ELECTROSTATIC SPRAY DRYING OF MICROORGANISMS
20230341182 · 2023-10-26
Assignee
Inventors
- Jimi Kjaersgaard Pettersson (Hoersholm, DK)
- Wendy Ossieur (Hoersholm, DK)
- Michelle Milling Madsen (Hoersholm, DK)
- Kim Nielsen (Hoersholm, DK)
Cpc classification
C12M35/02
CHEMISTRY; METALLURGY
F26B3/12
MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
International classification
F26B3/12
MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
C12M1/42
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to a process for electrostatic spray drying of a living microorganism, the process comprising the following steps: a. Providing a suspension, comprising a number of components, including a microorganism, a solvent and an additive; b. Applying an electrostatic charge to said suspension; c. Forming droplets of said suspension; d. Drying said droplets, thereby forming dried particles; and e. (Optionally) collecting the dried particles.
Claims
1. A process for electrostatic spray drying of a living microorganism, the process comprising: applying an electrostatic charge to a suspension comprising a live microorganism, a solvent, and an additive, wherein the suspension comprises at least 35% by weightwater; droplets of said suspension; said droplets, thereby forming dried particles; and optionally, collecting the dried particles.
2. The process according to claim 1, wherein the electrostatic charge is applied by an electrode in contact with said suspension, wherein said electrode has an electric potential difference, with respect to ground, below about 40 kV.
3. The process according to claim 2, wherein the electric potential difference, with respect to ground, of the electrode varies over time.
4. The process according to claim 1, wherein the microorganism has a lower effective dielectric property than one or both of the additive and the solvent.
5. The process according to claim 1, wherein themicroorganism comprises facultative anaerobic bacteria or strict anaerobic bacteria.
6. The process according to claim 1, whereinforming droplets is carriedwith a two-fluid nozzle.
7. The process according to claim 1, wherein forming droplets is carried out with an atomizing gasselected from one or more of an inert gas, a noble gas, and an alkane gas.
8. The process according to claim 1, wherein the suspension comprisesas at least 40% by weight water.
9. The process according to claim 1, wherein the additive comprises a drying protectant.
10. The process according to claim 9, wherein the dried particle comprises the drying protectant in an amount from about 50% by weight to about 90% by weight.
11. The process according to claim 1, wherein the dried particles have a size from about 10 micrometers to about 200 micrometers, measured as Dv50 values.
12. The process according to claim 1, wherein the size distribution of the dried particles is substantially unimodal.
13. The process according to claim 1, wherein the dried particles comprise a microorganism having a viability of at least 1.0 × 10E4 per gram, as determined by one or more of the most probable number (MPN), the number of colony forming units (CFU), and the number of viable cells as measured by flow cytometry.
14. A dried particle comprising living microorganisms embedded in a mass of additive, wherein the microorganisms are not present at the surface of the particle.
15. The particle according to claim 14, wherein the particle is not layered, and has a substantially continuous radial gradient of additive.
16. The process according claim 1, wherein the solvent has a higher dielectric constant than the additive, and the additive has a higher dielectric constant than the microorganism.
17. The process according to claim 9, wherein the additive comprises a drying protectant comprising one or more selected from cyclitols, monosaccharides, disaccharides, polysaccharides, magnesium stearate, peptides, proteins, sugar alcohols, hydrogenated starch hydrolysates, fatty acid esters, and alginates and salts thereof.
18. The process according to claim 9, wherein the additive comprises a drying protectant comprising one or more selected from inositol, dextrose, lactose, sucrose, trehalose, inulin, maltodextrin, starch, magnesium stearate, mannitol, sorbitol, and sodium alginate.
Description
DETAILED DESCRIPTION OF DRAWINGS
[0291] The invention will in the following be described in greater detail with reference to the accompanying drawings. The drawings are exemplary and are intended to illustrate some of the features of the presently disclosed methods for electrostatic spray drying of microorganisms and microorganisms embedded in dried particles, and are not to be construed as limiting to the presently disclosed invention.
[0292]
[0293]
[0294]
[0295]
[0296]
[0297]
[0298]
[0299]
[0300]
EXAMPLE 1 MANUFACTURING OF DRIED PARTICLES COMPRISING LACTOBACILLUS RHAMNOSUS AND OTHER REPRESENTATIVE SPECIES
[0301] An electrostatic spray drier was used for manufacturing of dried particles comprising microorganisms.
[0302] The feedstock was pumped from the feed container (7) through a peristaltic Watson Marlow pump (8) to a two-fluid nozzle (9) at the top of the electrostatic spray dryer. The atomized feedstock was dried co-current in the chamber (3) and was collected as dry powder in a glass container (10) at the end of the drying chamber.
[0303] The atomization was performed with pressurized N.sub.2, which entered from the main gas flow (1), passed through a heater (11) and entered the two-fluid nozzle (9) at a controlled atomization pressure. A voltage generator (12) controlled the Pulse Width Modulation (PWM), and it was in electric contact with the two-fluid nozzle such that an electric charge could be applied to the suspension.
[0304] The spray dryer comprised an oxygen level sensor (13), and was configured to shut down if the oxygen level got too high.
[0305] To obtain the suspension, Lactobacillus rhamnosus (LGG®) was grown in a fermenter (Infors, Switzerland) using MRS (Oxoid) and concentrated using cross flow filtration (Repligen, The Netherlands). The total solid content of the suspension was 33.51%.
[0306] Electrostatic spray drying was performed at continuous stirring of the suspension, with a main air flow of 25 m.sup.3/h N.sub.2. The supply gas pressure and temperature of N.sub.2 was 80 bar(g) and 20.8° C. respectively. The inlet temperature (T.sub.in) was kept at 80° C. and the outlet temperature (T.sub.out) was kept at 38.3° C.
[0307] Atomization of the suspension (feedstock) was performed with a two-fluid nozzle, where the atomization pressure (P.sub.nozzle) was 1.0 bar(g) N.sub.2 and the temperature was 50° C. The feed rate was kept at approximately 4.1 g/min.
[0308] The electrostatic charge was applied to the suspension, by the use of an electrode in contact with the suspension, located prior to the nozzle, by Pulse Width Modulation (PWM), wherein a 5 kV square pulse, with a pulse length of 1 s, was provided every third second, with a baseline of 1 kV. The oxygen level in the drying chamber was kept at 0.5% O.sub.2.
[0309] The electric charge, applied to the suspension by contacting the suspension with the electrode, enables partitioning of the components within the formed droplets, such as with respect to their polarity, as can be seen in
[0310] Following drying, the dried particles were collected in a container at the outlet end of the drying chamber of the electrostatic spray dryer. As a control a sample of the suspension was pelletized by dripping into liquid nitrogen, collecting the pellets, freeze-drying these and milling the dry pellets into a powder.
Viability Analysis of Dried Particles
[0311] The viability of the microorganisms embedded within the manufactured dried particles, as described above, was analyzed using standard plate counting, and further compared to other methods for drying of microorganisms.
[0312] Colonies formed from electrostatic spray dried particles and milled pellets material are shown in
[0313] The measured CFU/ml is shown in
[0314] Additionally, the MRS tubes where the serial dilutions were made in showed slower growth of LGG® for the milled product, compared to the electrostatic spray dried particles.
Flow Cytometry
[0315] Viability of the dried particles was measured using flow cytometry. The viability of the electrostatic dried material was compared to pelletized material, pelletized and freeze dried material, and milled material wherein the dried particles of each process had been formed from a similar suspension, comprising LGG®.
