2 -5000 micrometre, irrespective of the precise interior or exterior structure. In one embodiment, the microcapsule is having a size in the range of 0.2 to 5000 micrometre, such as 1 to 5000 micrometre. In another embodiment, the microcapsule is having a size (diameter) in the range of 100 to 2000 micrometre, such as 100 to 1000 micrometre. In a further embodiment, the microcapsule is having average size in the range of 300 to 600 micrometre.

The amount of the coating dispersion added to the particles is expressed in % weight gain (WG). In one embodiment, the amount of the coating dispersion corresponds to 30% to 800% of the weight of the microorganism, such as 50% to 500%, preferably 80% to 300%. In a further embodiment, the the amount of the coating dispersion is less than 300% of the weight of the microorganism, such as above 80%, but less than 300% of the weight of the microorganism, such as such as in the range of 80% to 210% of the weight of the microorganism, such as in the range of 100% to 210% of the weight of the microorganism. The inventors have surprisingly discovered that the survival of the microorganism after the the encapsulation drop significantly at a weight gain of 300% and in particulary at 400% (See Example 2, Table 3).

In yet a further embodiment, where method includes the addition of one ore more excipients, viscosity modifiers, emulsifiers, weight gain (WG) is measured as the the amount of the coating dispersion % of the encapsulated matter, i.e. microorganism(s) and any further ingredients.

The fluidized bed reactor comprises a spray nozzle for introducing coating material. The spray nozzle for introducing coating dispersion is preferably positioned at the bottom of the coating chamber.

The coating dispersion is introduced in the coating chamber under conditions where the one or more fatty alcohol(s) is in liquid form (molten form). In one embodiment, the melting point of said coating dispersion (and thus the melting point of said one or more fatty alcohol(s)) is above 18°C, such as above 20°C, for example above 25°C, preferably above 37°C.

In one embodiment, the fluidizing gas is having a temperature in the range of 15 to 45°C such as, 15 to 45°C, for example 15 to 40°C, such as 18 to 25, for example around 20°C during step (v). In another embodiment, the microorganism(s) is having a temperature in the range of 20 to 40°C, such as in the range of 20 to 35°C, for example in the range of 33 to 35°C during step (v).

In further embodiment, the temperature of said coating dispersion at the entry in the coating chamber through said spray nozzle is in the range of 80 to 130°C, such as 90 to 120°C, for example 90 to 110°C, such as around 100°C.

In yet a further embodiment, the said coating dispersion is introduced in coating chamber at a spray pressure in the range of 0.7 to 1.3 bar, such as 0.7 to 1.2 bar, for example 0.8 to 1.1 bar, for example 0.8 to 1.0 bar, such as 0.8 to 0.9 bar, for example around 0.8 bar.

In one embodiment, the coating dispersion is introduced in coating chamber at a spray rate in the range of 13 to 17 g/min, such as 13 to 17 g/min. In industrial scale application of the method, it may be advantageous to use a higher spray rate.

The microorganism may be introduced with one or more excipients or other actives selected from the group, but not limited to, consisting of silicon dioxide, maltodextrin, a vitamin, a prebioticand a taste modifying agent. In one embodiment, the microorganism is introduced in combination with the silicon dioxide in the range of 0.1 to 5% by weight, such as 0.1 to 1%, for example 1% or around 1%.

The coating dispersion may further comprise one or more emulsifiers. In one embodiment, the emulsifier selected from the group consisting of polysorbate, tween and acetylated monoglycides.

The coating dispersion may further comprise one or more viscosity modifiers. In one embodiment, the coating dispersion further comprises one or more viscosity modifiers selected from the group consisting of mixtures of triglycerides e.g. vegetable oil or vegetable fat (such as olive oil), ethyl cellulose, hydroxyl propyl cellulose, beewax, and shellac. In one embodiment, the coating dispersion further comprises a vegetable oil, such as 1 to 10% by weight of said vegetable oil, for example 1 to 10% by weight of olive oil, such as about 5% by weight of olive oil, such as 5% by weight of olive oil.

The ratio of microorganism to coating dispersion in the microcapsule by the method of the present invention may vary. In one embodiment, the microcapsule comprises 25-90% of said coating dispersion, such as 30-85% of said coating dispersion, for example 40-75% of said coating dispersion.

It may be advantageous to prepare the microencapsulated organism based on granulate of the microorganism. Accordingly, in one embodiment, the method includes a step of preparing a granulate by

(a) introducing said coating dispersion into the coating chamber, wherein said one or more fatty alcohol(s) is in (molten) liquid form;

(b) allowing the formation of a granulated microorganism.

In a further embodiment, the fluidizing gas is having a temperature in the range of 15 to 20°C, for example 15 to 19°C, such as 15 to 18, for example around 15°C during preparation of the granulate of the microorganism (step (a) and (b)).

In a further embodiment, the temperature of the coating dispersion at the entry in the coating chamber through said spray nozzle is in the range of 80 to 130°C, such as 90 to 120°C, for example 90 to 110°C, such as around 100°C during preparation of the granulate of the microorganism (step (a) and (b)).

In a further embodiment, the coating dispersion is introduced in coating chamber at a spray pressure in the range of 0.4 to 0.7 bar, such as 0.5 to 0.7 bar, for example 0.5 to 0.6 bar or for example around 0.6 bar during preparation of the granulate of the microorganism (step (a) and (b)).

In a further embodiment, the coating dispersion is introduced in coating chamber at a spray rate in the range of 13 to 17 g/min, such as 13 to 16 g/min, for example 14 to 16 g/min, such as 15 to 16 g/min, for example 15 g/min during preparation of the granulate of the microorganism (step (a) and (b)). In industrial scale application of the method, it may be advantageous to use a higher spray rate. The microorganism, preferably a bacterium, is typically in a lyophilized or spray dried form.

