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    • 专利摘要:
    The present invention relates to a method for preserving a sample of a set of aerobic and anaerobic bacteria other than lactic acid bacteria, in particular bacteria of the intestinal microbiota, preferably a sample of human stool, and a lyophilizate obtained by the process said process comprising the following successive steps wherein: a) a said sample of a set of said bacteria is prepared by dilution in an aqueous storage medium comprising a combination of protective agents comprising (a1) at least one antioxidant compound and (a2) at least one sugar, and (a3) milk proteins, and b) freezing said sample recovered in step b) in liquid nitrogen at about -196 ° C for no more than 1 minute, preferably for 30 to 50 seconds, and c) freeze-drying said sample thus frozen to obtain a lyophilisate.
    公开号:FR3067928A1
    申请号:FR1755796
    申请日:2017-06-23
    公开日:2018-12-28
    发明作者:Eric Chabriere；Didier Raoult；Jean-Christophe Lagier；Sara Bellali；Saber KHELAIFIA；Camille Gouiran
    申请人:Aix Marseille Universite；Centre National de la Recherche Scientifique CNRS；Institut National de la Sante et de la Recherche Medicale INSERM；Assistance Publique Hopitaux de Marseille APHM；Fondation Mediterranee Infection；
    IPC主号:
    专利说明:

    Method for preserving a sample of bacteria
    The present invention relates to a method for preserving a sample of a set of aerobic and anaerobic bacteria other than lactic acid bacteria, in particular bacteria of the intestinal microbiota, more particularly still a sample of human stool, and a lyophilisate obtained.
    Human stool is characterized by the presence of an intestinal microbiota comprising all of the microorganisms in the human digestive tract, in particular aerobic bacteria and strict anaerobic bacteria and methanogenic archaea such as Methanobrevibacter smithii, the latter being the most difficult to keep.
    The most efficient method to date of stool samples preserving the viability of bacteria is freezing, but this method of conservation is impractical and expensive because of the need to maintain the cold chain during transport and storage.
    Freeze-drying aims to remove the water contained in a product by freezing followed by sublimation to facilitate its conservation. The frozen products are thus dried under vacuum, without thawing.
    The lyophilization of human stools allows storage under conditions which avoid the cold chain and which allows them to be put into capsules to be administered for therapeutic use, in particular in the context of probiotic treatments or intestinal microbiota transplants by which microbes are absorbed. a saddle of a healthy subject by a patient with colitis or an infection by a multi-resistant bacteria such as Clostridium difficile. Ideally, an autologous fecal sample is taken from the patient before medical treatment, for example before an operation or a risky situation. The sample is kept. If the patient develops C. difficile infection, then the sample is extracted into a saline solution and filtered. The filtrate is lyophilized. The resulting lyophilisate (powder) is put into the form of enteric-coated capsules (resistant to stomach stomach acid) to restore the patient's anterior colic flora by competing with C. difficile. This procedure avoids the risk of introducing another pathogen from a donor who is not the patient. It also avoids the problem of administration by nasal tube to reach the patient's duodenum.
    Lyophilization, however, leads to the destruction or reduction of the viability of a considerable number of bacteria with a bacterial mortality ranging from 90% to 99.99% depending on their aerotolerant or aero-intolerant nature.
    There are known methods for lyophilizing bacteria aimed at improving their viability with protective agents, but these vary according to the type of bacteria and do not relate to the lyophilization of clinical stool samples.
    Lyophilization is a cold drying process generally used only for bacterial samples of bacteria very sensitive to heat such as lactic acid bacteria. In WO 2003/018778, the viability of lyophilized lactic acid bacteria is improved by adding protective agents comprising:
    - an antioxidant compound, and
    - a compound increasing the glass transition temperature of the lyophilisate, namely glycerol or a carbohydrate such as lactose, galactose, maltose, sucrose and glucose.
    According to the present invention, it has been demonstrated that these conditions are not sufficient to sufficiently improve the viability of bacteria of the entire intestinal microbiota, in particular in clinical samples of human stool.
    The object of the present invention is to provide a method for preserving the maximum viability of bacteria of the intestinal microbiota in human stool.
    According to the present invention, common single lyophilization conditions have been discovered in order to multiply by at least 100 the number of aerobic bacteria as well as viable anaerobic bacteria present in a sample of a set of said aerobic and anaerobic bacteria other than lactic acid bacteria, in particular a sample of human stool after lyophilization in comparison with the standard lyophilization process.
    More precisely, according to the present invention, these lyophilization conditions consist in:
    on the one hand, dilute the bacterial samples, in particular a stool sample in an aqueous preservation medium comprising a specific combination of protective agent (s), and
    - on the other hand, implement an initial very rapid freezing step in liquid nitrogen at about -196 ° C before carrying out the lyophilization process. The lyophilization process involves freezing followed by a dehydration step by sublimation. These conditions made it possible to obtain the best viability results compared with known methods of preservation by freezing at 80 ° C. or of lyophilization by simple addition of conventional lyoprotective agent.
