Lyophilized composition for preserving microbiota in its ecosystem

Abstract

The invention relates to a lyophilized composition comprising a microbiota in its ecosystem, in particular stools, and a mixture of cryoprotectant agents comprising maltodextrin and trehalose, a capsule comprising said lyophilized composition, the use thereof, and methods for the production thereof.

Claims

1. A lyophilized composition comprising a collected microbiota and a mixture of cryoprotectants, wherein said mixture of cryoprotectants comprises maltodextrin and trehalose in a maltodextrin/trehalose (M/T) weight ratio ranging from 35/65 to 45/55, wherein said microbiota is faecal microbiota contained in stool, and wherein said lyophilized composition comprises at least one viable bacterial population that show extreme oxygen sensitivity (EOS) after at least 6 months of storage and wherein said microbiota is unchanged compared with the microbiota existing naturally in the body and further comprises at least bacterial metabolites.

2. The lyophilized composition according to claim 1 in which: (i) the collected microbiota is faecal microbiota contained in stool, (ii) the stool weight proportion represents 15 to 35% of the lyophilized composition and the weight proportion of the mixture of cryoprotectants represents 85 to 65% of the lyophilized composition, and (iii) maltodextrin (M) and trehalose (T) are in a maltodextrin/trehalose (M/T) weight ratio of 40/60.

3. A gastro-resistant capsule comprising a lyophilized composition as defined in claim 1.

4. A gastro-resistant capsule according to claim 3, wherein said lyophilized composition is mixed with at least one flow excipient.

5. A method of treatment of a disease or a disorder related to an imbalance of the intestinal microbiota, comprising administering the lyophilized composition according to claim 1 to a subject in need thereof.

6. The method according to claim 5, wherein the lyophilized composition is administered as a relay for an oral antibiotic treatment, and wherein the subject has recurrent Clostridium difficile infections.

7. A method for the preparation of a gastro-resistant capsule of claim 3 comprising the following steps: a. collecting the microbiota; b. diluting said microbiota with a mixture of cryoprotectants comprising maltodextrin and trehalose in an M/T weight ratio ranging from 35/65 to 45/65; c. lyophilizing the mixture achieved in step b to produce a lyophilizate; d. pulverizing the lyophilizate obtained in step c into powder form; e. optionally adding of one or more excipients improving the flow of the lyophilizate; f. filling the envelope of the gastro-resistant capsule with said lyophilized mixture; and g. sealing the capsules.

8. The method according to claim 7, wherein said microbiota is faecal microbiota contained in stool and the mixture of cryoprotectants consists of the mixture of maltodextrin (M) and trehalose (T) with an M/T weight ratio of 40/60.

9. A method of treatment of a disease or a disorder related to an imbalance of the intestinal microbiota, comprising administering the gastro-resistant capsule according to claim 3 to a subject in need thereof.

10. The method according to claim 9, wherein the gastro-resistant capsule is administered as a relay for an oral antibiotic treatment, and wherein the subject has recurrent Clostridium difficile infections.

11. A method for preserving a microbiota in a lyophilized form, comprising: contacting the microbiota with a mixture of cryoprotectants, wherein said mixture of cryoprotectants comprises maltodextrin (M) and trehalose (T) in an M/T weight ratio ranging between 35/65 and 45/55; and lyophilizing the microbiota wherein the microbiota is faecal microbiota contained in stool.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Representation of major bacterial groups and yeasts recovered after growth in native stools and diluted in NaCl 9% or within the maltodextrin cryoprotectant mixture comprising 6.7% maltodextrine and 10% trehalose (MT). The results shown in FIG. 1 correspond to those obtained for stool 3.

(2) FIG. 2: Representation for the original stool of the viable bacterial load after reconstitution of the lyophilizate depending on the nature of the cryoprotectant: 5% maltodextrin (M), 5% trehalose (T5) 10% trehalose (T10), mixture of 6.7% maltodextrin and 10% trehalose (MT) or milk (L).

(3) FIG. 3: Representation of the evolution of the bacterial load over time depending on the nature of the cryoprotectant: 5% trehalose (T5), 10% trehalose (T10) and mixture of 6.7% maltodextrin and 10% trehalose (MT). J0 represents pre-lyophilization; M0 is post-lyophilization; M4 is 4 months post-lyophilization and M7 is 7 months post-lyophilization.