[0316] The flow cytometry results (
[0317] The number of intact cells of the freeze dried LGG® pellets and the milled pellets were measured, and found to be in the same range, The highest number of intact cells/g was acquired by the electrostatic spray drying process.
SEM Micrographs
[0318]
[0319] To investigate the interior of the particles, electrostatically spray dried particles were split such that their cross sections were revealed and thereafter analyzed by SEM,
[0320] It was also seen that there was a small layer of material on the surface of the particle, that was free of bacterial cells and the bacterial cells were therefore not exposed to the surroundings.
[0321] The fact that the bacterial cells were protected by a material layer, could result in a better stability of the electrostatic spray dried powder, compared to the other particles where some of the bacterial cells were exposed to the surroundings at the surface of the particles.
EXAMPLE 2 PELLETIZING, FREEZE DRYING, MILLING AND ELECTROSTATIC SPRAY DRYING OF B BREVE
[0322] The primary objective of this test was to characterize and compare electrostatic spray dried B. breve with pelletized, freeze dried and milled B. breve. The difference in viability of the dried powder from each process was evaluated.
Materials and Method
Electrostatic Spray Drying
[0323] Electrostatic spray drying was carried out identical to Example 1.
[0324] To obtain the suspension, Bifidobacterium breve was grown in a fermenter (Infors, Switzerland) using MRS (Oxoid) and concentrated using cross flow filtration (Repligen, The Netherlands).
Cross Flow Filtration
[0325] The fermentate was pumped directly from the fermenter to the cross flow filtration unit. The retentate was pumped back to the fermenter while the permeate was collected. The process was allowed to continue until the desired concentration was achieved or until the retentate was too viscous for further pumping.
[0326] Following concentration, the concentrate was measured to have a total solid content of 8.50% (w/w) (Mettler Toledo: 105° C. - 1 mg/50 s).
[0327] Additives, such as drying protectants, suitable for protecting microorganisms during cryogenic freezing were added to the suspension. These additives were added such that the ratio between the total solid content of the concentrate and total solid content of these additives were 1:4. The additive used was sucrose.
[0328] After the additives were added to the concentrate, total solids were measured again, but due to the large amounts of sugar added, it was decided to perform the total solids measurement at 60° C. instead of 105° C., in order not to burn the sugars and thereby getting a false result.
[0329] Total solid content of the suspension was measured to be 26.09% (w/w) (Mettler Toledo: 60° C. - 1 mg/140 s).
Pelletizing and Freeze Drying
[0330] A concentrated B. breve fermentate comprising drying protectant (sucrose) was pelletized and subsequently freeze dried.
[0331] Pelletizing was performed without atomization gas and the feed rate was controlled by the Watson Marlow pump, set to approximately 13.86 ml/min.
[0332] The pelletized material was collected by a 50 .Math.m sieve from Retsch and thereafter the collected pelletized material was transferred to a plastic container and kept cold on dry ice until it was transferred to an anaerobic glovebox.
[0333] For freeze drying, the pelletized material was evenly distributed to a metal freeze drying trays, and freeze dried (Martin Christ Gefriertrocknungsanlagen GmbH) for 46 hours and 10 minutes. The freeze dried material was loaded to a small aluminum bag, which subsequently was welded.
[0334] The freeze dried pellets: [0335] Water activity (a.sub.w) = 0.152 at 21.44° C. [0336] Residual moisture = 0.44% (Mettler Toledo: 60° C. - 1 mg/140 s)
[0337] It was not possible to measure particle size distribution on the freeze dried pellets, because they were too big.
Milling of Freeze Dried Pellets:
[0338] The milling was performed manually in a mortar for approximately 5 minutes.
[0339] Properties of the milled pellets: [0340] Water activity (a.sub.w) = 0.142 at 21.53° C. [0341] Residual moisture = 0.84% (Mettler Toledo: 60° C. - 1 mg/140 s) [0342] Mean particle size distribution (d.sub.50) = 150 .Math.m Span = 5.174
Electrostatic Spray Drying
[0343] Electrostatic spray drying was performed at continuous stirring of the suspension, comprising B. breve and drying protectant, with a main air flow of 25 m.sup.3/h N.sub.2. The supply gas pressure and temperature of N.sub.2 was 80 bar(g) and 21.5° C. respectively. The inlet temperature (T.sub.in) was kept at 80° C. and the outlet temperature (T.sub.out) was kept at 36° C.
[0344] Atomization of the suspension (feedstock) was performed with a two-fluid nozzle, where the atomization pressure (P.sub.nozzle) was 1.0 bar(g) N.sub.2 and the temperature was 50° C. The feed rate was kept at approximately 4.1 g/min. The drying chamber had a vacuum pressure of 0.3 kPa. The dried particles were collected at the outlet end of the drying chamber, and analyzed. As mentioned above, the total solid content of the B. breve concentrate comprising drying protectant, i.e. the suspension before drying, was 26.09%.
Results
Most Probable Number (MPN):
[0345] Growth was measured on the fermentate, concentrate, concentrate + drying protectant, frozen pellets, freeze dried pellets, milled pellets and electrostatic spray dried material.
[0346] The cell count pr. ml or cell count pr. gram of all the samples was an average of six analytical results.
TABLE-US-00001 Process step MPN pr. mL or pr. g Log.sub.10 MPN 95% confidence limits Lower Upper FM 3.9E+10 10.6 1.4E+10 1.1E+11 Concentrate 2.2E+11 11.4 9.1E+10 5.6E+11 Concentrate and drying protectant 2.2E+11 11.4 9.1E+10 5.6E+11 Frozen pellets 7.8E+10 10.9 2.6E+10 2.3E+11 Freeze dried pellets 2.9E+10 10.5 1.1E+10 7.9E+10 Milled pellets 4.0E+1 10.6 1.8E+10 9.0E+10 Electrostatic spray dried 5.8E+11 11.8 1.9E+11 1.8E+12
[0347] High MPN numbers were seen in all samples and all steps of the process. It was seen from the above table that MPN decreased during pelletizing. Electrostatic spray drying, on the other hand, resulted in high MPN, about an order of magnitude higher than the pelletized materials.
[0348] It was also seen that the MPN decreased during the freeze drying step and it was the milled pellets that had the highest MPN of the freeze dried samples.
Flow Cytometry
[0349] Flow cytometry was measured on the pre-fermentate, the frozen pellets, the freeze dried pellets, the milled pellets, and the electrostatic spray dried material.
TABLE-US-00002 Sample Damaged Intermediate Intact Total Intact% cells/ml or cells/g Pre-fermentate (PFM) 3.57.Math.10.sup.7 1.71.Math.10.sup.8 3.16.Math.10.sup.8 5.23.Math.10.sup.8 60.4 Pellets (frozen) 1.52.Math.10.sup.10 4.99.Math.10.sup.9 2.24.Math.10.sup.9 2.25.Math.10.sup.10 10.0 Freeze dried pellets 9.16.Math.10.sup.9 3.26.Math.10.sup.10 7.98.Math.10.sup.10 1.22.Math.10.sup.11 65.6 Milled pellets 1.11.Math.10.sup.10 3.44.Math.10.sup.10 7.40.Math.10.sup.10 1.20.Math.10.sup.11 61.6 Electrostatic spray dried 6.78.Math.10.sup.9 2.07.Math.10.sup.10 1.03.Math.10.sup.11 1.30.Math.10.sup.11 79.3
[0350] As it was seen from the flow cytometry results, there was a high number of total cells in all the analyzed samples.
[0351] It can be seen from the above table that the number of total cells/g in the dried powders were comparable. It was seen that there was a slight decrease in intact cells/g when the freeze dried pellets were milled. This was also seen in the CFU analysis.