    While rapidly frozen cells generally have a lower survival rate than slowly frozen cells, according to the present invention, it has surprisingly been discovered that - subject to compliance with a specific preservation medium referred to above, rapid freezing in the nitrogen (-196 ° C) increases the survival rate of said bacteria, in particular the microbiota of human stool by a factor of at least 100 compared to a slow freezing at -80 ° C before lyophilization.
    To do this, the present invention provides a method of preserving a sample of a set of aerobic and anaerobic bacteria other than lactic acid bacteria, in particular bacteria of the intestinal microbiota, comprising the following successive steps in which:
    a) a said sample of a set of said bacteria is prepared by dilution in an aqueous preservation medium comprising at least milk proteins, and
    b) freezing of said sample recovered in step b) in liquid nitrogen at approximately -196 ° C for not more than 2 minutes, preferably for 30 to 50 seconds, and
    c) lyophilization of said sample thus frozen is carried out in order to obtain a lyophilisate.
    In step b), said sample recovered in step b) is frozen in a container, by immersion of the container containing said sample in liquid nitrogen.
    In step c), freeze-drying involves sublimation consisting in dehydrating the frozen product by drying the ice in a vacuum under cold conditions without melting it, the water sublimes, that is to say that it passes directly from solid to gaseous state. This step therefore essentially consists here of establishing a high vacuum.
    More particularly, in step c) the frozen sample is placed in a lyophilizer for at least 12 hours at a temperature of at least 0 ° C. and at a high vacuum pressure of not more than 1 mbar.
    More particularly, in step a), a so-called clinical stool sample is prepared by diluting said stool in an aqueous preservation medium comprising at least milk proteins, and the dispersion obtained is filtered to at least remove the debris therefrom. larger than 2mm.
    Preferably, in step a), said sample is diluted in an aqueous preservation medium comprising at least the triple combination (al) of at least one antioxidant compound and (a2) two different sugars, and (a3) proteins. of milk.
    More particularly, the milk proteins are in the form of whole milk, preferably at a concentration by weight in the said storage medium of at least 5%, preferably 10%.
    More particularly still, said protective agents comprise a combination of antioxidant compounds chosen from ascorbic acid, uric acid and glutathione.
    More particularly still, said protective agents comprise a combination of antioxidant compounds consisting of ascorbic acid at a concentration of lg / L, uric acid at a concentration of 0.4g / L and glutathione at a concentration of 0, lg / L.
    More particularly still, said protective agents comprise at least one sugar chosen from sucrose (also referred to below as "sucrose") and trehalose.
    More particularly still, said protective agents comprise sucrose at a concentration by weight in the said preservation medium of at least 5%, preferably 10%.
    More particularly still, the said preservation medium is a buffer medium with a pH of 7 to 7.5 comprising in water the salts KCL, CaCl2, MgCl2, KH2PO4, Na2HPO4, NaCL and KOH as well as at least one said protective agent.
    More particularly still, the said conservation medium comprises:
    (al) a combination of antioxidant compounds consisting of ascorbic acid at a concentration of lg / L, uric acid at a concentration of 0.4g / L and glutathione at a concentration of 0, lg / L, and (a2) sucrose at a weight concentration in the said storage medium of 5%, and trehalose at a weight concentration in the said storage medium 10%, and (a3) milk proteins at a weight concentration in the says 10% conservation medium.
    More particularly still, the said sample comprises at least the following aerobic and anaerobic bacteria: Staphylococcus au reus, Enterobacter aerogenes, Escherichia coii, Klebsiella oxytoca, Streptococcus aga / actiae, Serratia marcescens, P rote us mirabilis, Staphylococcus epidermidis, Bacii Streptococcus pneumoniae, Staphylococcus hominis and Acinetobacter bau Tuesday.
    More particularly still, the said sample includes at least the following strict anaerobic bacteria: Bacteroides fragiiis, Bacteroides nordii, Bacteroides thetaiotaomicron, Ciostidium butyricum, Clostridium massiiioamazoniensis, Clostridium beijerinckii, Clostridium irreguiar Finegoidia magnaid avid, Magniidium magnaid avid, Magniidium magnaimidium, Propionidium magnaimidium, Propionidium magnioma avionidae
    The present invention therefore also relates to a lyophilisate of sample of a set of aerobic and anaerobic bacteria other than lactic acid bacteria, in particular bacteria of the intestinal microbiota, more particularly still a sample of human stool, obtained by a method according to the invention. invention as defined above, characterized in that it comprises:
    - at least one protective agent consisting of milk proteins and preferably at least the combination of protective agents consisting in (a1) at least one antioxidant compound and (a2) at least one sugar, and (a3) milk proteins , and
    - an amount of bacteria from the intestinal microbiota, preferably in number:
    - at least 10 8 CFU / mL bacteria of each of the categories of aerobic bacteria and anaerobic bacteria respectively cultured after 48 hours of storage at 4 ° C, and
    - at least 10 8 CFU / mL bacteria from each of the categories of aerobic bacteria and anaerobic bacteria respectively cultured after 6 weeks of storage at 4 ° C.
