Process for Producing a Soft Gel Capsule Comprising Viable Probiotic Bacteria and a Soft Gel Capsule Comprising Viable Probiotic Bacteria Having a Long Shelf Life

Abstract

The present invention provides a method for producing a soft gel capsule comprising uncoated probiotic bacteria, the method comprising gentle mixing the uncoated probiotic bacteria with at least one oil to obtain a soft gel capsule fill material at a temperature in the range of 5 to 15° C., encapsulating the fill material in a soft gel capsule made of a gelatin having a melting point in the range of 11 to 28° C.; and drying the soft gel capsule in one or more steps to a water activity of at the most 0.25 at a temperature of at the most 25° C., as well as soft gel capsules produced by this method. The present invention further provides a soft gel capsule comprising dried, uncoated, non-spore-forming probiotic bacteria wherein the soft gel capsule comprises at least E+09 viable bacteria after 24 months of storage at 25° C., e.g. at least 2 E+09 viable bacteria after 12 months of storage at 25° C.

Claims

1. A method for producing a soft gel capsule comprising uncoated probiotic bacteria, the method comprising: a) mixing the uncoated probiotic bacteria with at least one oil to obtain a soft gel capsule fill material at a temperature in the range of 5 to 15° C., b) encapsulating the fill material in a soft gel capsule made of a gelatin having a melting point in the range of 11 to 28° C.; and c) drying the soft gel capsule in one or more steps at a temperature of at the most 25° C. to a water activity of at the most 0.25.

2. A method according to claim 1, wherein the uncoated probiotic bacteria are non-spore-forming.

3. A method according to claim 1, wherein the at least one oil comprises omega 3s.

4. A method according to claim 1, wherein the at least one oil comprises vegetable oils.

5. A method according to claim 1, wherein the fill temperature is in the range of 6 to 14° C.

6. A method according to claim 1, wherein the gelatin has a bloom strength in the range of 100 to 300 bloom.

7. A method according to claim 1, wherein the probiotic bacteria are Lactobacilli, Lactococci, Pediococci, Streptococci or Bifidobacteria, or a mixture thereof.

8. A method according to claim wherein the probiotic bacteria are from the strain Bifidobacterium animalis subspecies lactis.

9. A method according to claim 1, wherein the soft gel capsule comprises one, two, three, four or more active ingredients selected from the group consisting of vitamins; minerals; and vegetable extracts.

10. A method according to claim 1, wherein the soft gel capsule is dried during shelf storage to an a.sub.w of at the most 0.2.

11. A method according to claim 10, wherein the soft gel capsule is stored during shelf storage in a resealable container in/on the inside wall(s) of which at least one channel former is embedded, at least over part of the area, together with at least one absorbent.

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. The method according to claim 3, wherein the omega 3 fatty acids are selected from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), fish oil, and krill oil.

19. The method according to claim 4, wherein the vegetable oils are selected from the group consisting of rapeseed oil, borrage oil/evening primrose oil, linseed oil, perilla oil, and garlic oil.

20. The method according to claim 9, wherein the vitamins are selected from the group consisting of vitamin A, D, E, K2, C, B2, B6, B12, biotin, niacin, and folic acid.

21. The method according to claim 9, wherein the minerals are selected from the group consisting of zinc, selenium, chromium, copper, calcium, and chloride.

22. The method according to claim 9, wherein the vegetable extracts are selected from the group consisting of cranberry extract/juice and royal jelly.

23. A soft gel capsule produced by the method according to claim 1.

24. A dietary supplemental, nutraceutical or pharmaceutical comprising the soft gel capsule according to claim 23.

Description

FIGURES

[0085] FIG. 1 shows CFU values of soft gel capsules produced in trial 2 during storage at 25° C./60% RH for up to 18 months. The soft gel capsules are packed in three different packaging systems. (.square-solid.) Aluminum foil+silicate bag, ⋄ blister card, (•) box+silicate bag.

[0086] FIG. 2 shows CFU values of soft gel capsules produced in trial 2 during storage at 8-12° C. for up to 18 months. The soft gel capsules are packed in three different packaging systems. (.square-solid.) Aluminum foil+silicate bag, ⋄ blister card, (•) box+silicate bag.

[0087] FIG. 3 shows CFU values of soft gel capsules produced in trial 4 packed in simulated bulk packaging systems and stored at 5° C. and 25° C./60% RH for up to 12 months. (.square-solid.) 5° C., and (•) 25° C./60% RH.

[0088] FIG. 4 shows CFU values of soft gel capsules produced in trial 4 packed in plastic vials with desiccant lining during storage at 5° C., 25° C./60% RH and 30° C./75% RH for up to 12 months. (≡) 5° C., (•) 25° C./60% RH , and ⋄ 30° C./75% RH.

[0089] FIG. 5 shows shows CFU values of soft gel capsules produced in trial 4 packed in simulated bulk packaging systems and stored at 5° C. and 25° C./60% RH for up to 24 months. (.square-solid.) 5° C., and (•) 25° C./60% RH.