(4) FIG. 4: Representation of the percentage of EOS bacteria at M0, depending on the nature of the cryoprotectant: maltodextrin 5% (M), trehalose 5% (T5), 10% trehalose (T10), mixture of 6.7% maltodextrin and 10% trehalose (MT). J0 represents pre-lyophilization; M0 is post-lyophilization.

(5) FIG. 5: Representation of the main bacterial groups found in the native stool and in the lyophilizates directly after their preparation, depending on the nature of the cryoprotectant: 5% trehalose (T5), 10% trehalose (T10) and mixture of 6.7% maltodextrin and 10% trehalose (MT). The results presented in FIG. 5 show those obtained for stool 3.

(6) FIG. 6: Representation of the main bacterial groups found in the native stool and in the lyophilizates after their preparation (M0), depending on the nature of the cryoprotectant: 5% trehalose (T5), 10% trehalose (T10) and mixture of 6.7% maltodextrin and 10% trehalose (MT). The results are presented immediately after lyophilization and after storage at 4° C. for 3 months (+3) and 8 months (+8). The results presented in FIG. 6 show those obtained for stool 3.

(7) FIG. 7: Representation of the impact of lyophilization (L72h) and the preservation of lyophilizates for 3 months (L3M) and 6 months (L6M) on the viability of the main bacterial groups in three stool samples (S4, S5, S6) compared with the native stool (NS). (A) Effect of different strictly anaerobic bacterial populations. (B) Effect on different optionally aerobic/anaerobic bacterial populations.

(8) FIG. 8: Representation of the impact of lyophilization (L72h) and the preservation of lyophilizates for 3 months (L3M) and 6 months (L6M) on the viability of bacteria extremely oxygen sensitive (EOS) in various stool samples (S4, S5, S6) compared with the native stool (NS).

(9) FIG. 9: Assessment of the evolution of bacterial metabolites by gas chromatography conducted on native stools (NS) and lyophilized stools (L72H).

(10) FIG. 10: Assessment of the anti-Clostridium difficile effect of native (S or NS) and lyophilized stools (L72h).

EXAMPLE 1

(11) 1. Materials and Methods

(12) 1.1. Faecal Sampling

(13) The selection of lyophilization conditions is based on the study of 3 stools provided by healthy independent volunteers and placed immediately after emission in the presence of a GENbagAnaer® system installed in the sample pot for incubation. The sample was sent straight to the laboratory after emission, within less than 1 hour. A fraction of each stool is preserved with GENbagAnaer® as a control for immediate analysis.

(14) 1.2. Choice of Cryoprotectants

(15) The cryoprotectants used for this study are: glycerol at 10% (G) reconstituted skimmed and pasteurized milk powder at 10% (L). maltodextrin, at a concentration of 5% (M). trehalose, at a concentration of 5% (T5) and 10% (T10). mixture of maltodextrin at a concentration of 6.7% and trehalose at a concentration of 10%, or in an M/T weight ratio of 40/60 (MT). NaCl solution at a concentration of 9% as a control, as it is generally used to dilute fresh faecal matter.

(16) Each solution was packaged in sterile pots and stored at +4° C. until use.

(17) 1.3. Preparation of the Faecal Lyophilizate

(18) The stools were diluted 10 times in the cryoprotectant solution and homogenized using an Ultraturrax®-type homogenizer fitted with a disposable stem. The suspended solutions were placed in lyophilization vials, with a volume corresponding to 3 ml for 300 mg of the native stool sample.

(19) For each condition tested, and for each of the 3 analysed stools, a suspension vial was immediately placed in the presence of a GENbagAnaer® system to assess the effect of different cryoprotectants on the viability of the bacteria in the microbiota of each diluted and non-lyophilized stool.

(20) The remaining vials are immediately placed at −80° C. After freezing for 30 minutes, the vials are placed in the lyophilizer (FreeZone®, 6 Liter, Freeze Dry System, Model 77530, Labcono Corporation, Kansas City, Mo., USA), in which the trap has already cooled for a 24-hour period under automatic cycle. At the end of this period of lyophilization, the vials are sealed under vacuum and secured with aluminium tops.

(21) For each stool and each condition of cryo-preservation, a series of vials is sent to a microbiology laboratory for analysis of the microbiota and 2 series are stored at 4° C. for stability studies for 3-4 months and 7-8 months.