In one embodiment, the microorganism a bacterium. In a preferred embodiment, the microorganism is a probiotic bacterium. In one embodiment, the microorganism is a bacterium selected from Lactobacillales. In another embodiment, the microorganism is a bacterium selected from the group consisting of a Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Lactococcus spp., Streptococcus spp., Aerococcus spp., Carnobacterium spp., Enterococcus spp., Oenococcus spp., Sporolactobacillus spp., Tetragenococcus spp., Vagococcus spp., and Weisella spp..

In a further embodiment, the microorganism is a Lactobacillus spp. selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus fermentum, Lactobacillus gasseri DSM 225583, Lactobacillus crispatus (DSM 32717; DSM 32718; DSM 32720; DSM 22566), Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus rhamnosus SP1 (DSM 21690), Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus rhamnosus DSM 22560, Lactobacillus reuteri (ATCC 55730), Lactobacillus reuteri (DSM 17938) and Lactobacillus johnsonii (NCC533; CNCM 1-1225), Lactobacillus jensenii DSM 22567.

In another embodiment, the microorganism is a Lactococcus ssp. selected from the group consisting of Lactococcus lactis, Lactococcus cremoris, Lactococcus diacetylactis.

In one embodiment, the microorganism is a bacterium selected from Bifidobacteriales. In another embodiment, the microorganism is a Bifidobacterium spp., such as a Bifidobacterium spp. selected from the group consisting of Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum and Bifidobacterium adolescentis, Bifidobacterium lactis BI-04, Bifidobacterium lactis CNCM 1-3446 (Bb12), Bifidobacterium longum NCC3001 , ATCC BAA-999 (BB536), Bifidobacterium breve Bb-03, Bifidobacterium breve M-16V, Bifidobacterium breve R0070 and Bifidobacterium infantis.

A second aspect of the present invention relates to a microcapsule comprising a microorganism obtainable by the method according to any of the preceding claims. The inventors of the present invention have discovered that the microcapsule obtained by the method of the present invention demonstrates improved tolerance to acids.

In one embodiment, the acid tolerance of the microencapsulated microorganism is improved by a minimum of 50% compared to the uncoated microorganism.

In one embodiment, the potency of the microencapsulated microorganism is higher or equal to 10E+09 CFU/gram. This is based on a starting material containing a minimum of 5E+10 CFU/gram.

A further aspect of the present invention relates to a microcapsule comprising a microorganism, wherein the surface of said microcapsule comprises one or more fatty alcohol (s).

Preferably, the fatty alcohol is a C12-C24 chain fatty alcohol, such as C12-C18. Thus, in one embodiment, one or more fatty alcohol(s) is independently selected from the group consisting of C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C23, and C24.

In one embodiment, the microcapsule comprising a microorganism may further comprise one or more excipients or other active ingredients selected from the group, but not limited to, consisting of silicon dioxide, maltodextrin, a vitamin, a mineral, a prebioticand a taste modifying agent. In one embodiment, the microorganism is introduced in combination with the silicon dioxide in the range of 0.1 to 5% by weight, such as 0.1 to 1%, for example 1% or around 1%.

The coating dispersion may further comprise one or more viscosity modifiers. In one embodiment, the coating dispersion further comprises one or more viscosity modifiers selected from the group consisting of mixtures of triglycerides e.g. vegetable oil or vegetable fat (such as olive oil), ethyl cellulose, hydroxyl propyl cellulose, beewax, and shellac. In one embodiment, the coating dispersion further comprises a vegetable oil, such as 1 to 10% by weight of said vegetable oil, for example 1 to 10% by weight of olive oil, such as 5% by weight of olive oil.

The ratio of microorganism to coating dispersion in the microcapsule by the method of the present invention may vary. In one embodiment, the microcapsule comprises 25-90% of the coating dispersion described herein, such as 30-85% of the coating dispersion, for example 40-75% of the coating dispersion.

The microorganism, preferably a bacterium, is typically in a lyophilized or spray dried form.

In a further embodiment, the microorganism is a Lactobacillus spp. selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus reuten, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus crispatus, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus rhamnosus SP1 (DSM 21690), Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus reuteri (ATCC 55730), Lactobacillus reuteri (DSM 17938) and Lactobacillus johnsonii (NCC533; CNCM 1-1225).

In one embodiment, the microorganism is a bacterium selected from Bifidobacteriales. In one embodiment, the microorganism is a Bifidobacterium spp., such as a Bifidobacterium spp. selected from the group consisting of Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum and Bifidobacterium adolescentis, Bifidobacterium lactis BI-04, Bifidobacterium lactis CNCM I-3446 (Bb12), Bifidobacterium longum NCC3001 , ATCC BAA-999 (BB536), Bifidobacterium breve Bb-03, Bifidobacterium breve M-16V, Bifidobacterium breve R0070 and Bifidobacterium infantis.

In one embodiment, the acid tolerance of the microorganism is at least 50% higher than the corresponding uncoated microorganism.

In yet a further aspect, the present invention provides a composition comprising the microcapsule comprising a microorganism obtained or obtainable by the method of the present invention. The composition may be provided in any suitable formulation. Preferably, the composition is provided in a formulation suitable for oral administration. In one embodiment, the composition is in the form of a powder, granulate, tablet or a capsule. In a preferred embodiment, the composition is formulated as a granulate.

The composition may also comprise a prebiotic that stimulates the proliferation of the microorganism in the Gl of the subject ingesting the composition. In one embodiment, the composition further comprises at least one prebiotic selected from the group consisting of sialo-oligosaccharides (SOS), fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), isomalto-oligosaccharides (IMO), xylooligosaccharides (XOS), arabino-xylo oligosaccharides (AXOS), mannan oligosaccharides (MOS), oligosaccharides of soy, glycosylsucrose (GS), lactosucrose (LS), sialyl-lactose (SL) Fucosyl-lactose (FL), Lacto-N-Neotetraose (LNNT), lactulose (LA), palatinose-oligosaccharides (PAO), malto-oligosaccharides, gums and/or hydrolysates thereof, pectins, starches, and/or hydrolysates thereof.