[0352] It can also be seen that the intact cells/g was higher for the electrostatic spray dried powder compared to the freeze dried powders (freeze dried pellets and milled pellets).
[0353] From the viability analysis it can be seen that the MPN and flow cytometry results were very well correlated with each other and the CFU results were approximately 1 log lower when comparing MPN to flow cytometry.
SEM Micrographs
[0354] From all the powder samples produced, SEM pictures were produced. SEM pictures were taken of the surface of the particles, but the particles were also cut in half and SEM pictures of inside of the pellets were taken.
[0355] The pellet surfaces differ between different pellets, wherein the number of cavities and the smoothness of the surface vary significantly. Broken off areas, typically sharp edges of the pellets, were abundant. Bacteria can be seen both inside and on the surface of the pellets.
[0356] The micrographs of cut pellets (
[0357] Milled pellets,
[0358] The SEM micrographs reveal that bacterial cells were present on all surfaces of the milled pellets, meaning that there was a higher number of bacteria cells that were exposed to the surroundings compared to the un-milled pellets, which could result in a decreased stability of the milled pellets.
EXAMPLE 3 PELLETIZING, FREEZE DRYING, MILLING AND ELECTROSTATIC SPRAY DRYING OF F PRAUSNITZII
[0359] The main objective of the performed tests was to characterize and compare the viability of electrostatic spray dried F. prausnitzii with the powder obtained from freeze dried pellets and milled freeze dried pellets.
Materials and Methods
Cross Flow Filtration
[0360] The fermentate was pumped directly from the fermenter to the cross flow filtration unit. The retentate was pumped back to the fermenter while the permeate was collected. This process was allowed to continue until the desired concentration was achieved or until the retentate was too viscous for further pumping.
Pelletizing and Freeze Drying
[0361] The liquid feed was pumped from a feed container through a Watson Maslow peristaltic pump to a nozzle. The liquid material was dripping from the nozzle into liquid nitrogen (LN.sub.2) placed under the nozzle. The container for LN.sub.2 was used to add extra LN.sub.2 to the frozen product container during the experiment. The pelletized material was thereafter filtered through a 50 .Math.m Retsch filter, where the frozen pellets were collected.
[0362] For freeze-drying, pelletized material was separated from the LN.sub.2 by the filter, the pellets were thereafter loaded onto freeze drying trays or glass petri dishes and subsequently freeze dried.
Electrostatic Spray Drying
[0363] Electrostatic spray drying was carried out identical to Example 1.
Experimental Work
[0364] A culture of F. prausnitzii was grown in a fermenter (Infors) using media as described in Duncan et al., Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen. nov., comb. nov. Int. J. Syst. Evol. Microbiol. 52, 2141-2146 (2002).
[0365] The fermentate was concentrated by Cross flow filtration (CFF), initiated at pH = 5.54 and was performed with a fermentate temperature of 37° C.
[0366] Cross flow filtration was slowly started with a feed flow of 180 ml/min. and within approximately 7 minutes the feed rate was gradually increased to 1000 ml/min.
[0367] At a feed flow of 1000 ml/min., inlet pressure (P.sub.in) was 0.37 bar(g), TMP was 0.21 bar(g) and permeate flow (Q.sub.permeate) was 120 ml/min. and the shear rate was 1450 s.sup.-1.
[0368] After approximately 9 minutes the back pressure valve was activated with a TMP setpoint of 0.50 bar(g).
[0369] Corresponding P.sub.in was 0.60 bar(g), Q.sub.permeate = 125 ml/min. and shear rate = 1080 s.sup.-1.
[0370] After additional 13 minutes the TMP setpoint was changed to 0.60 bar(g). Corresponding P.sub.in was 0.75 bar(g), Q.sub.permeate was 126 ml/min and the shear rate was 1080 s.sup.-1.
[0371] In the end of the concentration process, foam was seen in the fermenter, which was due to the high feed rate used.
[0372] Approximately 1 hours and 30 minutes after the start, P.sub.inlet had increased to 0.85 bar(g) and a concentration factor of at least 30x was reached.
[0373] Following concentration, the concentrate was measured to have a total solid content of 12.55% (Mettler Toledo: 105° C. - 1 mg/50 s)
[0374] Additives, such as drying protectants, suitable for protecting microorganisms during cryogenic freezing were added to the suspension. These additives (sucrose in the present case) were added such that the ratio between the total solid content of the concentrate and total solid content of these additives were 1:4.
[0375] TS of concentrate with drying protectant = 31.83% (Mettler Toledo: 60° C. - 1 mg/140 s)
Pelletizing and Freeze Drying
[0376] A concentrated F. prausnitzii fermentate comprising drying protectant was pelletized and freeze dried. Pelletizing was performed without atomization gas and the feed rate was controlled by the Watson Marlow pump, which was set to 2 RPM, which corresponds to approximately 13.86 ml/min. The pelletizing process took 16 minutes. The pelletized material was collected by a 50 .Math.m sieve from Retsch.
[0377] During the pelletizing process, it was seen that the frozen pellet size was very ununiform, and the size was varying very much. There were seen some very big pellets and there were also some small pellets.
[0378] The collected pelletized material was transferred to a plastic container and kept cold on dry ice until it was transferred to the anaerobic glovebox and thereafter loaded to a freeze drier (Martin Christ).
[0379] After 46 hours and 10 minutes the freeze drying was ended, and the freeze dried material was removed from the freeze drying tray.
[0380] The freeze dried material was loaded to a small aluminum bag, which subsequently was welded.
[0381] Properties of the freeze dried pellets: [0382] Water activity (a.sub.w) was measured at room temperature. [0383] Water activity (a.sub.w) = 0.078 [0384] Residual moisture = 0.41% (Mettler Toledo: 60° C. - 1 mg/140 s)
[0385] It was not possible to measure particle size distribution on the freeze dried pellets, because they were too big.
Milling of Freeze Dried Pellets:
[0386] The milling was performed manually in a mortar for approximately 5 minutes.
[0387] Properties of the milled pellets: [0388] Water activity (a.sub.w) = 0.074 [0389] Residual moisture = 0.94% (Mettler Toledo: 60° C. - 1 mg/140 s) [0390] Mean particle size distribution (d.sub.50) = 833 .Math.m [0391] Span = 2.547
[0392] It was seen from the particle size distribution that the mean particle size of the milled pellets was much larger compared to the freeze dried mean particle size, but the span was in the same range.
Electrostatic Spray Drying
[0393] During electrostatic spray drying the feed was kept under steering. F. prausnitzii concentrate comprising drying protectant was electrostatic spray dried.
[0394] Electrostatic spray drying was performed with a main air flow of 25 m.sup.3/h N.sub.2. The supply gas pressure and temperature of N.sub.2 was respectfully 80 bar(g) and 21.5° C. The inlet temperature (T.sub.in) was 80° C. and the outlet temperature (T.sub.out) was 36° C.
[0395] Atomization of the feed was performed with a two-fluid nozzle, where the atomization pressure (P.sub.nozzle) was 1.0 bar(g) N.sub.2 and the temperature was 50° C.
[0396] The feed rate was controlled by a peristaltic pump with and RPM of, which corresponds to approximately 4.8 g/min.
[0397] The electrostatic charge was applied to the suspension, by the use of an electrode in contact with the suspension, located prior to the nozzle. Pulse Width Modulation (PWM) was used, wherein a 5 kV square pulse, with a pulse length of 1 s, was provided every third second, with a baseline of 1 kV.