(22) 1.4. In Vitro Studies of the Viability of the Microbiota Depending on Lyophilization Conditions

(23) The first step of the microbiological study was the analysis of non-lyophilized diluted stools to test the effect of the dilution in the presence of the different cryoprotectants on maintaining the diversity of the microbiota and its viability. For this purpose, a certain amount of the dilutions made with 10% milk (L), 10% glycerol (G), 5% maltodextrin (M), 5% trehalose (T) 10% trehalose (T10) and a mixture of 6.7% maltodextrin and 10% trehalose, i.e. an M/T weight ratio of 40/60 (MT) were immediately placed, after the preparation of the faecal lyophilizate, under anaerobiosis and microbial cultures were made immediately.

(24) The second step of the process related to the study of lyophilizates. Analyses were conducted during the week after the preparation, and then after 3-4 months and 7-8 months of storage at 4° C. Each lyophilizate is reconstituted with 3 ml of sterile peptone water, which corresponds to the volume of diluted stool initially present in each vial.

(25) For the analysis of strictly aerobic and anaerobic microbiota, the non-lyophilized stools and the reconstituted lyophilizates were diluted 10 to 10 and seeded on selective and non-selective media (Rougé et al., 2010). The media incubated aerobically and in an anaerobic unit (N.sub.2/CO.sub.2/H.sub.2:80/10/10) at 37° C. are shown in Table 1.

(26) TABLE-US-00001 TABLE 1 The media used for the analysis of the microbiota for each culture Searched elements of Incubation/ the microbiota Agar base reading Total anaerobic Colombia + cysteine (Cc) + 37° C. population blood + neomycine Anaerobic unit (100 mg/ml) Reading and Bacteroides sp., Cc + Blood + Kanamycine (100 counting Prevotella sp. mg/ml) + vancomycine at 48 hours (7 mg/ml) and 5 days Clostridium CC + blood + difficile Clostridium difficile medium (CLO-M Biomérieux) Other Clostridium CC + mupirocine (100 mg/ml) + full milk (5 ml) + neutral red (40 mg/ml) Bifidobacterium sp. WCB (Wilking-Chalgren for Bifidobacteria) + mupirocine (100 mg/ml) Lactobacillus sp. MRS (Nan, Rogosa, Sharpe agar) Total aerobic Trypticase soy 37° C. population Aerobiosis Enterobacteria Drigalski Reading at Enterococcus sp. Enterococcosel 24 hours Staphylococcus sp. Chapman 110 Yeast Sabouraud Chloramphenicol

(27) The quantification of bacteria extremely oxygen sensitive (EOS), such as bacteria of the Clostridium leptum group was conducted out by differential culture in the presence or absence of oxygen. For this purpose, fresh stool samples (TO) and the various lyophilized samples rehydrated in peptone water were successively diluted in a very rich no selective culture medium (YBHI corresponding to a brain-heart medium enriched with yeast extract supplemented with cellobiose, maltose and cysteine). This medium was developed to allow the culture of bacteria extremely difficult to cultivate bacteria, such as the Faecalibacterium prausnitzii. Once the ad hoc dilutions in strict anaerobic conditions were completed, their ability to resist or not to the presence of oxygen was assessed by parallel cultures in 2 dishes, one of which was subjected to an aerobic atmosphere for 60 minutes while the other was not. This method allows to evaluate the percentage of strictly viable anaerobic bacteria in different samples at different times after lyophilization.

(28) 2. Results

(29) 2.1. Effect of Different Cryoprotectants on the Diversity and Viability of the Microbiota Before Lyophilization.

(30) A stool dilution in the various assessed cryoprotectants, and in particular in the MT mixture, had no significant impact on bacterial load, and didn't influence the equilibrium of the different main genera of cultivable microbiota, compared with the native stool or the stool diluted in 9% NaCl (which corresponds to the preparation used for FMT with fresh stools) (FIG. 1). The major groups of the microbiota remain viable.

(31) 2.2. Effect of Lyophilization on the Viability of the Microbiota Based on the Cryoprotectants Used

(32) The total bacterial load was assessed during the week following lyophilization, or at MO. The results are presented in FIG. 2.

(33) The three cryoprotectants tested, namely a 5% trehalose solution (T5), a 10% trehalose solution (T10) and a mixture of 6.7% maltodextrin and 10% trehalose (TM), allow to provide a stability greater than 90% of the total bacterial load of cultivatable bacteria during lyophilization; this result corresponds to a loss of about 1 log compared with the existing load in a native stool, or a stool diluted in 9% NaCl before lyophilization.

(34) The total bacterial load measured up to 7 months post-lyophilization has not change much (FIG. 3).