In one embodiment, the composition of the present invention is formulated as a pharmaceutical composition, which comprises at least one pharmaceutically acceptable excipient or carrier. In another embodiment, the composition is nutritional composition.

The composition may be formulated for administration as a once daily dose. The composition may thus be formulated accordingly, e.g. as a one daily dose unit. The composition may also be for administration as a twice daily dose, three times daily dose or even for administration several times daily. It follows that the composition may thus be formulated according to the dosage regimen. In one embodiment of the present invention, the composition is administrated once or twice daily.

In one embodiment, one dose of said composition comprises 10e3 to 10e12 colony forming units of said microorganism, such as 10e6 to 10e12 colony forming units, for example 10e7 to 10e11 colony forming units, such as 10e8 to 10e10 colony forming units. In one embodiment, the present invention provides a microencapsulated microorganism obtained by the method described below. The microencapsulated microorganism described in the present embodiment consists of freeze dried probiotic particles encapsulated in fatty alcohols. Particles more suitable for encapsulation may be produced by an initial granulation step, where, by adjusting droplet size and spray rate, the particles are joined into larger, more spherical granules. The particles consist of a matrix of freeze dried live probiotic bacteria and fatty alcohol, with an outer layer of fatty alcohol. The fatty alcohol, when applied in correct amounts, and when the coating layer is within a particular thickness range, will provide protection from gastric acid, while still being able to deliver live bacteria in the intestines. The fatty alcohols are mixed in a combination such that the encapsulation material will be solid at room temperature and body temperature.

In one embodiment, the method for preparing a microcapsule comprising a microorganism is defined by the below parameters. The application of fatty alcohols is made by hot-melt coating using a fluid bed, in this case a Innojet Ventilus 2.5 with a bottom mounted spray nozzle.

Freeze dried probiotic powder is introduced to the coating chamber, in some cases in combination with a small amount of silicon dioxide (such as 0-5%, for example 1%) or other excipients to reduce powder cohesiveness. Powder is fluidised, and molten fatty alcohols are sprayed into the fluidised powder from a bottom mounted spray nozzle. Inlet air temperature is kept below the melting point of the fatty acid, usually by keeping the product temperature within 20-45 Celcius. Spray rate and spray pressure are kept so that the droplet size has the right size to produce a granulate in the optional first step, and then altered to apply a coating layer to the particles using smaller droplets and a slightly higher process air temperature to obtain a desired gastric acid resistance.

The (optional) initial step is a granulation step, where larger droplets are applied. This is controlled through spray rate and spray pressure. The air flow should be high and the temperature low to ensure solidification. The coating step is carried out with different settings where the droplets size is decreased. After coating, a cooling step might be added to avoid particles sticking together and to the chamber. The air flow rate and the spray rate applied will be adjusted according to the size of the equipment. The amount of coating material added to the particles is expressed in % weight gain (WG).

When describing the embodiments (or items) of the present invention, the combinations and permutations of all possible embodiments have not been explicitly described. Nevertheless, the mere fact that certain measures are recited in mutually different dependent claims or described in different embodiments does not indicate that a combination of these measures cannot be used to advantage. The present invention envisages all possible combinations and permutations of the described embodiments.

The term “comprising”, “comprise” and “comprises” herein are intended by the inventors to be optionally substitutable with the terms “consisting of”, “consist of” and “consists of”, respectively, for every embodiment disclosed herein.

The invention is further described in the following non-limiting embodiments:

Embodiment 1. A method for preparing a microcapsule comprising a microorganism, preferably a bacterium, more preferably a probiotic bacterium, said method comprising the steps of:

(iii) fluidizing said microorganism using a fluidizing gas;

(iv) introducing a coating dispersion into the coating chamber, wherein said coating dispersion comprises one or more fatty alcohol(s) in (molten) liquid form;

(v) allowing formation of a microcapsule comprising said microorganism; wherein said fluidizing gas is having a temperature below the melting temperature of said fatty alcohol. Embodiment 2. The method according to embodiment 1 , wherein the fatty alcohol is a C12-C24 chain fatty alcohol.

Embodiment 3. The method according to any one of embodiments 1 and 2, wherein the microcapsule is having a size in the range of 0.2 to 5000 micrometre.

Embodiment 4. The method according to any of the preceding embodiments, wherein the microcapsule is having a size in the range of 100 to 2000 micrometre.

Embodiment 5. The method according to any of the preceding embodiments, wherein the microcapsule is having average size in the range of 300 to 600 micrometre.

Embodiment 6. The method according to any of the preceding embodiments, wherein the fluidized bed reactor comprises a spray nozzle for introducing coating material positioned at the bottom of the coating chamber.

Embodiment 7. The method according to any of the preceding embodiments, wherein said microorganism is introduced in combination with one or more excipients or actives, such as silicon dioxide, maltodextrin, a vitamin or a prebiotic.

Embodiment 8. The method according to any of the preceding embodiments, wherein said coating dispersion comprises one or more fatty alcohol(s) selected from the group consisting of cetyl alcohol (C16), stearyl alcohol (C18), myristyl alcohol (C14), lauryl alcohol (C12) and cetostearyl alcohol or any combination thereof.

Embodiment 9. The method according to any of the preceding embodiments, wherein said coating dispersion comprises cetyl alcohol, stearyl alcohol and myristyl alcohol.

Embodiment 10. The method according to any of the preceding embodiments, wherein said coating dispersion comprises cetyl alcohol and stearyl alcohol. Embodiment 11. The method according to any of the preceding embodiments, wherein said coating dispersion comprises stearyl alcohol.

Embodiment 12. The method according to any of the preceding embodiments, wherein said coating dispersion consists or consists essentially a fatty alcohol selected from the group consisting of cetyl alcohol (C16), stearyl alcohol (C18), myristyl alcohol (C14), lauryl alcohol (C12) and cetostearyl alcohol or any combination thereof.

