Process for the production of freeze dried micro-organisms and related compositions

Abstract

The present invention provides a process for the preparation of freeze dried microorganism composition, comprising the step of (i) subjecting a frozen composition comprising micro-organisms to a drying pressure of from 133 Pa [1000 mT] to 338 Pa [2540 mT] such that at the drying pressure the frozen composition is dried by sublimation of water present in the frozen composition to provide a freeze dried composition comprising the micro-organisms.

Claims

1. A process for the preparation of freeze dried micro-organism composition, comprising the step of: (i) subjecting a frozen composition comprising micro-organisms to a drying pressure of from 133 Pa [1000 mT] to 200 Pa [1500 mT] such that at the drying pressure the frozen composition is dried by sublimation of water present in the frozen composition to provide a freeze dried composition comprising the micro-organisms, wherein the micro-organisms are subjected to drying during a primary and a secondary drying phase and wherein the drying pressure is applied to the micro-organisms through the entire primary drying phase.

2. A process according to claim 1 wherein the micro-organisms are selected from the group consisting of yeasts, moulds, fungi, bacteria and mixtures thereof.

3. A process according to claim 1 wherein the micro-organisms are selected from one or more strains of bacteria.

4. A process according to claim 3 wherein the one or more strains of bacteria are selected from lactic acid bacteria.

5. A process according to claim 4 wherein the lactic acid bacteria are selected from Streptococcus thermophilus and Lactobacillus acidophilus.

6. A process according to claim 1 wherein the frozen composition is in the form of frozen pellets.

7. A process according to claim 1 wherein the process comprises the additional step of (i) freezing a composition comprising micro-organisms to provide the frozen composition of step (i).

8. A process according to claim 1 wherein the freeze dried micro-organism composition comprises the one or more strains of bacteria in an amount of 1E8 to 5E12 CFU/g of freeze dried micro-organism composition.

9. A process according to claim 1 wherein in step (i) the composition is subjected to a temperature of from 10 to 40 C.

10. A process according to claim 1 wherein in step (i) the composition is subjected to a temperature of approximately 25 C.

11. A process according to claim 1 wherein the drying pressure is applied to the frozen composition comprising micro-organisms for a period of from 24 to 72 hours.

12. A process according to claim 1 wherein the freeze dried composition is subsequently milled.

13. A process for the preparation of a food or feed, the process comprising (a) preparing a freeze dried micro-organism composition in accordance with claim 1; and (b) combining the freeze dried micro-organism composition with a foodstuff or feedstuff.

14. A freeze dried micro-organism composition prepared by a process as defined in claim 1.

15. A food or feed comprising (a) a freeze dried micro-organism composition as defined in claim 14; and (b) a foodstuff or feedstuff.

16. A method of preparing a freeze dried micro-organism composition having improved stability and/or improved cell count and/or increased density and/or improved dispersibility, the method comprising: applying a drying pressure of from 133 Pa [1000 mT] to 200 Pa [1500 mT] to a frozen composition comprising micro-organisms to dry the frozen composition by sublimation of water present in the frozen composition, wherein the micro-organisms are subjected to drying during a primary and a secondary drying phase and wherein the drying pressure is applied to the micro-organisms through the entire primary drying phase.

Description

(1) The present invention will now be described in further detail by way of example only with reference to the accompanying figures in which:

(2) FIG. 1 shows Scanning electron microscope (SEM) images of the interior of a conventionally freeze-dried pellet of a strain of S. thermophilus. (a) 54 magnification of dried pellet; (b) 540 magnification of dried pellet; (c) 2700 magnification of dried pellet. The images demonstrate a finely textured and homogeneous structure of the cell/protectant matrix.

(3) FIG. 2 represent Scanning electron microscope (SEM) images of the interior of a high-pressure freeze-dried pellet of the same strain of S. thermophilus. (a) 34 magnification of dried pellet; (b) 540 magnification of dried pellet; (c) 2700 magnification of dried pellet (what look like holes in this photo are pits where individual cells have been knocked out of place on this fractured surface).

(4) The images demonstrate a rearrangement of the structure of the cell/protectant matrix.

(5) The present invention will now be described in further detail in the following examples.

EXAMPLES

(6) Introduction

(7) Scanning electron microscopy (SEM) image comparisons of conventionally freeze-dried pellets and high-pressure freeze-dried pellets demonstrate real differences in the physical characteristics resulting from the two processes. Cryogenically frozen pellets from a lot of a strain of Streptococcus thermophilus were divided into two groups. Half the pellets were dried with a conventional freeze drying cycle using a 100 mTorr vacuum setting, the other half of the pellets were dried with a high-pressure cycle using a 1400 mTorr vacuum setting. FIG. 1 shows that conventionally freeze-dried bacterial pellets have a uniform friable structure consisting of narrow interstitial matrix and open channels, at times each no thicker than a single 1.0 m bacterium. FIG. 2 shows that the high-pressure freeze-dried bacterial pellets have a less organized matrix, in which the interior of a high-pressure dried pellet has been rearranged. Open spaces have been converted from narrow channels to an interconnected series of spherical voids, and bacteria have become encapsulated in thick bands of matrix.

(8) Scanning electron microscopy was performed on a JEOL JCM 5000 NeoScope SEM. Samples were prepared for SEM imaging by coating with 4-6 nm gold plate using a Cressington 108 sputter coater.

(9) Experimental

(10) Streptococcus thermophilus was produced by batch fermentation. Cells were washed by the addition of tap water to the cell fermentate on a 1:1 basis, and cells were concentrated by centrifugation to achieve a cell density of approximately 3.0E+11 cfu/ml to 4.0E+11 cfu/ml. A standard solution of trehalose, a known protectant, was added to the cell concentrate. The cell concentrate/protectant combination was thoroughly mixed, and the mixture was dripped into liquid nitrogen to form frozen pellets. Frozen pellets were stored at 85 C. until the drying experiments were performed.