(35) Furthermore, these three cryoprotectants allow efficient preservation of EOS bacteria during lyophilization (FIG. 4).

(36) 2.3. Effect of Lyophilization on the Diversity of the Microbiota Depending on the Different Cryoprotectants Used

(37) Among the three cryoprotectants tested above, the cryoprotectant mixture of 6.7% maltodextrin and 10% trehalose (or in a weight ratio of 40/60) is the one that gives the best results maintaining the main genera constituting the faecal microbiota alive, despite a decrease in certain strict anaerobic genera of about 1 to 2 log, for stool 3 (FIG. 5) (and up to 4 log for other stools—not shown). Conversely, the 10% glycerol solution is not very protective both for aerobic genera and anaerobic genera (result not shown).

(38) Strictly anaerobic genera (Clostridium sp. and Bacteroides sp.) remain dominant when the study is conducted within one week after lyophilization (FIG. 6). Preservation at 4° C. of the lyophilizate has little impact, and shows an additional loss of around 1.5 log for Bacteroides sp. after 3 months of storage at 4° C. and a loss of 1 log at 8 months for Clostridium sp. (FIG. 6). In any case, the dominant genera of the microbiota remain dominant during the preservation.

(39) The results obtained with stool 2, rich in Bifidobacterium sp. showed the stability of that group, for up to 6 months post-lyophilization in the MT mixture (result not shown).

(40) Taken together, these results show that the MT mixture is the one that allows the best preservation of lyophilized faecal microbiota, both in terms of viability and of diversity and for a period of up to 6 months after lyophilization.

EXAMPLE 2

(41) 1. Materials and Methods

(42) Pursuant to the method described in example 1, three additional faecal samples identified as S4, S5 and S6 were diluted in the presence of a cryoprotectant mixture of 6.7% maltodextrin and 10% trehalose (or in an M/T weight ratio of 40/60) and then lyophilized.

(43) 2. Results

(44) 2.1. Stability of the Bacterial Ecosystem in Lyophilizates After 6 Months of Storage

(45) 2.1.1. Analysis of the Bacterial Viability and Diversity in Lyophilizates After 6 Months of Storage

(46) The strictly anaerobic population was analysed based on the count of representative bacteria groups: Bifidobacterium spp. and Bacteroides spp.

(47) Regarding the Bifidobacterium spp. genus, it remains an integral part of the dominant microbiota and no significant changes during the 6-month preservation period.

(48) For the Bacteroides spp., genus, there were inter-individual variations resulting in a loss of about 1.5 to 4 log over a 6 month-period.

(49) The optional aerobic-anaerobic populations from the different samples were analysed based on the count of bacterial groups Lactobacillus spp., Enterococcus spp. and Enterobacteriaceae. The stability and viability of these genera and bacterial groups post lyophilization throughout the 6-month preservation period are confirmed.

(50) These results show that the lyophilization process mostly retains the viability of the various target bacterial populations without inducing changes in the overall balance between the major bacterial groups, and that preservation remains stable over a 6-month preservation period (FIG. 7).

(51) 2.1.2. Study of Bacteria Extremely Oxygen Sensitive

(52) One of the specific features of faecal microbiota is the presence of bacteria extremely oxygen sensitive (EOS) that can be affected by the lyophilization process.

(53) These EOS bacteria are detected both for the native stool and in faecal lyophilizates in all analysed samples (FIG. 8). When these EOS bacteria are expressed as a percentage relative to the total quantity of anaerobic bacteria (CFU/ml), the level remains stable throughout the preservation period (3 to 6 months depending on the sample) and the quantity of strictly anaerobic bacteria is not affected by the preservation process. These results show that the lyophilization does not affect bacterial populations that show extreme oxygen sensitivity.

(54) 2.2. Stability of the Functions of Bacterial Ecosystem in the Lyophilizates

(55) 2.2.1. Detection of Metabolites in the Lyophilizates

(56) The production of bacterial metabolites, i.e. short-chain fatty acids, was assessed in the 72 h lyophilizate compared with the native stool. Out of 3 analysed samples, lyophilization induces preservation of fermentation products with the concentration of short-chain fatty acids increased 2 to 4 fold for acetate and propionate, depending on the stool samples, and 2 to 6 fold for butyrate. Bacterial metabolites, although volatile, are present in the lyophilizates. The increase of their concentration would be a consequence of lyophilization that causes a concentration of the metabolites (FIG. 9).