[0398] During the entire test the oxygen level in the chamber was 0.5% oxygen. The drying chamber had a vacuum pressure of 0.3 kPa. After the test was ended, dried particles were collected at the outlet end of the drying chamber and analyzed. As mentioned above, the total solid content of the F. prausnitzii concentrate with drying protectant, i.e. the suspension before drying, was 31.83%.
[0399] Properties of the electrostatic spray dried product: [0400] Water activity (a.sub.w) = 0.245 at 21.78° C. [0401] Residual moisture = 3.67% (Mettler Toledo: 60° C. - 1 mg/140 s) [0402] Mean particle size distribution (d.sub.50) = 28.0 .Math.m [0403] Span = 2.461
Results
Particle Analytics
[0404] For all the produced samples, residual moisture (R.sub.M%), water activity (a.sub.w), particle size distribution (d.sub.50 and span) were measured.
[0405] Viability was also measured for all produced samples.
[0406] Viability was measured by CFU (Colony Forming Units), MPN (Most Probable Number) and flow cytometry.
TABLE-US-00003 Sample a.sub.w R.sub.M% d.sub.50 Span Freeze-dried pellets 0.078 0.40 - - Milled pellets 0.074 0.94 833 .Math.m 2.547 Electrostatic spray dried 0.245 3.67 28 .Math.m 2.461
[0407] It was seen from the analytical results in the table above, that the water activity was higher for the electrostatic spray dried powder than for the pellets and milled pellets. Mean particle size distribution was large for the milled pellets.
[0408] From the Malvern data measurements it was seen that the mean particle size distribution of the milled pellets was higher than the mean particle size distribution of the electrostatic spray dried powder and the span of the powders were in the same range.
Most Probable Number (MPN):
[0409] Growth was measured on the fermentate, concentrate, concentrate + drying protectant, pelletized material and electrostatic spray dried material.
[0410] The cell count pr. ml or cell count pr. gram of all the samples was an average of six analytical results.
TABLE-US-00004 Process step MPN pr. mL or pr. g Log.sub.10 MPN 95% confidence limits Lower Upper Fermentate 2.9*10.sup.9 9.5 1.1*10.sup.9 7.9*10.sup.9 Concentrate 1.2*10.sup.11 11.0 4.2*10.sup.10 3.3*10.sup.11 Concentrate and drying protectant 1.3*10.sup.9 9.1 5.2*10.sup.8 3.3*10.sup.9 Frozen pellets 1.7*10.sup.8 8.2 6.6*10.sup.7 4.6*10.sup.8 Freeze dried pellets 3.7*10.sup.8 8.6 1.5*10.sup.8 1.1*10.sup.9 Milled pellets 5.3*10.sup.7 7.7 2.0*10.sup.7 1.7*10.sup.8 Electrostatic spray dried 2.2*10.sup.8 8.3 8.9*10.sup.7 5.4*10.sup.8
[0411] From the MPN results it could be seen that the viability decreased 87% during pelletizing. It could also be seen that the freeze dried pellets and the electrostatic spray dried powder was in the same range, with the freeze dried pellets slightly higher. From the MPN results it was seen that the viability decreased 69% during the milling step.
Colony Forming Units (CFU)
[0412] For all CFU measured samples, no forging colonies was observed, strongly indicating that no contaminations were in the analyzed samples. In the table below the analytical results can be seen.
TABLE-US-00005 Process step CFU count (cells/mL) Log FM 3.07.Math.10.sup.8 8.5 Concentrate 1.02.Math.10.sup.9 9.0 Concentrate + drying protectant 4.97.Math.10.sup.7 7.7 Pellets (frozen) 8.27.Math.10.sup.6 6.9 Freeze dried pellets 1.86.Math.10.sup.6 6.3 Milled freeze dried pellets 6.90.Math.10.sup.5 5.8 Electrostatic spray drying 1.77.Math.10.sup.6 6.2
[0413] As it was seen from the CFU results, CFU/mL decreased 83% during pelletizing. It was also seen that the CFU/mL was in the same range for freeze dried pellets and electrostatic spray dried powder. During milling of the pellets there was a 63% decrease in CFU/mL, which also correlates with the results seen from the MPN analysis.
Flow Cytometry
[0414] Flow cytometry was measured on the fermentate, pelletized material and electrostatic spray dried material.
TABLE-US-00006 Sample Damaged Intermediate Intact Total Intact% cells/ml or cells/g Fermentate (PFM) 1.46.Math.10.sup.8 1.63.Math.10.sup.8 1.50.Math.10.sup.9 1.81.Math.10.sup.9 82.9 Pellets (frozen) 2.84.Math.10.sup.10 5.58.Math.10.sup.9 7.48.Math.10.sup.8 3.47.Math.10.sup.10 2.2 Freeze dried pellets 8.05.Math.10.sup.10 9.00.Math.10.sup.8 4.00.Math.10.sup.8 8.18.Math.10.sup.10 0.49 Milled pellets 9.12.Math.10.sup.10 8.16.Math.10.sup.8 2.04.Math.10.sup.8 9.22.Math.10.sup.10 0.21 Electrostatic spray dried 7.12.Math.10.sup.10 7.65.Math.10.sup.8 4.93.Math.10.sup.8 7.25.Math.10.sup.10 0.68
[0415] As it was seen from the flow cytometry results in the table above, there was a high number of total F. prausnitzii cells in all the analyzed samples.
[0416] It was seen from the above table that the number of total cells/g in the electrostatic spray dried powder was slightly lower than that of the milled powder and the freeze dried pellets.
[0417] The electrostatic spray dried powder had the highest number of intact cells/g, and also the highest intact percentage, which was correlated to the number of total cells/g in the individual sample. It was seen that the viability decreased 49% when the freeze dried pellets were milled. This was in correlation with what was also observed in the MPN and CFU analysis.
EXAMPLE 4 PELLETIZING, FREEZE DRYING, MILLING AND ELECTROSTATIC SPRAY DRYING OF B THETAIOTAOMICRON
[0418] The primary objective of this test was to characterize and compare electrostatic spray dried Bacteroides thetaiotaomicron with pelletized, freeze dried and milled B. thetaiotaomicron. The characterization of the products included measurements of the respective viabilities of the dried powder from each process.
Materials and Methods
[0419] Cross flow filtration was carried out identical to Example 3. Two separate fermenters were used. Pelletizing and Freeze Drying were carried out identical to Example 3. Electrostatic spray drying was carried out identical to Example 1.
Experimental Work
[0420] A culture of B. thetaiotaomicron was grown in a fermenter (Infors) using media as described in Taketani et al., A Phase-Variable Surface Layer from the Gut Symbiont Bacteroides thetaiotaomicron, mBio. 2015 Sep-Oct; 6(5): e01339-15. The fermentate was concentrated by Cross Flow Filtration in Fermenter A or Fermenter B as described below.
Fermenter A
[0421] Cross flow filtration was initiated at pH = 5.20 and was performed with a fermentate temperature of 37° C. Total solids of the fermentate before concentration were 5.82% (Mettler Toledo: 105° C. - 1 mg/50 s).
[0422] Cross flow filtration was slowly started with a feed flow of 280 ml/min. and within approximately 4 minutes the feed rate was gradually increased to 1680 ml/min. At a feed flow of 1680 ml/min., inlet pressure (P.sub.in) was 0.07 bar(g), TMP was 0.11 bar (g) and permeate flow (Q.sub.permeate) was 152 ml/min. and the shear rate was 2500 s.sup.-1. After 13 minutes the back pressure valve was activated with a TMP setpoint of 0.30 bar (g). Corresponding P.sub.in was 0.24 bar (g), Q.sub.permeate = 200 ml/min. and shear rate = 2475 s.sup.-1. The concentration was stopped after approximately 1 hours and 37 minutes.