Embodiment 13. The method according to any of the preceding embodiments, wherein said coating dispersion consists or consists essentially of cetyl alcohol and stearyl alcohol.

Embodiment 14. The method according to any of the preceding embodiments, wherein said coating dispersion comprises 0 to 40 wt% cetyl alcohol, 25 to 100 wt% stearyl alcohol and 0 to 20 wt% myristyl alcohol of the total weight of the coating dispersion.

Embodiment 15. The method according to any of the preceding embodiments, wherein said coating dispersion comprises a mixture of cetyl alcohol and stearyl alcohol.

Embodiment 16. The method according to any of the preceding embodiments, wherein said coating dispersion comprises a mixture of cetyl alcohol, stearyl alcohol and myristyl alcohol.

Embodiment 17. The method according to any of the preceding embodiments, wherein said coating dispersion comprises a mixture of cetyl alcohol and stearyl alcohol in the ratio 3 to 7.

Embodiment 18. The method according to any of the preceding embodiments, wherein said coating dispersion comprises a mixture of cetostearyl alcohol, and myristyl alcohol in the ratio 9 to 1. Embodiment 19. The method according to any of the preceding embodiments, wherein said coating dispersion further comprises one or more emulsifiers, such an emulsifier selected from the group consisting of polysorbate, tween and acetylated monoglycides.

Embodiment 20. The method according to any of the preceding embodiments, wherein said coating dispersion further comprises one or more viscosity modifiers selected from the group consisting of mixtures of triglycerides, for example vegetable oil or vegetable fat (such as olive oil), ethyl cellulose, hydroxyl propyl cellulose, beewax, and shellac.

Embodiment 21. The method according to any of the preceding embodiments, wherein melting point of said coating dispersion is above 18°C, such as above 20°C, for example above 25°C, preferably above 37°C.

Embodiment 22. The method according to any of the preceding embodiments, wherein the resulting coated microorganism comprises 25-90% of said coating dispersion.

Embodiment 23. The method according to any of the preceding embodiments, wherein said fluidizing gas is having a temperature in the range of 15 to 45°C such as, 15 to 45°C, for example 15 to 40°C, such as 18 to 25, for example around 20°C during step (v).

Embodiment 24. The method according to any of the preceding embodiments, wherein the microorganism(s) is having a temperature in the range of 20 to 40°C, such as in the range of 20 to 35°C, for example in the range of 33 to 35°C during step (v).

Embodiment 25. The method according to any of the preceding embodiments, wherein the temperature of said coating dispersion at the entry in the coating chamber through said spray nozzle is in the range of 80 to 130°C, such as 90 to 120°C, for example 90 to 110°C, such as around 100°C. Embodiment 26. The method according to any of the preceding embodiments, wherein said microorganism is in a lyophilized or spray dried form.

Embodiment 27. The method according to any of the preceding embodiments, wherein said granulate is prepared by

(b) allowing the formation of a granulated microorganism.

Embodiment 28. The method according to any of the preceding embodiments, wherein said fluidizing gas is having a temperature in the range of 15 to 20°C, for example 15 to 19°C, such as 15 to 18, for example around 15°C during step (a) and (b).

Embodiment 29. The method according to any of the preceding embodiments, wherein the temperature of said coating dispersion at the entry in the coating chamber through said spray nozzle is in the range of 80 to 130°C, such as 90 to 120°C, for example 90 to 110°C, such as around 100°C during step (a) and (b).

Embodiment 30. The method according to any of the preceding embodiments, wherein said microorganism is a bacterium, preferably a probiotic bacterium.

Embodiment 31. The method according to any of the preceding embodiments, wherein said microorganism is a bacterium selected from Lactobacillales or Bifidobacteriales.

Embodiment 32. The method according to any of the preceding embodiments, wherein said microorganism is a bacterium selected from the group consisting of a Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Lactococcus spp., Streptococcus spp., Aerococcus spp., Carnobacterium spp., Enterococcus spp., Oenococcus spp., Sporolactobacillus spp., Tetragenococcus spp., Vagococcus spp., and Weisella spp.. Embodiment 33. The method according to any of the preceding embodiments, wherein said microorganism is a bacterium selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus rhamnosus SP1 (DSM 21690), Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus reuteri (ATCC 55730), Lactobacillus reuteri (DSM 17938) and Lactobacillus johnsonii (NCC533; CNCM 1-1225).

Embodiment 34. The method according to any of the preceding embodiments, wherein said microorganism is a Lactococcus ssp. selected from the group consisting of Lactococcus lactis, Lactococcus cremoris, Lactococcus diacetylactis.

Embodiment 35. The method according to any of the preceding embodiments, wherein said microorganism is a Bifidobacterium spp.

Embodiment 36. The method according to any of the preceding embodiments, wherein said microorganism is a bacterium selected from the group consisting of Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum and Bifidobacterium adolescentis, Bifidobacterium lactis BI-04, Bifidobacterium lactis CNCM I-3446 (Bb12), Bifidobacterium longum NCC3001 , ATCC BAA-999 (BB536), Bifidobacterium breve Bb-03, Bifidobacterium breve M-16V, Bifidobacterium breve R0070 and Bifidobacterium infantis.

Embodiment 37. A microcapsule comprising a microorganism obtainable by the method according to any of the preceding embodiments.

Embodiment 38. A composition comprising the microcapsule comprising a microorganism according to embodiment 37.

Embodiment 39. The composition according to embodiment 38, wherein one dose of said composition comprises 10e3 to 10e12 colony forming units of said microorganism, such as 10e6 to 10e12 colony forming units, for example 10e7 to 10e11 colony forming units, such as 10e7 to 10e11 colony forming units.

Embodiment 40. A microcapsule comprising a microorganism, wherein the surface of said microcapsule comprises one or more fatty alcohol(s).

Embodiment 41. The microcapsule according to embodiment 40, wherein the fatty alcohol is C12-C24 chain fatty alcohol, preferably C12-C18.