    Other characteristics and advantages of the present invention will emerge more clearly on reading the detailed description which follows, given in an illustrative and nonlimiting manner, with reference to the appended drawings in which:
    - Figures IA and IB illustrate the results of differences in the quantity of live bacteria detected by the differences in log numbers of CFUs of aerobic bacteria (Figure IA) and anaerobic bacteria (Figure IB) after different freezing protocols used P1, P2 and P3 on stool samples without a protective agent in the storage medium;
    FIGS. 2A and 2B illustrate the results of the quantity of living bacteria detected anaerobically at 48 hours (FIG. 2A) and anaerobically after 6 weeks of storage of the lyophilisate at 4 ° C. (FIG. 2B), by the log CFU / mL, on stool samples with different conditions of storage media: A = fresh stool in saline water, B = lyophilisate in PBS medium, C = Lyophilisate with PBS medium containing antioxidants only, D = Lyophilisate with PBS medium containing powdered milk alone, E = lyophilisate with PBS medium containing antioxidants + sucrose + powdered milk, F = freezing of fresh saddle in PBS medium without protective agent;
    FIGS. 3A and 3B illustrate the results of the quantity of bacteria detected aerobically at 48 hours (FIG. 3A) and aerobically after 6 weeks of storage of the lyophilisate at 4 ° C. (FIG. 3B), by the log CFU / mL, on stool samples with the same media conditions A, B, C, D, E and F;
    - Figure 4 represents graphs illustrate the results of quantity of living bacteria detected in a sample after lyophilization of a cocktail of 11 anaerobic bacteria by the CFU / ml log with the same conditions of conservation medium A, B, C, D , E and F;
    - Figures 5A and 5B illustrate the results of differences in the amount of live bacteria detected by the differences in log numbers of CFU of aerobic bacteria (Figure 5A) and anaerobic bacteria (Figure 5B) on stool samples with different protective agents n ° 1 to 15 in the conservation medium;
    - Figures 6A and 6B illustrate the comparative results of differences in the quantity of live bacteria detected by the differences in log numbers of CFU of aerobic bacteria (Figure 6A) and anaerobic bacteria (Figure 6B) on stool samples under 4 conditions 1 to 4 with different protective agents No. 14 and 15 according to two different conditions of freezing before lyophilization.
    In FIGS. 5A-5B and 6A-6B, the cases "0" on the abscissa correspond to the results on fresh stools.
    A) MATERIALS AND METHODS
    1-Bacterial Ion s tested
    Faeces samples were collected from healthy donors, that is, individuals not suffering from infection, who had not taken an antibiotic in the month prior to sampling, and who were not suffering from known gastrointestinal pathology.
    In parallel, 29 bacterial strains of the digestive flora were selected from the international collection of microorganisms
    CSUR (Rickettsia Unity Strain Collection) from URMITE (WDCM 875) (Table 1 below).
    The strains of bacteria are stored in a form suitable for subsequent freezing in tubes containing freezing broth containing glycerol, porous beads called cryobeads.
    Table 1: list of the 29 reference bacterial strains
    Bacterium Atmosphere CSUR N ° Bacteroides nordii anaerobic P1040 Propionibacterium avidum anaerobic P872 Clostridum irregular anaerobic P1913 Clostridum massilioamazoniensis anaerobic P1360 Clostridum butyricum anaerobic P607 Clostridum beijerinckii anaerobic P883 Bacteroides thetaiotaomicron anaerobic P766 Propionibacterium acnes anaerobic P742 Finegoldia magna anaerobic P588 Haemophilus influenzae anaerobic P1075 Bacteroides fragilis anaerobic P863 Staphylococcus aureus aerobics P749 Enterobacter aerogenes aerobics P989 Escherichia coli aerobics P729 Klebsiella oxytoca aerobics P992 Streptococcus agalactiae aerobics P1030 Serratia marcescens aerobics P833 Enterococcus faecalis aerobics P720
    Proteus mirabilis aerobics P2049 Pseudomonas aeruginosa aerobics P211 Streptococcus mitis aerobics P1123 Staphylococcus epidermidis aerobics P748 Morganella morganii aerobics P752 Citrobacter freundii aerobics P731 Enterobacter cloacae aerobics P713 Bacillus circulans aerobics P655 Streptococcus pneumoniae aerobics P825 Staphylococcus hominis aerobics P842 Acinetobacter baumanii aerobics P981
    2-Experimental freezing / freeze-drying protocol
    g. stool in 150cm3 in an aqueous liquid conservation medium using a mixer (BOSCH Ultracompact 400W device) for 10 minutes. This sample is then sieved using a dedicated food sieve (with 2mm pores) to remove food debris and the filtrate is collected in glass vials containing conservation medium (2mL) (shell vial type, REF 223683 , Wheaton Millville, New Jersey, USA).