Fermenter B
[0423] Cross flow filtration (CFF) was initiated at pH = 5.12 and was performed with a fermentate temperature of 37° C. Total solids of the fermentate before concentration were 5.82% (Mettler Toledo: 105° C. - 1 mg/50 s).
[0424] Cross flow filtration was slowly started with a feed flow of 280 ml/min. and within approximately 4 minutes the feed rate was gradually increased to 1680 ml/min. At a feed flow of 1680 ml/min., inlet pressure (P.sub.in) was 0.06 bar(g), TMP was 0.17 bar (g) and permeate flow (Q.sub.permeate) was 190 ml/min. and the shear rate was 2450 s.sup.-1. After 12 minutes the back pressure valve was activated with a TMP setpoint of 0.30 bar (g). Corresponding P.sub.in was 0.27 bar (g), Q.sub.permeate = 195 ml/min. and shear rate = 2446 s.sup.-1. The concentration was finished after approximately 1 hours and 11 minutes.
[0425] The collected concentrate from fermenter A and fermenter B was mixed, in total a concentration factor of 25.4 was reached. Total solids (TS) of concentrate = 11.72% (Mettler Toledo: 105° C. - 1 mg/50 s)
[0426] Additives, drying protectants, suitable for protecting microorganisms during cryogenic freezing were added to the suspension. These additives (sucrose in the present case) were added such that the ratio between the total solid content of the concentrate and total solid content of these additives were 1:4. TS of concentrate with drying protectant was 31.09% (Mettler Toledo: 60° C. - 1 mg/140 s).
Pelletizing and Freeze Drying
[0427] A concentrated B. thetaiotaomicron fermentate comprising drying protectants was pelletized and freeze dried. Pelletizing was performed without atomization gas and the feed rate was controlled by the Watson Marlow pump, which was set to 2 RPM, which corresponds to approximately 13.86 ml/min. The pelletized material was collected by a 50 .Math.m sieve from Retsch.
[0428] The collected pelletized material was transferred to an aluminum bag and kept cold on dry ice until it was transferred to the anaerobic glovebox and thereafter loaded to a freeze drier (Martin Christ).
[0429] After 46 hours and 10 minutes the freeze drying was ended, and the freeze dried material was removed from the freeze drying tray. The freeze dried material was loaded to a small aluminum bag, which subsequently was sealed by welding.
[0430] Properties of the freeze dried pellets: [0431] Water activity (a.sub.w) = 0.131 at 23.00° C. [0432] Residual moisture = 0.21% (Mettler Toledo: 60° C. - 1 mg/140 s)
[0433] It was not possible to measure particle size distribution on the freeze dried pellets, because they were too big.
Milling of Freeze Dried Pellets:
[0434] The milling was performed manually in a mortar for approximately 5 minutes.
Properties of the Milled Pellets:
[0435] Water activity (a.sub.w) = 0.216 at 22.0° C. [0436] Residual moisture = 1.18% (Mettler Toledo: 60° C. - 1 mg/140 s) [0437] Mean particle size distribution (d.sub.50) = 195 .Math.m [0438] Span = 3.880
[0439] As was seen from the obtained water activity, a.sub.w increased considerably during milling of the pellets.
Electrostatic Spray Drying
[0440] During electrostatic spray drying the feed was kept under steering.
[0441] B. thetaiotaomicron concentrate with drying protectant was electrostatic spray dried.
[0442] Electrostatic spray drying was performed with a main air flow of 25 m.sup.3/h N.sub.2. The inlet temperature (T.sub.in) was 80° C. and the outlet temperature (T.sub.out) was in the range of 37.0° C. to 38.5° C. Atomization of the feed was performed with a two-fluid nozzle, where the atomization pressure (P.sub.nozzle) was 2.0 bar(g) N.sub.2 and the atomization temperature was 50° C. The feed rate was controlled by a peristaltic pump with RPM in the range of 6 to 8, which corresponds to approximately 3.5 g/min to 5.5 g/min.
[0443] A two-fluid nozzle was used. The electrostatic charge was applied to the suspension, by the use of an electrode in contact with the suspension, located inside the nozzle prior to the nozzle tip where the liquid suspension is atomized into droplets. Pulse Width Modulation (PWM) was used, wherein a 5 kV square pulse, with a pulse length of 1 s, was provided every third second, with a baseline of 1 kV.
[0444] During the entire test the oxygen level in the chamber was 0.5% oxygen. The chamber had a vacuum pressure of 0.31 kPa. After the test was ended, dried particles were collected at the outlet end of the drying chamber and analyzed.
Properties of the Electrostatic Spray Dried Product:
[0445] Water activity (a.sub.w) = 0.140 [0446] Residual moisture = 1.85% (Mettler Toledo: 60° C. - 1 mg/140 s) [0447] Mean particle size distribution (d.sub.50) = 8.61 .Math.m [0448] Span = 5.219
Results
Particle Analytics
[0449] For all the produced samples, residual moisture (R.sub.M%), water activity (a.sub.w), particle size distribution (d.sub.50 and span) were measured. In addition, viability was measured for all produced samples by MPN (Most Probable Number) and flow cytometry.
TABLE-US-00007 Sample a.sub.w R.sub.M% d.sub.50 Span Pellets 0.131 0.21 - - Milled pellets 0.216 1.18 195 .Math.m 3.880 Electrostatic spray dried 0.140 1.85 8.61 .Math.m 5.219
[0450] The table above shows that the measured water activity was much higher for the milled pellets than for the other samples. Milling increased the water activity considerably. It was not possible to measure the mean particle size of the freeze dried pellets, because the pellets were too large for the Malvern Mastersizer 3000 analytical equipment.
Most Probable Number (MPN):
[0451] Growth was measured on the fermentate, concentrate, concentrate + drying protectant, pelletized material, freeze dried pellets, milled pellets, and electrostatic spray dried material.
[0452] The cell count pr. ml or cell count pr. gram of all the samples was an average of six analytical results.
TABLE-US-00008 Process step MPN [pr. mL or pr. g] Log.sub.10 MPN Standard deviation Fermenter A 2.8E+09 9.4 1.0E+08 Fermenter B 1.5E+09 9.2 2.0E+09 Concentrate 5.0E+10 10.7 4.0E+10 Conc. + drying protectant 1.3E+10 10.1 1.3E+10 Frozen pellets 5.5E+06 6.7 6.4E+06 Freeze dried pellets 1.2E+06 6.1 1.5E+06 Milled pellets 4.8E+04 4.7 4.2E+03 Electrostatic spray dried 4.7E+07 7.7 3.1E+04
[0453] From the performed MPN tests it could be seen that the viability of B. thetaiotaomicron decreased during the freezing step; the viability was decreased by 3.4 log during pelletizing. In addition, during the freeze drying step, the viability of the pellets was reduced 0.6 log. It was also seen that the viability of the freeze dried pellets was decreased by 1.4 log during the milling of the pellets.
[0454] From the MPN results it was seen that the electrostatic spray dried powder had the highest viability, followed by the freeze dried pellets. The electrostatic spray dried powder had a viability that was 1.6 log higher compared to the viability of the freeze dried pellets and 3.0 log higher compared to the milled pellets.
Flow Cytometry
[0455] Flow cytometry was measured on the fermentate, concentrate, pelletized material, freeze dried pellets, milled pellets and electrostatic spray dried material.