(57) 2.2.2. Detection of the Anti-Clostridium difficile Effect in the Lyophilizates

(58) The anti-Clostridium difficile effect in native stools and in lyophilizates was analysed in vitro in the presence of two strains of Clostridium difficile, including one toxinogen strain (strain 630) and one non-toxinogen strain (strain PCD1). When the growth of the strains is inhibited, a pale circle appears around the inoculation area: this is the inoculation circle.

(59) Various controls, known for their anti-Clostridium difficile effects, were systematically used as positive controls. Acetic acid (AA) as well as lactic acid (AL) was therefore used at different concentrations (300, 400, 500 mM). In each dish, an inhibition circle was observed in the presence of acetic acid and/or lactic acid.

(60) Pure strains were used as control, i.e. a Lactobacillus strain and a Bifidobacterium (CB) strain, which do not cause inhibition circles to appear.

(61) In the presence of native stools (S or NS) and in the presence of stools that were frozen at −80° C. for 72 hours (T1, T2, and T3), no inhibition circle is visible whereas an inhibition circle is visible in the presence of lyophilizates (FIG. 10).

(62) The results of these various experiments reveal the presence of an inhibition area for acetic acid and lactic acid, and for the lyophilizates of all of the analysed stools. The inhibitory capacity of in vitro development of C. difficile by a stool is therefore potentiated by lyophilization.

(63) All the results shown in example 2 confirm the stability and viability of the microbiota in the lyophilizate over a period of at least 6 months and that the functional capacities of the bacteria are maintained within the lyophilizate, in particular with the preservation of bacterial metabolites of interest and an anti-Clostridium difficile effect in an in vitro study model.

EXAMPLE 3

(64) 1. Materials and Methods

(65) 1.1. Stool Lyophilizates

(66) Four stools from healthy donors were processed with the method described in example 1 for the purpose of obtaining four faecal lyophilizates: S4, S5, S6 and S7, where S7 representing an additional faecal sample compared with example 2.

(67) 1.2. Choice of the Excipients

(68) Diluents

(69) The selection of diluents is based on their inert nature and, in particular, on the absence of specific intestinal effects.

(70) The selected diluents are a mineral excipient, dicalcic phosphate (Anhydrous Encompress®, JRS Pharma, Rosenberg, Germany) and nebulized maltodextrins (Emdex®, JRS Pharma Rosenberg Germany) where the latter type of excipient is already in the composition of the lyophilizate. The density and the flow time of these both pure excipients are respectively of 0.79 g/mL and 3 seconds 18/100 g for anhydrous Encompress®, and 0.68 g/mL and 6 seconds 03/100 g for Emdex®.

(71) Lubricants

(72) Three flow lubricants were selected: talcum (Cooper, France), hydrophilic colloidal silica (Aérosil® 200 Pharma, Kraemer & Martin GmbH, St-Augustin, Germany), and a hydrophobic colloidal silica (Aérosil®, R972, Degussa-Hüls, Courbevoie, France).

(73) 1.3. Measuring the Flow Time and Mixing the Lyophilizates to the Excipients

(74) This test was conducted according to monograph 2.9.16 of the European Pharmacopoeia, first on the lyophilizates alone, and then after inclusion of a flow lubricant used at 0.5% and, when necessary, of a diluent tested in the 75/25, 50/50 and 25/75 weight ratio. These mixtures were achieved manually.

(75) The density of the final mixtures featuring a good fluidity was measured for all the mixtures, before being reported by calculation to 1 mL.

(76) 1.4. Making Capsules with the Mixtures Featuring a Good Flow.

(77) Nº100 capsules associated with a significant volume of 0.95 mL and of a size compatible xith relatively easy ingestion (23.3 mm) in length) were selected. They were filled manually with a capsule filler and mixtures featuring a good flow.