    Samples of bacterial strains are similarly stored in glass vials containing conservation medium (2 ml) (shell vial type, REF 223683, Wheaton Millville, New Jersey, USA). The samples were then frozen either for 5 at -80 ° C or in liquid nitrogen for 30 sec to 2 min, then placed in a DELTA 1-24 LSC freeze dryer (Martin Christ Gefriertrocknungsanlagen GmbH, DE) for 36 h at a temperature from 0 ° C, and at a reduced pressure of 0.63 mbar. The lyophilisates obtained were stored at room temperature.
    3-Protocol of viability studies
    3.1 Enumeration
    The viability of the microorganisms was estimated by the method of Colony Forming Units (CFU) after decimal dilutions and spreading of stool samples and samples of bacterial strains tested on Columbia agar COS agars (BioMerieux, Craponne France). The dilution is expressed in CFU / mL. For each dilution, three Petri dishes are inoculated and the dishes are incubated at 37 ° C for 48 hours under two conditions: aerobic and anaerobic (anaerobic pregnant or anaerobic generators).
    This work was carried out from:
    - Fresh bacterial samples and fresh stool samples, and
    - Lyophilisates rehydrated in sodium diphosphate buffer (PBS) at 25 ° C.
    The results obtained from fresh stools (OJ of the program) are used as a reference and compared to those obtained from the same sample previously frozen in different storage media and then, if necessary, lyophilized.
    The total number of viable microorganisms was evaluated after aerobic and anaerobic culture on petri dishes, before and after lyophilization. The loss of viable microorganisms compared to fresh stool is expressed as the logarithmic difference in CFUs lost after the various freezing protocols used throughout the experiment.
    3.2 Identification of bacteria
    In order to identify the bacteria which have survived the lyophilization process, the colonies thus cultivated are identified exhaustively by MALDI-TOF mass spectrometry using a MICROFLEX mass spectrometer (Bruker Daltonics). Each deposit is covered with 2 ml of matrix solution (α-saturated cyano-4hydroxyciannamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid) as described in the literature (ref.l). This analysis is performed using according to the manufacturer's recommendations. The spectra thus generated are compared with the database of the Bruker base combined with the specific base of the laboratory of La Timone hospital. An isolate is considered correctly identified by MALDITOF if its identification score is greater than 1.9. For the unidentified colonies, after 3 passages in MALDI-TOF, sequencing of the 16S RNA is carried out (ref. 2).
    (ref.l): Seng P, Drancourt M, Gouriet F, La Scola B, Fournier P-E, Rolain JM, et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis Off Publ Infect Dis Soc Am. 2009 Aug 15; 49 (4): 543-51.
    (ref.2): Morel AS, Dubourg G, Edouard S, Prudent E, Gouriet F, Casalta J et al. 2014. Complementarity between targeted real-time specifies PCR and conventional broad-range 16S rDNA PCR in the syndrome-driven diagnosis of infectious diseases. Eur J Clin Microbiol Infect Dis 34: 561-570.
    4) Conservation media
    Several aqueous pre-lyophilization preservation media have been tested to increase the survival of microorganisms during drying. Different combinations of protective agents have been used (Table 2 below). The samples diluted in the preservation medium are then frozen for 12 hours, then lyophilized.
    Various combinations of the following protective agents have been tested:
    - glycerol alone,
    - antioxidants alone,
    - milk only,
    - binary combinations: sugar + antioxidants, milk + sugar, milk + antioxidants, antioxidants + glycerol, sugar + glycerol,
    - the ternary combination: milk + antioxidants + sugar.
    After various comparative tests, the most effective antioxidant compounds selected included the following ternary combination:
    - 0.4g / L of uric acid (S / ÿ / 77a-Aldrich, Lyon, France),
    - Ig / L of ascorbic acid (Inresa Medical, Bartenheim, France),
    - 0.1g / L of L-Glutathion (S / ÿ / 77a-Aldrich, Lyon, France),
    The protective agents were used in saline water or saline water + sodium diphosphate buffer PBS (see table 3 below).
    The saline water included:
    - 0.3 / 0.6g KOH (BDH LABORATORY SUPPLIES, Poole, United Kingdom),
    - 0.1g CaCI2 (Inresa Medical, Bartenheim, France),
    - 0.1g MgCI2 (Inresa Medical, Bartenheim, France)
    The milk tested was skim milk or whole milk powder. The best results were obtained with whole milk powder (Régilait, Lyon, France) at a weight concentration of 10%.
    Different sugars have been tested (lactose, glucose, trehalose and sucrose) at different concentrations. The best results have been obtained with sucrose (also referred to below as "sucrose") and / or trehalose (Inresa Medical, Bartenheim, France) at a concentration by weight of 5 to 10%.