Embodiment 42. The microcapsule according to embodiment 40 or 41, wherein melting point of said fatty alcohol is above 18°C, such as above 20°C, for example above 25°C, preferably above 37°C.

Embodiment 43. The microcapsule according to any one of embodiments 40 to 42, wherein said one fatty alcohol is selected from the group consisting of cetyl alcohol (C16), stearyl alcohol (C18), myristyl alcohol (C14), lauryl alcohol (C12) and cetostearyl alcohol or any combination thereof.

Embodiment 44. The microcapsule according to any one of embodiments 40 to 43, wherein said one or more fatty alcohol(s) comprises cetyl alcohol, stearyl alcohol and myristyl alcohol.

Embodiment 45. The microcapsule according to any one of embodiments 40 to 44, wherein said one or more fatty alcohol(s) comprises stearyl alcohol.

Embodiment 46. The microcapsule according to any one of embodiments 40 to 45, wherein said one or more fatty alcohol(s) comprises 0 to 40 wt% cetyl alcohol, 25 to 100 wt% stearyl alcohol and 0 to 20 wt% myristyl alcohol of the total weight of the coating dispersion.

Embodiment 47. The microcapsule according to any one of embodiments 40 to 46, wherein said one or more fatty alcohol(s) comprises a mixture of cetyl alcohol and stearyl alcohol. Embodiment 48. The microcapsule according to any one of embodiments 40 to 47, wherein said one or more fatty alcohol(s) comprises a mixture of cetyl alcohol, stearyl alcohol and myristyl alcohol.

Embodiment 49. The microcapsule according to any one of embodiments 40 to 48, wherein said one or more fatty alcohol(s) comprises a mixture of cetyl alcohol and stearyl alcohol in the ratio 3 to 7.

Embodiment 50. The microcapsule according to any one of embodiments 40 to 49, wherein said one or more fatty alcohol(s) comprises a mixture of cetostearyl alcohol, and myristyl alcohol in the ratio 9 to 1.

Embodiment 51. The microcapsule according to any one of embodiments 40 to 50, wherein the surface of said microcapsule comprises one or more emulsifiers, such an emulsifier selected from the group consisting of polysorbate, tween and acetylated monoglycides.

Embodiment 52. The microcapsule according to any one of embodiments 40 to 51, wherein the surface of said microcapsule comprises one or more comprises one or more viscosity modifiers selected from the group consisting mixtures of triglycerides, for example vegetable oil or vegetable fat (such as olive oil), ethyl cellulose, hydroxyl propyl cellulose, and beewax.

Embodiment 53. The microcapsule according to any one of embodiments 40 to 52, wherein said microorganism is present in the form of a mixture of microorganism and one or more excipients, such as silicon dioxide or maltodextrin.

Embodiment 54. The microcapsule according to any one of embodiments 40 to 53, wherein the microcapsule is having a size in the range of 0.2 to 5000 micrometre.

Embodiment 55. The microcapsule according to any one of embodiments 40 to 54, wherein the microcapsule is having a size in the range of 100 to 2000 micrometre. Embodiment 56. The microcapsule according to any one of embodiments 40 to 55, wherein the microcapsule is having average size in the range of 300 to 600 micrometre.

Embodiment 57. The microcapsule according to any one of embodiments 40 to 56, wherein said microorganism is a bacterium selected from Lactobacillales.

Embodiment 58. The microcapsule according to any one of embodiments 40 to 57, wherein said microorganism is a bacterium selected from the group consisting of a Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Lactococcus spp., Streptococcus spp., Aerococcus spp., Carnobacterium spp., Enterococcus spp., Oenococcus spp., Sporolactobacillus spp., Tetragenococcus spp., Vagococcus spp., and Weisella spp..

Embodiment 59. The microcapsule according to any one of embodiments 40 to 58, wherein said microorganism is a bacterium selected from the group consisting of Lactobacillus rhamnosus, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus casei, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus rhamnosus GG (ATCC 53103), Lactobacillus rhamnosus SP1 (DSM 21690), Lactobacillus rhamnosus CGMCC 1.3724, Lactobacillus reuteri (ATCC 55730), Lactobacillus reuteri (DSM 17938) and Lactobacillus johnsonii (NCC533; CNCM 1-1225).

Embodiment 60. The microcapsule according to any one of embodiments 40 to 59, wherein said microorganism is a Lactococcus ssp. selected from the group consisting of Lactococcus lactis, Lactococcus cremoris, Lactococcus diacetylactis.

Embodiment 61. The microcapsule according to any one of embodiments 40 to 60, wherein said microorganism is a Bifidobacterium spp.

Embodiment 62. The microcapsule according to any one of embodiments 40 to 61, wherein said microorganism is a bacterium selected from the group consisting of Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium mfantis, Bifidobacterium animahs, Bifidobacterium bifidum and Bifidobacterium adolescentis, Bifidobacterium Lactis BI-04, Bifidobacterium lactis CNCM 1-3446 (Bb12), Bifidobacterium longum NCC3001 , ATCC BAA-999 (BB536), Bifidobacterium breve Bb-03, Bifidobacterium breve M-16V, Bifidobacterium breve R0070 and Bifidobacterium infantis.

Embodiment 63. The microcapsule according to any one of embodiments 40 to 62, wherein said microcapsule comprises said microorganism in lyophilized or spray dried form or in a granulated form.

Embodiment 64. The microcapsule according to any one of embodiments 40 to 63, wherein the acid tolerance of the microorganism is improved by a minimum of 50% compared to the uncoated microorganism.

Embodiment 65. The microcapsule according to any one of embodiments 40 to 64, wherein the potency of the microorganism is higher or equal to 10E+09 CFU/gram, wherein the starting material containing a minimum of 5E+10 CFU/gram of said microorganism.

Embodiment 66. The microcapsule according to any one of embodiments 40 to 65, wherein the coating dispersion comprises a taste modifying agent.