TABLE-US-00009 Sample Damaged Intermediate Intact Total Intact% cells/ml or cells/g Fermenter A 1.7(±0.03) E+09 1.5(±0.03) E+08 2.1(±0.03) E+09 4.0(±0.01) E+09 52.1 Fermenter B 1.9(±0.07) E+09 1.9(±0.03) E+08 1.9(±0.05) E+09 4.1(±0.11) E+09 47.6 Concentrate 5.6(±0.11) E+10 1.4(±0.04) E+10 4.0(±0.07) E+10 1.1 (±0.02) E+11 36.2 Pellets (frozen) 9.5(±1.00) E+10 4.7(±0.50) E+09 2.7(±0.30) E+09 1.0(±0.10) E+11 2.7 Freeze dried pellets 1.1(±0.97) E+11 2.7(±2.40) E+09 2.4(±3.14) E+08 1.1(±0.96) E+11 0.15 Milled pellets 9.1(±9.76) E+10 3.9(±4.33) E+09 2.5(±3.35) E+08 9.2(±9.69) E+10 0.17 Electrostatic spray dried 7.8(±1.70) E+10 2.3(±0.50) E+09 7.1(±0.50) E+08 8.1(±1.70) E+10 0.9
[0456] As it was seen from the flow cytometry results, provided above, there was a high number of total B. thetaiotaomicron cells in all the analyzed samples. It was seen that the number of total cells/g through the whole process was quite high and it was seen that the total number of cells/g were in the same range for all the analyzed samples through the full process.
[0457] Like the MPN results it was seen that the viability decreased during the freezing step. It was seen that during pelletizing the viability decreased 1.2 log. It was also seen that there was a viability loss during freeze drying of the pellets. The viability of the pellets was decreased 1.0 log during freeze drying.
[0458] A decrease in viability was not seen during milling. It was also seen that the number of intact cells of the dried powders; e.g. freeze dried pellets, milled pellets and electrostatic spray dried powder were in the same range. The electrostatic spray dried powder had the highest viability and the viability was 0.5 log higher compared to the freeze dried pellets and the milled pellets.
EXAMPLE 5 PELLETIZING, FREEZE DRYING, MILLING AND ELECTROSTATIC SPRAY DRYING OF E HALLII
[0459] The primary objective of this test was to characterize and compare electrostatic spray dried E. hallii with pelletized, freeze dried and milled E. hallii.
Materials and Methods
[0460] Cross flow filtration was carried out identical to Example 3. Two separate fermenters were used. Pelletizing and Freeze Drying were carried out identical to Example 3. Electrostatic spray drying was carried out identical to Example 1.
Experimental Work
[0461] A culture of E. hallii was grown in a fermenter (Infors) using media as described in Engels et al., The Common Gut Microbe Eubacterium hallii also Contributes to Intestinal Propionate Formation, Front. Microbiol., 19 May 2016. The fermentate was concentrated by Cross Flow Filtration in Fermenter A or Fermenter B as described below.
Fermenter A
[0462] Cross flow filtration was initiated at pH = 7.50 and was performed with a fermentate temperature of 26° C.
[0463] Cross flow filtration was slowly started with a feed flow of 300 ml/min. and within approximately 8 minutes the feed rate was gradually increased to 1020 ml/min.
[0464] At a feed flow of 1020 ml/min., inlet pressure (P.sub.in) was 0.42 bar(g), TMP was 0.32 bar(g) and permeate flow (Q.sub.permeate) was 60 ml/min. and the shear rate was 1564 s.sup.-1. After 20 minutes the back pressure valve was activated with a TMP setpoint of 0.50 bar(g). Corresponding P.sub.in was 0.60 bar(g), Q.sub.permeate = 54 ml/min. and shear rate = 1581 s.sup.-1. After approximately 2 hours and 44 minutes, the concentrate was further concentrated by centrifugation .
Fermenter B
[0465] Cross flow filtration was initiated at pH = 7.30 and was performed with a fermentate temperature of 20° C. Cross flow filtration was slowly started with a feed flow of 800 ml/min. and within approximately 2 minutes the feed rate was gradually increased to 1020 ml/min. At a feed flow of 1020 ml/min., inlet pressure (P.sub.in) was 0.40 bar(g), TMP was 0.33 bar(g) and permeate flow (Q.sub.permeate) was 65 ml/min. and the shear rate was 1563 s.sup.-1. After 24 minutes the back pressure valve was activated with a TMP setpoint of 0.50 bar(g). Corresponding P.sub.in was 0.62 bar(g), Q.sub.permeate = 40 ml/min. and shear rate = 1604 s.sup.-1. Following CFF the concentrate had a concentration factor of almost 30x.
[0466] The concentrates from fermenter A and B was mixed giving a total concentration factor of 25.12x and with a total solids of concentrate of 8.03% (Mettler Toledo: 105° C. - 1 mg/50 s)
[0467] Additives, drying protectants, suitable for protecting microorganisms during cryogenic freezing were added to the suspension. These additives (sucrose in the present case) were added such that the ratio between the total solid content of the concentrate and total solid content of these additives were 1:4. TS of concentrate with drying protectant was 25.15% (Mettler Toledo: 60° C. - 1 mg/140 s)
Pelletizing and Freeze Drying
[0468] A concentrated E. hallii fermentate comprising drying protectants was pelletized and freeze dried. Pelletizing was performed without atomization gas and the feed rate was controlled by the Watson Marlow pump, which was set to 2 RPM, which corresponds to approximately 13.86 ml/min. The pelletized material was collected by a 50 .Math.m sieve from Retsch.
[0469] The collected pelletized material was transferred to an aluminum bag and kept cold on dry ice until it was transferred to the anaerobic glovebox and thereafter loaded to a freeze drier (Martin Christ). After 46 hours and 10 minutes the freeze drying was ended, and the freeze dried material was removed from the freeze drying tray. The freeze dried material was loaded to a small aluminum bag, which subsequently was sealed by welding.
Properties of the Freeze Dried Pellets
[0470] Water activity (a.sub.w) = 0.024 at 22.89° C. [0471] Residual moisture (RM) = 0.44% (Mettler Toledo: 60° C. - 1 mg/140 s)
[0472] It was not possible to measure particle size distribution on the freeze dried pellets, because they were too big.
Milling of Freeze Dried Pellets:
[0473] The milling was performed manually in a mortar for approximately 5 minutes.
Properties of the Milled Pellets:
[0474] Water activity (a.sub.w) = 0.075 at 22.91° C. [0475] Residual moisture (RM)= 1.26% (Mettler Toledo: 60° C. - 1 mg/140 s) [0476] Mean particle size distribution (d.sub.50) = 243 .Math.m [0477] Span = 1.985
Electrostatic Spray Drying
[0478] During electrostatic spray drying the feed was kept under steering. E. hallii concentrate with drying protectant was electrostatic spray dried.
[0479] Electrostatic spray drying was performed with a main air flow of 25 m.sup.3/h N.sub.2. The inlet temperature (T.sub.in) was 80° C. and the outlet temperature (T.sub.out) was approximately 36.6° C.
[0480] Atomization of the feed was performed with a two-fluid nozzle, where the atomization pressure (P.sub.nozzle) was 2.0 bar(g) N.sub.2 and the atomization temperature was 50° C. The feed rate was controlled by a peristaltic pump with and RPM of 8, which corresponds to approximately 4.8 g/min.
[0481] A two-fluid nozzle was used. The electrostatic charge was applied to the suspension, by the use of an electrode in contact with the suspension, located inside the nozzle prior to the nozzle tip where the liquid suspension is atomized into droplets. Pulse Width Modulation (PWM) was used, wherein a 5 kV square pulse, with a pulse length of 1 s, was provided every third second, with a baseline of 1 kV.