    Table 2: composition of the conservation media tested
    Middleofretention Composition 1 150 mL saline water 2 150 mL PBS + 3 150 mL PBS + antioxidants 4 150 mL PBS + sugar 5 150 mL PBS + antioxidants + glycerol 6 150 mL PBS + sugar + antioxidants 7 150 mL PBS + glycerol + sugar 8 150 mL saline water + Milk 9 150 mL PBS + antioxidants + Milk 10 150 mL PBS + sugar + Milk 11 150 mL PBS + antioxidants + Milk + sugar
    The composition of the PBS medium was as indicated in Table 3 below.
    Table 3: Conservation medium
    Concentration in 1 liter (pH 7.3 ± 0.2) KCI 0.2g CaCl2 0.1g MgCl2 0.1g KH2PO4 0.2g Na2HPO4 1.15g NaCl 3g KOH 0.6g
    B) RESULTS AND DISCUSSION
    We evaluated:
    i) the influence of the freezing method, i.e.
    -80 ° C compared to liquid nitrogen (-196 ° C) before lyophilization and ii) the impact of protective agents in the preservation medium.
    1) Impact of freezing
    For both the bacteria samples taken separately and for the stool samples, the comparative results whatever the storage medium are always better with rapid freezing in liquid nitrogen and then lyophilization in comparison with freezing at -80 ° C. for 12 H then lyophilization. Rapid freezing in liquid nitrogen followed by freeze-drying results in less loss of CFUs.
    Figures IA and IB illustrate the log numbers of CFUs of aerobic bacteria (Figure IA) and anaerobic bacteria (Figure IB) respectively lost after the different freezing protocols used on 6 stool samples without protective agents in the preservation medium:
    - Pl = freezing at -80 ° C for 12 H then lyophilization,
    - P2 = rapid freezing in liquid nitrogen then lyophilization,
    - P3 = freezing without freeze-drying (fresh stools).
    FIGS. 1A-1B illustrate that the preservation of the bacterial viability after lyophilization without protective agents remains, however, significantly less effective than after freezing at -80 ° C. for 48 hours without lyophilization.
    On the other hand, rapid freezing in nitrogen (-196 ° C) makes it possible to increase the survival rate of said bacteria, in particular the microbiota of human stool by a factor of 100 (2 log) at least compared to freezing. slow at -80 ° C before lyophilization.
    2) Impact of the protective agents tested.
    The presence of glycerol in the storage medium has no positive impact on viability.
    The presence of antioxidants and sugar alone or in binary combination has a positive impact but which does not make it possible to improve the viability compared to samples frozen without lyophilization.
    On the other hand, both for the samples of bacteria taken separately and for the samples of the stools, that is to say containing all the microbiota of the intestinal flora, there appears overall a synergy for the preservation medium comprising milk and a medium of ternary composition. protective agents comprising: milk + antioxidants + sucrose and / or trehalose which makes it possible to reach a level of viability greater than or equal to that with a preservation medium with milk alone and with that for frozen stools without lyophilization.
    FIGS. 2A and 2B illustrate the results of the quantity of bacteria detected put in culture in anaerobiosis after 48 hours of storage of the lyophilisate at 4 ° C. (FIG. 2A) and in anaerobiosis after 6 weeks of storage of the lyophilisate at 4 ° C. (FIG. 2B ) on 6 stool samples with 5 different medium conditions: A = fresh sample in saline water (here fresh stool), B = lyophilisate in PBS medium, C = Lyophilisate with PBS medium containing antioxidants only, D = Lyophilisate with PBS medium containing powdered milk alone, E = lyophilisate with PBS medium containing antioxidants + sucrose + powdered milk, F = freezing of the fresh sample in PBS medium without protective agent.
    Figures 3A and 3B illustrate the results of the quantity of bacteria detected by aerobic culture after 48 hours of lyophilisate storage at 4 ° C (Figure 3A) and aerobic after 6 weeks of lyophilisate storage at 4 ° C (Figure 3B) on 6 stool samples with the same 5 different media conditions A, B, C, D, E and F.
    As nitrogen freezing was found to be more efficient in the previous chapter, all the lyophilization steps in FIGS. 2A-2B and 3A-3B are preceded by a step of freezing with liquid nitrogen at 196 ° C.
    The results of Tables 3A and 3B below quantified in Log CFU / mL show that the preservation medium for the most efficient lyophilization technique for stool also remains the best for the conservation of samples of non-lactic bacteria taken separately for both bacteria. aerobic (Figure 3A) than anaerobic (Figure 3B).