[0090] FIG. 6 shows CFU values of soft gel capsules produced in trial 4 packed in plastic vials with desiccant lining during storage at 5° C., 25° C./60% RH and 30° C./75% RH for up to 24 months. (.square-solid.) 5° C., (•) 25° C./60% RH , and ⋄ 30° C./75% RH.

EXAMPLES

[0091] General:

[0092] All trial batches have a batch size of 65,000 capsules.

[0093] Fill Material:

[0094] The oil and oil soluble components are charged by vacuum to a stainless steel mixing vessel. The components are mixed at 12° C., and added silicon dioxide. The mixture is further mixed and homogenized until the silicon dioxide is dispersed. Bifidobacterium animalis subsp. lactis is charged by vacuum and the complete mixture is mixed (without homogenization). The finished fill material is then discharged through a sieve with a sieve opening between 0.7 and 1.2 mm to a pre-cooled (12° C.) transfer tank. The transfer tank is then connected to an encapsulating machine.

[0095] Gelatin Solution

[0096] The batch size of the gelatin solution is 100 kg. Gelatin, glycerol and water are weighed into stainless steel production vessel equipped for heating and stirring. The mixture is heated, stirred and evacuated until a stable viscosity is achieved. The gelatin is then filtered (150 microns) into a preheated stainless steel container. Coloring agents are then added and slowly mixed until homogenous. The gelatin is held at 50-68° C. during encapsulation.

[0097] Drying

[0098] The first drying is performed in rotary tumble dryers at 19.0 to 20.3° C., where the lubricant of the gelatin ribbons is removed by cleaning towels in the dryers. Final drying is done by spreading the capsules on trays in a single layer, and placing the trays in air-conditioned cabinets at controlled humidity of 14-23% RH and temperature of 21-24° C. Drying is terminated when a shell hardness of 9-11 N is reached. The typical drying time is 31-130 h, with an average of 100 hours.

[0099] Inspection and Packaging

[0100] The soft gel capsules are inspected and sorted to remove deformed capsules, capsules not properly sealed and other errors and packaged in bulk packages of 3500 soft gel capsules containing probiotic bacteria with 30 g of desiccant in an aluminum bag and heat sealed.

[0101] In Table 2 the composition of the batches produced in five different trials are shown.

TABLE-US-00002 TABLE 2 Composition of five different batches produced Ingredients in mg per Trial No capsule, 10 oval 1 2 3 4 5 Bifidobacterium 21.875 120.000 50.000 50.000 50.000 animalis subsp. lactis Particle size, <730 <166 average, μm by microscopy Particle size, 139 139 223 average, μm by (un- laser scattering filtered) 115 (filtered) D90 (90% of 291 291 465 batch), mm (un- filtered) 231 (filtered) D-a-Tocopherol 9.000 9.000 9.000 9.000 9.000 1000 Medium Chain 500 Triglycerides Total fish oil 550.000 500.000 500.000 500.00 Silicon dioxide 25.125 25.000 9.300 31.000 31.000 Fish gelatin 139.20 139.20 140.00 140.00 140.00 Glycerine 63.86 63.86 67.49 69.96 67.49 99.5% Water 16.94 16.94 5.75 4.82 5.75 Lemon Oil 3.50 3.50 3.50 Colouring 4.36 3.26 1.72 3.26 agent* Total weight of 606.0 654.0 568.3 590.0 590.0 fill Total weight of 826.0 878.0 788.3 810.0 810.0 capsule *Iron Oxide, Titanium oxide, riboflavin

[0102] During the production of the two first trial batches, the temperature was closely monitored throughout the encapsulation process in order to identify where reduction of number of CFU took place.

[0103] The soft gels produced in trial 1 showed significant loss of viable cells through the production process. Changing the processing conditions and repeating the trial gave a significantly more stable production process. The results are shown below in Table 3:

TABLE-US-00003 TABLE 3 Comparison of the temperatures and CFU values during the first two trials Trial no. 1 2 Fill Fill CFU/ Temp./ CFU/ Temp./ Sample Sampling point capsule ° C. capsule ° C. Mixing Upper part 1.39 E+09 20 1.90 E+10 7.5 vessel Mixing Lower part 1.64 E+09 20 7.20 E+09 7.5 vessel Capsules Beginning of 1.84 E+04 20 1.31 E+08 7.5 encapsulation Capsules Middle of 5.10 E+05 20 1.24 E+08 7.5 encapsulation Capsules End of 1.70 E+05 20 4.97 E+07 7.5 encapsulation Capsules Rotary dryer, start 2.12 E+05 20 2.62 E+08 20 Capsules Rotary dryer, middle 1.64 E+05 20 3.00 E+08 20 Capsules Rotary dryer, end 5.64 E+05 20 4.91 E+08 20 Capsules Tray drying, start 4.73 E+05 19 2.94 E+09 20 Capsules Tray drying, middle 1.03 E+08 19 2.62 E+09 20 Capsules Tray drying, end 7.27 E+04 19 2.49 E+09 20

[0104] The impact of the reduction in fill temperature from 20° C. to 7.5° C. is evident, the survival of the probiotic bacteria is significantly better.