Embodiment 67. The method or microcapsule according to any one of proceeding embodiments, wherein the amount of the coating dispersion on the microcacpsule corresponds to 80% to 300% of the weight of the microorganism, such as 80% and above but less than 300%, such as in the range of 80% to 210% of the weight of the microorganism.

Examples

Based on initial studies fatty alcohols have been identified as a suitable encapsulation material for probiotics. Tested fatty alcohols include, but is not limited to, cetostearyl alcohol, a mixture of cetyl and stearyl alcohol, a mixture of cetyl, stearyl and myristyl alcohol. The number of carbons in the fatty alcohol structure will determine the melting point of the coating material which should (preferably) be above the body temperature/physiological conditions (37°C) and lower than 90°C (equipment based). A mixture of fatty alcohols is advantageous since this may provide a viscosity modifying effect of the microencapsulation material. The rationale for using fatty alcohols for probiotics is that the material is hydrophobic, which is an advantage in combination with water-sensitive products such as probiotics. In contrast to other fatty like structures (fats, waxes, fatty acids) the chemical structure of fatty alcohols does not contain an acid- or ester as functional group but an alcohol group. Alcohols are not, in oppose to esters or acids, protonated at low pH values or degraded enzymatically under gastric conditions which is why the hydrophobic property is expected to be retained in vivo. Moreover fatty alcohols are tasteless which allow them to be incorporated into finished formulations such as tablets or powders without compromising taste.

The fatty alcohol material is applied using a fluid-bed system. The material can be applied using varied process parameters to obtain agglomerate growth/granulation or a protective outer layer/coating respectively. It is possible to adjust the parameters during the process (without discontinuing the process) to first achieve larger particles, where the fatty alcohol and probiotic material form a matrix, and thereafter a protective layer of fatty alcohol. The final particle interval will vary depending on the starting material but is typically within 100-2000pm with a mean particle size of 300-600pm. Using this technology, the bacteria tolerate the process well since the cooling of the melted material happens rapidly when in contact with the probiotic particle surface so that the bacteria do not experience a high temperature. The mechanical impact is low since the movement of the particles is obtained using a stream of air.

The achieved qualities tested include: Improvement of acid tolerance Unaffected influence of the long-term stability of the probiotic e.g. in a food supplement formulated as a granulate powder The amount of coating material needed can be reduced using a viscosity modifier.

The best results have been obtained using a mixture of triglycerides (e.g. olive oil). Typically a minimum of 100% WG needs to be added (i.e. 50% coating in the final particles) to achieve the above qualities.

To reduce adhesion to the fluid-bed equipment an anti-adhesion agent such as silicon dioxide can be added (<1%), however using optimized equipment models could eliminate the need of such agents.

The technology has been tested on three bacteria strains; Lactobacillus rhamnosus BIFOLAC™GG, Bifidobacterium longum BB536 and Lactobacillus acidophilus BIFOLAC™5 (referred to as respectively L. rhamnosus, B. longum and L. acidophilus in below examples).

Typical process parameters (based on 1 litre chamber setup):

0-30% WG (so-called granulation step)

Heating of the fatty alcohol to > 80°C. Airflow 25-30m³/h, dosing rate 15g/min, nozzle spray pressure 0.6bar. By this the average product temperature is max 35°C.

30%-end WG (so-called coating step)

Airflow 30-45m³/h, dosing rate 6.5g/min, nozzle spray pressure 0.8bar. By this the average product temperature is max 40°C.

Example 1

Background

Microencapsulation of L. rhamnosus and B. longum using cetostearyl or a mixture of cetyl and stearyl alcohol was tested. The results for L. rhamnosus was compared to a commercially available product comprising L. rhamnosus R0343 and a protective matrix (BIO-SUPPORT™; Lallemand).

Metode Acid tolerance: Samples were analyzed by suspending the encapsulated powder into 0.1M HCI-KCI buffer pH 1.2 for one to two hours (37°C), then neutralizing the powder to pH 6.8 and measuring the CFU potency by a standardized method. pH was controlled at start and end to 1.5-1.7. As reference the CFU potency of the sample was measured without previous acid treatment. The two CFU potencies were compared to determine the log reduction from acid treatment.

WG: The material weight gain of samples was analyzed using GC-MS.

Results

Table 1

The results for B. longum are disclosed in Table 1 and Figure 1. The data demonstrate that the uncoated bacteria is reduced by more than seven log units after acid treatment for one hour. The CFU potency in the encapsulated samples are more than 30,000 times more concentrated at the same sampling time.

Table 2

The results for L. rhamnosus are disclosed in Table 2 and Figure 2. The data demonstrate that the untreated bacteria drops more than six log units after acid treatment for one hour. The CFU potency in the encapsulated samples are more than 5,000 times more concentrated at the same time and extended testing to two hours shows that the bacteria is unaffected by the acidic conditions. For comparison L. rhamnosus with the BIO-SUPPORT™ protective matrix has a potency that is more than 1 ,000,000 times lower after one hour compared to coating with fatty alcohols.

Conclusion

• Using fatty alcohols for microencapsulation provides the tested bacteria with improved acid tolerance compared to the untreated bacteria. In the case of

L. rhamnosus a 100% acid stable product is obtained with >500% weight gain and more than 1E+05 times more viable bacteria after two hours acid treatment. In the case of B. longum there are more than 1E+04 times more viable bacteria at >203% weight gain (one hour acid treatment) compared to the untreated bacteria.

• Coating with fatty alcohols results in improved viability and acid tolerance of L. rhamnosus compared to the marketed protective technology, BIOSUPPORT™ (Lallemand), i.e. 1 ,000,000 times higher potency after 1 hour treatment in acid.

• The acid tolerance property can be obtained using either cetostearyl alcohol or a mix of cetyl and stearyl alcohol.