    Table 3A: AEROBIC BACTERIA (LOG CFU / mL)
    AT B VS D E F Staphylococcusaureus 9 6.949 4,808 8 9 8.347 Enterobacteraerogenes 9 8.453 5,155 8.155 8.779 8.55 Escherichia coli 10 5,255 7.779 8.261 9.588 8.477 Klebsiellaoxytoca 9 5,398 .................................................. ......................4,699 7,125 7.949 6,398 Streptococcusagalactiae 9 7 8,097 8.477 8 8.301 Serratiamarcescens 9 4,431 5.574 8,096 9 8.176 P rot eus mirabilis 9 8.477 7,699 9 9 8 Staphylococcusepidermidis 9 6 7,398 8.041 9 8 Bacilluscirculons 8 6.602 7,301 7,301 7.7 7 Streptococcuspneumoniae 8.301 6,096 7,846 8.301 8.778 8,738 Staphylococcushominis 9 8 8,398 8.812 8.853 8.787 Acinetobacterbaumanii 9 7.289 8.041 8 8.544 8
    Table 3B
    Anaerobes (Log CFU / mL) AT B VS D E F Bacteroides nordii 8 0 5 4,699 7 6,633 Propionibacteriuma vidum 10 7.889 8.977 9,698 9,176 9.477 C / ostridum irreguiar 8 6.875 6,368 5,301 7.65 7,699 Ciostridummassiiioamazoniensis 6 3.8 4,202 5.875 5,414 5 Ciostridumbutyricum 9 7,699 7.477 8.621 8.155 7 Ciostridumbeijerinckii 9 7.588 8.602 8,085 8.7 8,199 Bacteroidesthetaiotaomicron 9 0 0 3 4 5.7 Propionibacteriumacnes 9 6.796 8.301 8.041 8.544 8.729 Finegoidia magna 9 6,103 7.666 8.155 8.653 8,478 Bacteroides fragiiis 9 2 4 5,951 6.5 7,801
    Finally, the graphs in FIG. 4 illustrate that the ternary medium E (lyophilisate with antioxidants + sucrose + powdered milk) previously described has the best results for the lyophilization of a cocktail of the following 11 anaerobic bacteria in comparison with the same other conditions of conservation medium A, B, C, D and F above: Bacteroides fragiiis, Bacteroides nordii, Bacteroides thetaiotaomicron, Ciostidium butyricum, Clostridium massiiioamazoniensis, Clostridium beijerinckii, Clostridium irreguiar Finegoidia magna, Propionibioni acida.
    In all cases, it is observed that lyophilisates comprising:
    - at least 10 8 CFU / mL bacteria of each of the categories of aerobic bacteria and anaerobic bacteria respectively cultured after 48 hours of storage at 4 ° C, and
    - at least 10 8 CFU / mL bacteria from each of the categories of aerobic bacteria and anaerobic bacteria respectively cultured after 6 weeks of storage at 4 ° C, and
    - more generally, rapid freezing in nitrogen (-196 ° C) makes it possible to increase the survival rate of said bacteria of the intestinal microbiota by a factor of at least 100 compared to a slow freezing at -80 ° C before lyophilisation.
    In FIGS. 5A and 5B, the results were compared on two stools obtained from two healthy donors from the following conservation media No. 1 to 15 with the following protective agents:
    1- 10% sucrose
    2- trehalose 5%
    3- 10% milk
    4- antioxidants
    5- 10% milk + antioxidants
    6- 10% milk + 5% trehalose
    7- 10% milk + 10% sucrose
    8- trehalose 5% + sucrose 10%
    9- 5% trehalose + antioxidants
    10- 10% sucrose + antioxidants
    11- 10% sucrose + trehalose 5% + antioxidants
    12- milk 10% + trehalose 5% + sucrosel0%
    13- 10% milk + 5% trehalose + antioxidants
    14- 10% milk + 0% sucrosel + antioxidants
    15- 10% milk + 10% sucrose + 5% trehalose + antioxidants
    Freezing was carried out at -80 ° C for 5 hours.
    Lyophilization was carried out in the DELTA 1-24 lyophilizer
    LSC-CHRIST at 0.63 mbar pressure, and plateau temperature at 0 ° C for 36 hours followed by 6 h at +30 ° C (according to the method of Staley et al. Am J Gastro 2017).
    The viability results are reported in Figures 5A and 5B and Table 4 below (average values).