[0482] During the entire test the oxygen level in the chamber was 0.5% oxygen. The chamber had a vacuum pressure of 0.3 kPa. After the test was ended, dried particles were collected at the outlet end of the drying chamber and analyzed.
Properties of the Electrostatic Spray Dried Product:
[0483] Water activity (a.sub.w) = 0.216 at 22.90° C. [0484] Residual moisture = 1.09% (Mettler Toledo: 60° C. - 1 mg/140 s) [0485] Mean particle size distribution (d.sub.50) = 8.77 .Math.m [0486] Span = 4.711
Results
Particle Analytics
[0487] For all the produced samples, residual moisture (RM%), water activity (a.sub.w), particle size distribution (d.sub.50 and span) were measured. In addition, viability was measured for all produced samples by MPN (Most Probable Number) and flow cytometry.
TABLE-US-00010 Sample a.sub.W RM% d.sub.50 Span Pellets 0.024 0.44 - - Milled pellets 0.075 1.26 243 .Math.m 1.985 Electrostatic spray dried 0.216 1.09 8.77 .Math.m 4.711
[0488] From the table above, it can be seen that the water activity was higher for the electrostatic spray dried powder than the pellets and milled pellets. It was also seen that the a.sub.w increased substantially during milling of the pellets. It was not possible to measure the mean particle size of the freeze dried pellets, because the pellets were too large for the Malvern Mastersizer 3000 analytical equipment.
Most Probable Number (MPN):
[0489] Growth was measured on the fermentate, concentrate, concentrate + drying protectant, frozen pellets, freeze dried pellets, milled pellets and electrostatic spray dried material.
[0490] The cell count pr. ml or cell count pr. gram of all the samples is an average of six analytical results.
TABLE-US-00011 Process step MPN [pr. mL or pr. g] Log.sub.10 MPN Standard deviation Fermenter A 6.2E+08 8.8 1.3E+08 Fermenter B 2.0E+07 7.3 1.3E+07 Concentrate 1.5E+10 10.2 1.6E+10 Conc. + drying protectant 4.6E+09 9.7 1.2E+09 Frozen pellets 8.5E+06 6.9 4.9E+06 Freeze dried pellets 1.1E+06 6.0 5.6E+05 Milled pellets 2.0E+04 4.3 1.8E+04 Electrostatic spray dried 1.8E+07 7.3 6.3E+06
[0491] From the MPN results it was concluded that the viability decreased by 2.8 log during pelletizing. In addition, the viability decreased 2.6 log during the milling step. The electrostatic spray dried powder had 0.4 log higher viability compared to the freeze dried pellets. The viability of the electrostatic spray dried material was 3.0 log higher compared to the milled pellets.
Flow Cytometry
[0492] Flow cytometry was measured on the fermentate, concentrate, concentrate with drying protectant, pelletized material, freeze dried material, milled pellets, and electrostatic spray dried material.
TABLE-US-00012 Sample Damaged Intermediate Intact Total Intact% cells/ml or cells/g Fermenter A 2.6(±0.3) E+07 9.1(±0.3) E+07 3.8(±0.0) E+08 5.0(±0.05) E+08 76.00 Fermenter B 4.2(±0.0) E+07 9.1(±0.0) E+07 3.8(±0.04) E+08 5.1(±0.04) E+08 74.51 Concentrate 2.9(±0.3) E+08 6.0(±0.5) E+08 2.0(±0.2) E+09 2.9(±0.3) E+09 68.97 Pellets (frozen) 4.3(±0.2) E+08 1.0(±0.0) E+09 2.8(±0.1) E+07 1.5(±0.02) E+09 1.87 Freeze dried pellets 4.0(±0.6) E+09 4.5(±0.4) E+08 6.1(±1.2) E+07 4.4(±0.6) E+09 1.39 Milled pellets 8.9(±0.1) E+09 4.5(±0.1) E+08 9.7(± 10) E+06 9.3(±0.2) E+09 0.10 Electrostatic spray dried 1.8(±0.2) E+09 3.2(±0.3) E+09 1.8(±0.0) E+07 5.0(±0.5) E+09 0.36
[0493] As can be seen from the table above, there was a high number of total E. hallii cells in all the analyzed samples. The number of total cells/g in the dried powders were comparable for all the produced powders, with the freeze dried pellets had the highest number of intact cells/g, followed by the electrostatic spray dried powder. There was a viability loss of 0.8 log during the milling of the freeze dried pellets, similar to the MPN analysis. The viability of the electrostatic spray dried powder was 0.8 log lower compared to the freeze dried pellets, but 0.3 log higher compared to the milled pellets.
EXAMPLE 6 PELLETIZING, FREEZE DRYING, MILLING AND ELECTROSTATIC SPRAY DRYING OF A MUCINIPHILA
[0494] The primary objective of this test was to characterize and compare electrostatic spray dried Bacteroides thetaiotaomicron with pelletized, freeze dried and milled A. muciniphila. The characterization of the products included measurements of the respective viabilities of the dried powder from each process.
Materials and Methods
[0495] Cross flow filtration was carried out identical to Example 3. Two separate fermenters were used. Pelletizing and Freeze Drying were carried out identical to Example 3. Electrostatic spray drying was carried out identical to Example 1.
Experimental Work
[0496] A culture of A. muciniphila was grown in a fermenter (Infors) using media as described in Gómez-Gallego et al., Benef Microbes, 2016 Sep;7(4):571-84. The fermentate was concentrated by Cross Flow Filtration in Fermenter A or Fermenter B as described below.
Fermenter A
[0497] Cross flow filtration (CFF) was initiated at pH = 5.80 and was performed with a fermentate temperature of 37° C. Total solids of the fermentate before concentration were 7.32% (Mettler Toledo: 105° C. - 1 mg/50 s). Cross flow filtration was slowly started with a feed flow of 280 ml/min. and within approximately 7 minutes the feed rate was gradually increased to 1680 ml/min. At a feed flow of 1680 ml/min., inlet pressure (P.sub.in) was 0.17 bar(g), TMP was 0.22 bar(g) and permeate flow (Q.sub.permeate) was 170 ml/min. and the shear rate was 2480 s.sup.-1.
[0498] After 27 minutes the back pressure valve was activated with a TMP setpoint of 0.30 bar(g). Corresponding P.sub.in was 0.26 bar(g), Q.sub.permeate = 165 ml/min. and shear rate = 2471 s.sup.-1. After 1 hour and 2 minutes the TMP setpoint was increased to 0.40 bar(g). Corresponding P.sub.in was 0.38 bar(g), Q.sub.permeate = 95 ml/min. and shear rate = 2595 s.sup.-1.
[0499] A concentration factor of 30x was achieved after approximately 1 hours and 26 minutes.
Fermenter B
[0500] Cross flow filtration (CFF) was initiated at pH = 5.77 and was performed with a fermentate temperature of 37° C. Cross flow filtration was slowly started with a feed flow of 280 ml/min. and within approximately 7 minutes the feed rate was gradually increased to 1680 ml/min. At a feed flow of 1680 ml/min., inlet pressure (P.sub.in) was 0.17 bar(g), TMP was 0.22 bar(g) and permeate flow (Q.sub.permeate) was 160 ml/min. and the shear rate was 2493 s.sup.-1. After 20 minutes the back pressure valve was activated with a TMP setpoint of 0.30 bar(g). Corresponding P.sub.in was 0.25 bar(g), Q.sub.permeate = 150 ml/min. and shear rate = 2509 s.sup.-1. After 56 minutes the TMP setpoint was increased to 0.40 bar(g). Corresponding P.sub.in was 0.37 bar(g), Q.sub.permeate = 98 ml/min. and shear rate = 2593 s.sup.-1. A concentration factor of 30x was achieved after approximately 1 hours and 28 minutes.