Example 2

Background

Microencapsulation of L. rhamnosus using a mixture of cetyl and stearyl alcohol (3:7) was tested. The product is evaluated with respect to process loss at different applied weight gains.

Method

Proces loss (bacteria recovery): The CFU potency of samples was determined by a standardized method. Potency was normalized with respect to % bacteria powder per gram and compared to the potency of the untreated bacteria to calculate the process loss. Bacteria content was calculated theoretically from the applied coating amount.

Results

Table 3

WG = Weight gain *Determined from actual weight gain (measurement of fatty alcohol content by GC-MS).

The results are disclosed in Table 3 and Figure 3. It is observed that the survival after the process is not dropping until 300% weight gain. The lower CFU potency at 300% weight gain could also reflect that the bacteria are encapsulated too efficiently.

Conclusion • The process conditions of the encapsulation technology ensures a high bacteria survival.

Example 3

Background Microencapsulated products were manufactured of B. longum and L. acidophilus using a coating of cetyl and stearyl alcohol in the ratio 3:7 with 5% olive oil. Products had been stored six months at 10°C at the time of the below analyses.

Method

Acid tolerance: Samples were analyzed by suspending sample material into 0.1M HCI-KCI buffer pH 1 .2 for 20, 40, and 60 minutes (37°C). pH was controlled at all time points (1.5-1.7). The suspension was then neutralized to pH 6.8 and the CFU potency was measured using a standardized method. As reference the CFU potency of the same powder without previous acid treatment was determined. The two CFU potencies were compared to determine the log reduction from acid treament.

Weight gain: Material weight gain was determined by weighing the cooled, melt-off residue by a standardized method.

Results

Table 4

1) Based on few colonies. Table 5

The results are disclosed in Table 4, Table 5, Figure 4 and Figure 5. It is observed that the untreated B. longum is reduced by almost six log units after 20 minutes acid treatment. At the same time point there is 5,000 times more bacteria in the microencapsulated sample. After 60 minutes acid treatment the potency of the microencapsulated sample is 10,000 times higher with a potency of 2.4E+08 CFU/g (See Figure 4).

It is observed that the untreated L. acidophilus is reduced by more than four log units after 20 minutes acid treatment. At the same time point there is 350 times more bacteria in the microencapsulated sample. After 60 minutes acid treatment the potency of the microencapsulated sample is close to 500 times higher with a potency of 1.2E+08 CFU/g (See Figure 5).

Conclusion

• Both tested bacteria demonstrate a marked improvement in acid tolerance when microencapsulated with fatty alcohols. For B. longum the potency is 10,000 times higher in the encapsulated samples compared to the untreated bacteria after 60 minutes in acid (pH 1.5-1.7). For L. acidophilus the potency is close to 500 times higher in the microencapsulated samples compared to the untreated raw material after 60 minutes acid treatment (pH 1.5-1.7). Despite that the untreated bacteria contains a higher CFU count at beginning the potency of microencapsulated samples with cetyl and stearyl alcohol is higher already after 20 minutes treatment and maintain a potency of > 1.0E+08 CFU/g after 60 minutes acid treatment.

Example 4

Background

Two different bacteria i.e. B. longum and L. acidophilus were encapsulated using either cetostearyl alcohol or cetykstearyl alcohol 3:7 with 5% olive oil and the resulting acid tolerance was compared to a untreated reference. The products were investigated as the encapsulated bacteria alone or added to a nutritional supplement product, i.e. a granulate powder in a stick pack.

Method

Acid protocol: Samples were analyzed by suspending sample material into 0.1M HCI-KCI buffer pH 1.2 for time intervals up to 60 minutes (37°C). pH was controlled at all time points (1.5-1.7). The suspension was then neutralized to pH 6.8 and the CFU potency was measured using a standardized method. As reference the CFU potency of the sample without previous acid treatment was determined. The two CFU potencies were compared to determine the log reduction from acid treament.

Weight gain: The actual weight gain was determined using either GC-MS (batch from 2017) or by weighing the amount of cooled melt-off residue by a standardized method (batch from 2019).

Results: The results to B. longum are presented in Table 6 and Figure 6 and 7

By plotting the log reduction as a function of weight gain it is illustrated that there is a clear correlation (higher amount of coating results in a better acid tolerance), supporting the barrier effect of the material.

It is further illustrated by the data that the barrier properties are maintained throughout 26 months storage at respectively 10°C and 25°C. Table 7

*) Batch 91911401 **) Batch 91910301 + 91910302 and stick pack 9913101

***) Batch 1709208

Results: The results to L. acidophilus_are presented in Table 7 and Figure 8. The data support that there is a correlation between the amount of coating material and the resulting acid tolerance supporting the acid barrier properties of the technology. Conclusion

• For both tested bacteria, a correlation between the amount of coating material and the resulting acid tolerance has been demonstrated. This supports that encapsulation of bacteria with fatty alcohols has a protective effect and benefit on the acid tolerance of the bacteria.

Example 5

Background

B. longum was encapsulated using either cetostearyl alcohol or cetykstearyl alcohol 3:7 with 5% olive oil. The long-term stability of the coated product was investigated of the encapsulated bacteria alone as well as added to a nutritional supplement product, i.e. a granulate powder in a stick pack. The stability of the encapsulated bacteria was compared to the stability of the untreated bacteria in a granulate powder in a stick pack.

Method

Potency: Samples were analyzed using a standardized method.

Results The results are presented in Table 8 and Figure 9 and 10

It is observed that the encapsulated bacteria has the same stability profile as the untreated reference in a nutritional supplement. The encapsulated material in a nutritional product displays a similar stability profile after seven months storage (See Figure 9).

Comparison of the results from the same nutritional product at different temperatures with encapsulated B. longum further supports that minimal degradation is anticipated to take place in the product, otherwise a temperature dependent reduction in potency would be observed. The highest potency after seven months is observed in the stick pack stored at 25°C/60%RH (See Figure 10). The data support that the encapsulation technology does not comprise the stability of the bacteria in a food supplement product. The fastmelt product is anticipated to provide a declaration of 1 E+09 CFU/stick pack of B. longum.