(11) 100 gr aliquots of frozen pellets of Streptococcus thermophilus were dried in a Virtis Genesis 35 EL freeze drier at various pressures:

(12) for example 1: 100, 1425 mT and 2010 mT; and

(13) for example 2: 100, 1000 and 1400 mT.

(14) The layer of frozen pellets varied from 1.6 to 3.8 cm. For all the experiments, the shelf temperature was controlled at 25 C. Drying was held until the temperature of the pellets was stable and close to 25 C. The frozen pellets were dried for a period such that at the end of the freeze drying cycle freeze dried pellets were provided having an Aw below 0.15.

(15) Material was then milled using a lab scale mill (Jupiter Family Grain Mill) to provide a powder with a particle size smaller than 30 mesh. The freeze dried pellets and freeze dried powder were then characterized with a series of test. The water content of the dried pellets was assessed by measuring the activity of water (Aw) using an Hygrolab Aw meter (Rotronic). The cell concentration of the dried pellets was measured by resuscitating the cells in a peptone buffer for 2 minutes under agitation then plated using an Ellikers media (DIFCO). Incubation was performed at 38 C. for 48 h. Storage stability of the freeze dried pellets was assessed with an accelerated shelf-life test which consists of placing for 14 days the freeze dried material into an incubator set at 38 C. and measuring the cell counts after that period of time. Then a survival percent was calculated by dividing the cell counts obtained after 14 days by the initial cell counts.

(16) The bulk density of the powder was calculated by measuring the weight of a 10 ml freeze dried powder. The tap density was measured by taping the graduated cylinder until a constant volume was achieved. The tap density was calculated by dividing the weight by the volume obtained after taping. The dispersibility of the freeze dried powder into water was assessed by dropping a spoonful of powder (2 gr) into 100 ml of water at room temperature, then observing if the powder was floating at the surface or sinking quickly after 1minute and 2 minutes. Then the water was mixed for 10 seconds and the solution was observed and the quantity of residual powder floating or decanting at the bottom was visually estimated. A dispersibility index was then calculated with a scale from 1 to 5, with an index of 5 representing a highly dispersable powder (like powder sinking to the bottom in less than 1 minute and no particles floating or at the bottom of the container after mixing) and an index of 1 representing a poorly dispersible powder (no powder sinking within 2 minutes and most particles floating or at the bottom after mixing)

Example 1

(17) A first set of data was collected showing that the bulk and tap density increases as a function of the drying pressure. In addition it can be noted that the cell counts for a drying pressure of 1425 and 2010 mT was higher than when drying pressure was set at 100 mT.

(18) TABLE-US-00001 Drying pressure Bulk density Tap density (mTorr) Aw CFU/gr (g/ml) (g/ml) 100 0.004 5.10E+11 0.20 0.33 1425 0.092 8.54E+11 0.44 0.58 2010 0.02 7.80E+11 0.58 0.74

Example 2

(19) In the second series of experiment, stability and dispersibility were evaluated. The testing of culture dried by tray was also tested in parallel (the material dried by tray was done in an industrial setting). The data show that not only the drying at a pressure in accordance with the present invention increased the density of the powder, but also improved the dispersibility, cell counts and stability when compared to the powder coming from a pellet drying process at 100 mT. The density and dispersibility are of powder dried in accordance with the present invention also approached the density and dispersibility of the powder with the tray process.

(20) In this experiment we obtained freeze dried micro-organism composition having an improved stability, an improved cell count, an increased density and an improved dispersibility. Indeed: stability was increased of at least 9 fold in comparison with the standard pellet freeze drying process; cell count was increased of at least 3 fold in comparison with the standard pellet freeze drying process; density was increased at least 2 fold in comparison with the standard pellet freeze drying process; and dispersibility was increased at least 1.5 fold in comparison with the standard pellet freeze drying process.

(21) TABLE-US-00002 Drying Shelf Percent cell survival Drying pressure temperature Cell after 14 days at Bulk process (mTorr) (deg C.) Aw count 38 C. density Tap density Dispersibility Pellets 100 25 0.008 2.6E11 8 0.25 0.31 1.5 Pellets 1000 25 0.071 .sup.7E11 65 0.50 0.64 2.3 Pellets 1400 25 0.075 6.5E11 63 0.47 0.57 2.1 Tray 100 Variable 0.046 5.5E11 37.5 0.57 0.71 3.5

Example 3

(22) Lactobacillus acidophilus was produced by batch fermentation and concentrated by centrifugation. Then trehalose and phosphate were mixed with the concentrated culture in standard amounts to lyoprotect the strain, and the mixture was dripped into liquid nitrogen to form frozen pellets. Frozen pellets were stored at 85 C. until the drying experiments were performed.

(23) 100 gr aliquots of frozen pellets of Lactobacillus acidophilus were dried in a Virtis Genesis 35 EL freeze drier at various pressures: 100 mT, 450 mT, 700 mT, 1000 mT and 1250 mT with a shelf temperature of 15 C. The layer of frozen pellets was approximately 2 cm. Drying was held until the temperature of the pellets was stable and close to 15 C. Aw's of freeze dried material are reported in the following Table.

(24) TABLE-US-00003 Drying pressure (mTorr) Aw 100 0.07 450 0.124 700 0.136 1000 0.181 1250 0.21

(25) In conclusion, this example shows that a strain protected with a phosphate based protectant has an Aw rising as a function of the applied pressure.

(26) All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, biology, food science or related fields are intended to be within the scope of the following claims.