    Table 4: Percentage of viability bacterial after lyophilization Saddle 1 Anaerobes aerobic Saddle 2 Anaerobes aerobic (%viability) (% viability) (%viability) (%viability) l-Sucrose 10% 91.18 87.16 91.43 78.37 2-Trehalose 5% 93,14 88.54 90.12 78.84 3-Milk 10% 90.61 89.29 83 : 79.46 4-Antioxidants 86.16 87.81 92.48 77.86 5-Milk 10% + antioxidants 92.56 88.52 87,88 79.63 6-Milk 10% + trehalose 5% 89.23 90.97 89.74 81.27 7-Milk 10% + sucrose 10% 92.72 91.60 89.76 82.73 8-Trehalose 5% + sucrose 10% 92.03 87.65 88.41 . 77.43 9-Trehalose 5% + antioxidants 91.95 83.11 84.87 73,28 10-Sucrose 10% + antioxidants 90.38 93.73 87.95 85.38 11-Sucrose 10% + trehalose 5% + antioxidants 91.35 95,20 96.07 86.47 12-Milk 10% + trehalose 5% +sucrosel0% 93.13 87.65 96.95 76.82
    13-Milk 10% + trehalose 5%
    + antioxidants14-Milk 10% + sucrosel0% 89.95 90.90 92.46 82.82 + antioxidants 92.10 88.59 95.32 77.47 15- Milk 10 ° / o + sucrosel 0 ° / o + trehalose 5 % + antioxidants 97.34 98.34 97.37 89.31
    Table 5: composition of the conservation media tested
    Solution 1
    SI = 10% sucrose Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom sucrose 10% 100g Ref 57501-1, Inresa Medical, Bartenheim, France
    Solution 2
    S2 = trehalose 5% Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Trehalose 5% 50g Ref 194756, MP biomedicals LLC illkirch, France
    Solution 3
    S3 = Milk 10% Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Semi-skimmed milk 10% 100g Ruled, Lyon, France
    Solution 4
    S4 = antioxidants Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom CaCI 2 0.1g Ref 10035, Inresa Medical, Bartenheim, France MgCl 0.1g Ref 77911, Inresa Medical, Bartenheim, France KOH 0.3 / 0.6g Ref 102105W, BDH LABORATORY SUPPLIES, Poole, United Kingdom Ascorbic acid ig Ref 50817, Inresa Medical, Bartenheim, France Uric acid 0.4g Ref U2625, Sigma-Aldrich, Lyon, France glutathione 0.1g Ref G4251, Sigma-Aldrich, Lyon, France
    Solution 5
    S5 = 10% milk + antioxidants Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Semi-skimmed milk 10% 100g Ruled, Lyon, France CaCI 2 0.1g Ref 10035, Inresa Medical, Bartenheim,France MgCl 0.1g Ref 77911, Inresa Medical, Bartenheim,France KOH 0.3 / 0.6g Ref 102105W, BDH LABORATORY SUPPLIES, Poole, United Kingdom Ascorbic acid ig Ref 50817, Inresa Medical, Bartenheim,France Uric acid 0.4g Ref U2625, Sigma-Aldrich, Lyon, France glutathione 0.1g Ref G4251, Sigma-Aldrich, Lyon, France
    Solution 6
    S6 = LaitlO% + trehalose 5% Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Semi-skimmed milk 10% 100g Ruled, Lyon, France Trehalose 5% 50g Ref 194756, MP biomedicals LLC illkirch, France
    Solution 7
    S7 = LaitlO% + sucrose10% Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Semi-skimmed milk 10% 100g Ruled, Lyon, France sucrosel0% 100g Ref 57501-1, Inresa Medical, Bartenheim,France
    Solution 8
    S8 = sucrosel0% + trehalose5% Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom sucrosel0% 100g Ref 57501-1, Inresa Medical, Bartenheim,France Trehalose 5% 50g Ref 194756, MP biomedicals LLC illkirch, France
    Solution 9
    S9 = Trehalose5% + antioxidants Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Trehalose 5% 50g Ref 194756, MP biomedicals LLC illkirch, France CaCI 2 0.1g Ref 10035, Inresa Medical, Bartenheim, France MgCl 0.1g Ref 77911, Inresa Medical, Bartenheim, France KOH 0.3 / 0.6g Ref 102105W, BDH LABORATORY SUPPLIES, Poole, United Kingdom Ascorbic acid ig Ref 50817, Inresa Medical, Bartenheim, France Uric acid 0.4g Ref U2625, Sigma-Aldrich, Lyon, France glutathione 0.1g Ref G4251, Sigma-Aldrich, Lyon, France
    Solution 10
    S10 = 10% sucrose + antioxidants Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Sucrose 10% 100g Ref 57501-1, Inresa Medical, Bartenheim,France CaCI 2 0.1g Ref 10035, Inresa Medical, Bartenheim, France MgCl 0.1g Ref 77911, Inresa Medical, Bartenheim, France KOH 0.3 / 0.6g Ref 102105W, BDH LABORATORY SUPPLIES, Poole, United Kingdom Ascorbic acid ig Ref 50817, Inresa Medical, Bartenheim, France Uric acid 0.4g Ref U2625, Sigma-Aldrich, Lyon, France glutathione 0.1g Ref G4251, Sigma-Aldrich, Lyon, France
    Solution 11