[0501] The collected concentrate from fermenter A and fermenter B was mixed, in total a concentration factor of 30.4x was reached. Total solids of concentrate was 9.85% (Mettler Toledo: 105° C. - 1 mg/50 s).
[0502] Additives, drying protectants, suitable for protecting microorganisms during cryogenic freezing were added to the suspension. These additives (sucrose in the present case) were added such that the ratio between the total solid content of the concentrate and total solid content of these additives were 1:4. TS of concentrate with drying protectant was 27.85% (Mettler Toledo: 60° C. - 1 mg/140 s).
Pelletizing and Freeze Drying
[0503] Concentrated A. muciniphila fermentate comprising drying protectants was pelletized and freeze dried. Pelletizing was performed without atomization gas and the feed rate was controlled by the Watson Marlow pump, which was set to 2 RPM, which corresponds to approximately 13.86 ml/min. The pelletized material was collected by a 50 .Math.m sieve from Retsch.
[0504] The collected pelletized material was transferred to an aluminum bag and kept cold on dry ice until it was transferred to the anaerobic glovebox and thereafter loaded to a freeze drier (Martin Christ). After 46 hours and 10 minutes the freeze drying was ended, and the freeze dried material was removed from the freeze drying tray.
[0505] The freeze dried material was loaded to a small aluminum bag, which subsequently was sealed by welding.
Properties of the Freeze Dried Pellets
[0506] Water activity (a.sub.w) = 0.089 [0507] Residual moisture = 0.25% (Mettler Toledo: 60° C. - 1 mg/140 s)
[0508] It was not possible to measure particle size distribution on the freeze dried pellets, because they were too big.
Milling of Freeze Dried Pellets
[0509] The milling was performed manually in a mortar for approximately 5 minutes.
Properties of the Milled Pellets
[0510] Water activity (a.sub.w) = 0.206 [0511] Residual moisture = 1.25% (Mettler Toledo: 60° C. - 1 mg/140 s) [0512] Mean particle size distribution (d.sub.50) = 808 .Math.m [0513] Span = 2.633
Electrostatic Spray Drying
[0514] A. muciniphila concentrate with drying protectant was electrostatic spray dried, during electrostatic spray drying the feed was kept under steering.
[0515] Electrostatic spray drying was performed with a main air flow of 25 m.sup.3/h N2.
[0516] The inlet temperature (T.sub.in) was 80° C. and the outlet temperature (T.sub.out) was approximately 38.5° C. Atomization of the feed was performed with a two-fluid nozzle, where the atomization pressure (P.sub.nozzle) was 2.0 bar(g) N2 and the atomization temperature was 50° C. The feed rate was controlled by a peristaltic pump with and RPM of 6, which corresponds to approximately 3.5 g/min.
[0517] A two-fluid nozzle was used. The electrostatic charge was applied to the suspension, by the use of an electrode in contact with the suspension, located inside the nozzle prior to the nozzle tip where the liquid suspension is atomized into droplets. Pulse Width Modulation (PWM) was used, wherein a 5 kV square pulse, with a pulse length of 1 s, was provided every third second, with a baseline of 1 kV.
[0518] After the test was ended, dried particles were collected at the outlet end of the drying chamber and analyzed.
Properties of the Electrostatic Spray Dried Product:
[0519] Water activity (a.sub.w) = 0.167 [0520] Residual moisture (R.sub.m%)= 2.01 % (Mettler Toledo: 60° C. - 1 mg/140 s) [0521] Mean particle size distribution (d.sub.50) = 6.63 .Math.m [0522] Span = 4.693
Results
Particle Analytics
[0523] For all the produced samples, residual moisture (R.sub.M%), water activity (a.sub.w), particle size distribution (d.sub.50 and span) was measured. Viability was also measured for all produced samples, by MPN (Most Probable Number) and flow cytometry.
TABLE-US-00013 Sample a.sub.w R.sub.M% d.sub.50 Span Pellets 0.089 0.25 - - Milled pellets 0.206 1.25 808 .Math.m 2.633 Electrostatic spray dried 0.167 2.01 6.63 .Math.m 4.693
[0524] The table above shows that the water activity increased significantly during milling of the pellets. It was not possible to measure the mean particle size of the freeze dried pellets, because the pellets were too large for the Malvern Mastersizer 3000 analytical equipment.
Most Probable Number (MPN):
[0525] Growth was measured on the fermentate, concentrate, concentrate + drying protectant, frozen pellets, freeze dried pellets, milled pellets and electrostatic spray dried material. The cell count pr. ml or cell count pr. gram of all the samples is an average of six analytical results.
TABLE-US-00014 Process step MPN [pr. mL or pr. g] Log.sub.10 MPN Standard deviation Fermenter A 6.5E+08 8.8 1.7E+08 Fermenter B 2.9E+08 8.5 0.0E+00 Concentrate 8.2E+09 9.9 4.0E+09 Conc. + drying protectant 1.0E+10 10.0 4.2E+09 Frozen pellets 4.2E+10 10.6 1.4E+09 Freeze dried pellets 3.1E+10 10.5 6.9E+09 Milled pellets 7.5E+10 10.9 3.0E+10 Electrostatic spray dried 9.6E+10 11.0 5.1E+10
[0526] From the MPN results in the table above, it can be seen that a high viability was achieved and that there was no viability loss during the freezing step. The viability of the dried powders; e.g. freeze dried pellets, milled pellets and electrostatic spray dried powder were in the same range. Electrostatic spray dried powder had the highest viability and the viability was 0.5 log higher compared to the powder produced by freeze drying of pellets.
Flow Cytometry
[0527] Flow cytometry was measured on the fermentate, concentrate, concentrate with drying protectant, pelletized material, freeze dried pellets, milled pellets and electrostatic spray dried material.
TABLE-US-00015 Sample Damaged Intermediate Intact Total Intact% cells/ml or cells/g Fermenter A 3.9(±1.2) E+07 2.9(±0.2) E+08 7.0(±0.1) E+09 7.3(±0.1) E+09 95.4 Fermenter B 7.8(±1.5 E+07 1.3(±0.06) E+08 4.1(±0.2) E+09 4.3(±0.2) E+09 95.2 Concentrate 1.3(±0.2) E+09 6.8(±0.5) E+09 1.3(±0.2) E+11 1.4(±0.2) E+11 94.3 Pellets (frozen) 1.0(±0.2) E+09 9.8(±0.6) E+09 1.1(±0.1) E+11 1.2(±0.1) E+11 90.9 Freeze dried pellets 2.3(±0.4) E+09 2.5(±0.4) E+10 1.4(±0.1) E+11 1.7(±0.1) E+11 84.2 Milled pellets 2.5(±0.1) E+09 2.7(±0.1) E+10 1.6(±0.05) E+11 1.9(±0.06) E+11 84.7 Electrostatic spray dried 3.5(±0.5) E+09 8.3(±1.7) E+09 2.4(±0.1) E+11 2.5(±0.1) E+11 95.3
[0528] As can be seen from the flow cytometry results, provided in the table above, there was a high number of intact A. muciniphila cells in all the analyzed samples. The number of total cells/g in the dried powders were quite high and they were comparable for all the produced powders, similar to the MPN analysis.
[0529] As in the MPN analysis it was seen that the number of intact cells of the dried powders; e.g. freeze dried pellets, milled pellets and electrostatic spray dried powder were in the same range. Electrostatic spray dried powder had the highest viability and the viability was 0.3 log higher compared to the powder produced from freeze drying of pellets.