(78) 2. Results

(79) 2.1. Study of the Flow Characteristics and the Density of the Lyophilizates

(80) Table 1 shows the results of the tests conducted on the lyophilizates

(81) TABLE-US-00002 TABLE 1 Flow of raw faecal lyophilizates, and with mixtures containing a flow lubricant and/or diluent, and density of the final volumes. FLOW (AVERAGE OUT OF 3 WEIGHT MEASUREMENTS TESTED MIXTURES RATIO FOR X G) DENSITY S4 Raw faecal lyophilizate Infinite Not determined Faecal 99.5/0.5  Infinite lyophilizate/talcum Faecal lyophilizate- 75/25 Infinite talcum*/Emdex ® 50/50 4s16 (taps) 25/75 0s69 for 13.70 g 13.70 g/26 mL (0s69/0s68/0s71) (0.53 g/mL  S5 Raw faecal lyophilizate — Infinite Not determined Faecal 99.5/0.5  Infinite lyophilizate/Aérosil ® 200 Faecal 75/25 0s84/0s37/Infinite lyophilizate/Aérosil ® (taps) 200*/Anhydrous 50/50 0s53/0s31/Infinite Emcompress ® 25/75 0s57 for 10.86 g 10.86 g/17 mL (0s56/0s48/0s68) (0.64 g/mL) S6 Raw faecal lyophilizate — Infinite Not determined Faecal 99.5/0.5  0s30 fir 1.466 g  1.466 g/4.5 mL lyophilizate/Aérosil ® (0s35/0s25/0s31) (0.33 g/mL) R927 S7 Raw faecal lyophilizate — Infinite Not determined Lyophilizate/Aérosil ® 99.5/0.5  0s26 for 3.296 g 3.296 g/7 mL  R927 (0s25/0s25/0s27) (0.47 g/mL) *at a weight ratio of 99.5/0.5

(82) None of the tested raw faecal lyophilizates tested alone has any flow properties. It is therefore impossible to put them in capsules as they are.

(83) Despite their role of flow lubricants, talcum and Aérosil® 200 used at 0.5% did not allow to improve the fluidity of the lyophilizates (S5 and S5).

(84) The Emdex® and anhydrous Emcompress® diluents are efficient in improving the flow. The improvement occurs with the ratio 50/50 for the lyophilizate S4-talcum/Emdex® mixtures, and with the ratio 75/25 for the lyophilizate S5-Aérosil® 200/anhydrous Emcompress® mixture. At a ratio of 25/75 between the lyophilizate and the diluent, the mixture becomes fluid with a flow time of 0s69 for 13.70 g (S4) and 0s57 for 10.86 g (S5).

(85) Finally, the Aérosil® R972 flow lubricant used alone at 0.5% has led to a significantly improvement of the fluidity of the lyophilizates S6 (0s30 for 1.466 g) and S7 (0s26 for 3.296 g).

(86) The density of the mixtures ranges from 0.53 to 0.64 g/mL for “lyophilizate/flow lubricant/diluent” mixtures and from 0.33 to 0.47 g/mL for the “lyophilizate/flow lubricant” mixtures.

(87) 2.2. Making Capsules of the Mixtures

(88) Table 2 shows the number of prepared capsules for each sample, and the mass of the lyophilizate integrated in each capsule. The theoretical mass of fresh matter initially implemented is reported in the last column.

(89) TABLE-US-00003 TABLE 2 Characteristics of capsules prepared with lyophilized samples. THEORETICAL NUMBER OF MASS OF FRESH PREPARED MIXTURE LYOPHILIZATE MATTER IN n CAPSULES MASS/CAPSULE MASS/CAPSULE CAPSULES S4 - talcum/ 20 500 mg 125 mg 1754 mg Emdex ® S5 - Aérosil ® 200/ 18 603 mg 151 mg 3000 mg Anhydrous Emcompress ® S6 - Aérosil ® 5 293 mg 293 mg 1792 mg R972 S7 - Aérosil ® 7 471 mg 471 mg 5000 mg R972

(90) The case of samples S6 and S7 is the most favourable since the dilution of the lyophilizate is insignificant, because only 0.5% flow lubricant was added. However, there is a rather important variability observed between two samples that, with the same volume, can represent 293 mg (S6) or 471 mg (S7) of dry matter. This stems from the density difference between the two samples.

(91) With regard to samples S4 and S5, the dilution of the lyophilizate is more important because of the adding of 75% diluent, in addition to the 0.5% of flow lubricant. With the same volume, the mass of the lyophilizate varies from 125 mg (S4) to 151 mg (S5) of dry matter, with respect to the density of each sample.

(92) This study shows that powdered faecal microbiota lyophilizates do not have a fluidity property and therefore can not be put into capsules alone.

(93) However, it is possible to improve the flow properties of the lyophilizates by mixing, either with 75% diluent (Emdex® maltodextrins, anhydrous dicalcic phosphate, anhydrous Emcompress®) or with 0.5% of flow lubricant (hydrophobic colloidal silica, Aérosil® R972).

BIBLIOGRAPHY

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