Conclusion

The data support that the encapsulation technology does not compromise the stability of the bacteria alone or in a nutritional supplement product. The investigated nutritional supplement product is anticipated to provide a declaration of 1 E+09 CFU/stick of B. longum after 24 months at 25°C/60%RH.

Table 6

1) Measured using different acid protocol without pH-control. Subsequent simu ations of conditions reflect a pH of 1.5-2.0 throughout

60min. 2) Based on few colonies. Abbreviations: T26; Measured after 26 months storage at the indicated temperature.

Table 8

Claims

1. A method for preparing a microcapsule comprising a microorganism, such as a bacterium, preferably a probiotic bacterium, said method comprising the steps of:

(iii) fluidizing said microorganism using a fluidizing gas;

(iv) introducing a coating dispersion into the coating chamber, optionally wherein the fluidized bed reactor comprises a spray nozzle for introducing coating material positioned at the bottom of the coating chamber; wherein said coating dispersion comprises one or more fatty alcohol(s) in (molten) liquid form;

2. The method according to claim 1, wherein the fatty alcohol is a C12-C24 chain fatty alcohol, such as C12-18.

3. The method according to any one of claims 1 or 2 , wherein the amount of the coating dispersion on the microcacpsule corresponds to 80% to 300% of the weight of the microorganism, such as 80% and above but less than 300%, such as in the range of 80% to 210% of the weight of the microorganism.

4. The method according to to any of the preceding claims, wherein the microcapsule is having a size in the range of 0.2 to 5000 micrometre, such as a size in the range of 100 to 2000 micrometre, for example an average size in the range of 300 to 600 micrometre .

5. The method according to any of the preceding claims, wherein said coating dispersion comprises, consists or consists essentially of one or more fatty alcohol(s) selected from the group consisting of cetyl alcohol (C16), stearyl alcohol (C18), myristyl alcohol (C14), lauryl alcohol (C12) and cetostearyl alcohol or any combination thereof.

6. The method according to any of the preceding claims, wherein said coating dispersion comprises cetyl alcohol, stearyl alcohol and myristyl alcohol, or said coating dispersion comprises cetyl alcohol and stearyl alcohol, such as a mixture of cetyl alcohol and stearyl alcohol in the ratio 3 to 7, or said coating dispersion comprises stearyl alcohol, or said coating dispersion comprises a mixture of cetyl alcohol, stearyl alcohol and myristyl alcohol, such as a mixture of cetostearyl alcohol, and myristyl alcohol in the ratio 9 to 1 , or wherein said coating dispersion consists or consists essentially of cetyl alcohol and stearyl alcohol.

7. The method according to any of the preceding claims, wherein melting point of said coating dispersion is above 18°C, such as above 20°C, for example above 25°C, preferably above 37°C.

8. The method according to any of the preceding claims, wherein said fluidizing gas is having a temperature in the range of 15 to 45°C such as, 15 to 45°C, for example 15 to 40°C, such as 18 to 25, for example around 20°C during step (v).

9. The method according to any of the preceding claims, wherein the temperature of said coating dispersion at the entry in the coating chamber through said spray nozzle is in the range of 80 to 130°C, such as 90 to 120°C, for example 90 to 110°C, such as around 100°C.

10. The method according to any of the preceding claims, wherein said granulate is prepared by

(b) allowing the formation of a granulated microorganism.

11. The method according to any of the preceding claims, wherein said microorganism is a bacterium selected from Lactobacillales or Bifidobacteriales such as a bacterium selected from the group consisting of a Lactobacillus spp., Leuconostoc spp., Pediococcus spp., Lactococcus spp., Streptococcus spp., Aerococcus spp., Carnobacterium spp., Enterococcus spp., Oenococcus spp., Sporolactobacillus spp., Tetragenococcus spp., Vagococcus spp., and Weisella spp...

12. A microcapsule comprising a microorganism obtainable by the method according to any of the preceding claims.

13. A microcapsule comprising a microorganism, such as a bacterium, wherein the surface of said microcapsule comprises one or more fatty alcohol(s), such as a C12- C24 chain fatty alcohol, for example a fatty alcohol is selected from the group consisting of stearyl alcohol (C18), cetyl alcohol (C16), myristyl alcohol (C14), lauryl alcohol (C12) and cetostearyl alcohol or any combination thereof.

14. The microcapsule according to claim 12 or 13, wherein the fatty alcohol is a C12-C24 chain fatty alcohol, such as C12-18.

15. The microcapsule according to any one of claims 12 to 14, wherein the amount of the coating dispersion on the microcacpsule corresponds to 80% to 300% of the weight of the microorganism, such as 80% and above but less than 300%, such as in the range of 80% to 210% of the weight of the microorganism.

16. The microcapsule according to any one of claims 12 to 15, wherein said one or more fatty alcohol(s) comprises cetyl alcohol, stearyl alcohol and myristyl alcohol or a mixture of cetyl alcohol and stearyl alcohol, such as a mixture of cetyl alcohol and stearyl alcohol in the ratio 3 to 7, or a mixture of cetyl alcohol, stearyl alcohol and myristyl alcohol, or a mixture of cetostearyl alcohol, and myristyl alcohol such as in the ratio 9 to 1.

17. The microcapsule according to any one of claims 12 to 16, wherein the microcapsule is having a size in the range of 0.2 to 5000 micrometre, such as a size in the range of 100 to 2000 micrometre, for example an average size in the range of 300 to 600 micrometre.

18. The microcapsule according to any one of claims 12 to 17, wherein the acid tolerance of the microorganism is improved by a minimum of 50% compared to the uncoated microorganism and/or the potency of the microorganism is higher or equal to 10E+09 CFU/gram, wherein the starting material containing a minimum of 5E+10 CFU/gram of said microorganism.