[0105] Looking more into the details of the results, Table 3 shows a drastic reduction of CFU/soft gel capsule during the encapsulation in trial 1 and that even at the low fill temperature in trial 2, the cell count is reduced through the encapsulation process. During drying the CFU/capsule is increasing, the reason being that during drying water is lost, so that the total concentration of bacteria increases (most clearly for trial 2).

[0106] This temperature was used for the subsequent trials except for some variations in the temperatures for the fill material (12-14° C.). In the subsequent trials only a minimal production loss was found. As an example, in trial number 4 probiotic bacteria were added corresponding to a calculated amount of 3.0 E+10 CFU/soft gel capsule if all the cells would be available after the soft gel capsule production. The CFU value found when the stability study was started was 9E+09 CFU/soft gel capsule. These two values confirm the minimal loss of probiotic bacteria observed when manufacturing soft gel capsules comprising probiotic bacteria applying the improved process.

[0107] The batch of trial 2 was distributed into different packaging systems having different amounts of drying media availability and stability studies were initiated at different temperatures. The packaging systems were a) an aluminum foil bag containing 100 soft gel capsules and a 1 g silica bag, b) a blister card (PVC/PVDC foil, 10 soft gel capsules per blister card and c) an aluminum bottle with PE plug and PP cap with ALU/PE liner containing 60 soft gel capsules and a 0.5 g silica bag. The samples were stored at 8-12° C. and at 25° C./65% RH for up to 18 months. The results are provided in FIGS. 1 and 2.

[0108] The results in FIG. 1 show the importance of having an active desiccant present in the packaging system for the probiotic soft gel capsules.

[0109] The results in FIG. 2 demonstrates that if the storage temperature is low—in the range of 8-12° C.—the effect of desiccant material present in the packaging is less important for the CFU values of the soft gel capsules comprising probiotic bacteria.

[0110] The soft gel capsules from trial 4 have been included in various stability studies. In FIGS. 3 and 4 and FIGS. 5 and 6 some of the results are presented.

[0111] The stability as bulk soft gel capsules has been studied. In FIGS. 3 and 5 the total cell count during storage at 5° C. and 25° C. in simulated bulk packaging systems are shown (aluminum foil pouch each containing 30 soft gel capsules and 3.2 g desiccant material). The balance between the number of soft gel capsules and amount of desiccant material is not appropriate in order to make the soft gel capsules stable at 25° C. As evident from the figure the number of viable cells starts to decrease already after 1 month of storage at 25° C. In Table 4 the water activities measured during bulk storage are presented.

TABLE-US-00004 TABLE 4 Water activities during bulk storage Water activity Months 5° C. 25° C./60% RH 0 0.24 0.24 1 0.21 0.22 2 0.23 0.22 3 0.20 0.23 6 0.18 0.20 12 0.22 — 24 0.20 —

[0112] The bulk and final packaging data demonstrate the importance of controlling the water activity of the soft gel capsules during storage in order to make them as stable as possible.

[0113] The stability of the soft gel capsules in final packaging has been studied. 30 probiotic soft gel capsules were packed in plastic vials Active Vial, M-3009-336, Capitol Specialty Plastics Inc., 2039 McMillan Street Auburn, Alabama, USA with a desiccant lining (4.3 g molecular sieve).

[0114] The storage conditions were 5° C., 25° C./60% RH and 30° C./75% RH. The stability study is on-going and the CFU values after 12 months of storage are available as shown in FIG. 4. The CFU values after 24 months of storage are also available as shown in FIG. 6. It is evident that the samples stored at 5° C. or 25° C./60% RH have a low log loss whereas the log loss for samples stored at 30° C./75% RH is higher.

[0115] In Table 5 the corresponding water activities measured during storage are presented. The water activity decreases during the first month of storage due to the desiccant lining in the plastic vial and this has a stabilizing effect on the CFU value.

TABLE-US-00005 TABLE 5 Water activities during storage in plastic vials Water activity Appearance Time/ 25° C./ 30° C./ 5° C., 25° C./60% RH, Months 5° C. 60% RH 75% RH and 30° C./75% RH 0 0.24 0.24 0.24 At all sampling at the three 1 0.20 0.14 0.12 storage conditions the soft gel 2 0.22 0.16 0.13 capsule are red-brown, and no 3 0.18 0.14 0.09 leakages are observed 5 0.14 0.12 0.12 6 0.12 0.06 0.08 9 0.14 0.08 0.19 12 0.14 0.09 0.10 18 0.18 0.04 0.10 24 0.10 0.07 0.17

REFERENCES

[0116] Karim A. A. and Bhat, Rajeev, “Fish gelatin: properties, challenges, and prospects as an alternative to mammalian gelatins”, Food Hydrocolloids 23 (2009) 563-576

[0117] Lachmann, L., et al., eds. Lea & Febiger, Philadelphia, Pa., pp. 398-412 (1986)

[0118] Podczeck, Fridrun and Jones, Brian E., Pharmaceutical Capsules, 2004, pp. 195-204 Singh et al., “Microencapsulation: A promising technique for controlled drug delivery”, Res Pharm Sci. 2010 July-December; 5(2): 65-77

[0119] CA 2 675 892

[0120] WO2012/021432