    Sll = sucrose10% + trehalose 5% + antioxidants Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Tech nolog ies, Paiseley, United Kingdom Sucrose 10% 100g Ref 57501-1, Inresa Medical, Bartenheim, France Trehalose 5% 50g Ref 194756, MP biomedicals LLC illkirch, France CaCI 2 0.1g Ref 10035, Inresa Medical, Bartenheim, France MgCl 0.1g Ref 77911, Inresa Medical, Bartenheim, France KOH 0.3 / 0.6g Ref 102105W, BDH LABORATORY SUPPLIES, Poole, United Kingdom Ascorbic acid ig Ref 50817, Inresa Medical, Bartenheim, France Uric acid 0.4g Ref U2625, Sigma-Aldrich, Lyon, France glutathione 0.1g Ref G4251, Sigma-Aldrich, Lyon, France
    Solution 12
    S12 = Milk 10% + trehalose 5% + sucrose 10% Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference
    PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Semi-skimmed milk 10% 100g Ruled, Lyon, France Sucrose 10% 100g Ref 57501-1, Inresa Medical, Bartenheim,France Trehalose 5% 50g Ref 194756, MP biomedicals LLC illkirch, France
    Solution 13
    S13 = Milk 10% + trehalose 5% + antioxidants Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Semi-skimmed milk 10% 100g Ruled, Lyon, France Trehalose 5% 50g Ref 194756, MP biomedicals LLC illkirch, France CaCI 2 0.1g Ref 10035, Inresa Medical, Bartenheim, France MgCl 0.1g Ref 77911, Inresa Medical, Bartenheim, France KOH 0.3 / 0.6g Ref 102105W, BDH LABORATORY SUPPLIES, Poole, United Kingdom Ascorbic acid ig Ref 50817, Inresa Medical, Bartenheim, France Uric acid 0.4g Ref U2625, Sigma-Aldrich, Lyon, France glutathione 0.1g Ref G4251, Sigma-Aldrich, Lyon, France
    Solution 14
    S14 = Milk 10% + sucrose 10% + + antioxidants Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Semi-skimmed milk 10% 100g Ruled, Lyon, France Sucrose 10% 100g Ref 57501-1, Inresa Medical, Bartenheim,France CaCI 2 0.1g Ref 10035, Inresa Medical, Bartenheim, France MgCl 0.1g Ref 77911, Inresa Medical, Bartenheim, France KOH 0.3 / 0.6g Ref 102105W, BDH LABORATORY SUPPLIES, Poole, United Kingdom Ascorbic acid ig Ref 50817, Inresa Medical, Bartenheim, France Uric acid 0.4g Ref U2625, Sigma-Aldrich, Lyon, France glutathione 0.1g Ref G4251, Sigma-Aldrich, Lyon, France
    Solution 15
    S15 = Milk 10% + sucrosel0% +trehalose 5% + + antioxidants Concentration in 1 liter of medium (pH 7.3 ± 0.2) Reference PBS HE Ref 14190-094,Life Technologies, Paiseley, United Kingdom Semi-skimmed milk 10% 100g Ruled, Lyon, France Sucrose 10% 100g Ref 57501-1, Inresa Medical, Bartenheim, France Trehalose 5% 50g Ref 194756, MP biomedicals LLC illkirch, France CaCI 2 0.1g Ref 10035, Inresa Medical, Bartenheim, France MgCl 0.1g Ref 77911, Inresa Medical, Bartenheim, France
    KOH 0.3 / 0.6g Ref 102105W, BDH LABORATORY SUPPLIES, Poole, United Kingdom Ascorbic acid ig Ref 50817, Inresa Medical, Bartenheim, France Uric acid 0.4g Ref U2625, Sigma-Aldrich, Lyon, France glutathione 0.1g Ref G4251, Sigma-Aldrich, Lyon, France
    The viability results showed that the presence of each of the cryoprotective agents alone (milk; trehalose; sucrose) or with antioxidants greatly improves the survival of the bacteria after lyophilization. However, conservation is maximum in the presence of the 3 cryoprotective agents plus the antioxidants (medium No. 15). Medium No. 15 provided better protection of bacteria after freeze-drying, whether anaerobically (~ 97%) or aerobically (> 90%).
    FIGS. 6A and 6B illustrate the comparative viability results as a function of the conditions of freezing conditions before lyophilization (pre-lyophilization step). The results were compared on two stools from two healthy donors from 2 different lyophilization media and according to two different freezing modes.
    The two media tested included the following protective agents:
    1- Milk 10% + sucrosel0% + antioxidants
    2- Milk 10% + sucrose 10% + trehalose 5% + antioxidants.
    The two freezing modes included:
    - freezing at -80 ° C for 5 hours, and
    - rapid freezing in nitrogen (-195.79 ° C) for 1-2 min.
    The subsequent lyophilization was identical in all cases, namely at a pressure of 0.63 mbar, and temperature of the plate at 0 ° C. for 36 hours followed by 6 h at +30 ° C. in the lyophilizer DELTA 124 LSC-CHRIST.
    In FIGS. 6A and 6B and in Table 5 (average values) the results of viability are reported for the following 4 conditions n ° 1 to 4 (on the abscissa):
    - condition n ° l = freezing at -80 ° C / freeze-drying medium n ° 14 (Milk 10% + sucrosel0% + antioxidants),
    - condition n ° 2 = freezing at -80 ° C / freeze-drying medium n ° 15 (Milk 10% + sucrose 10% + trehalose 5% + antioxidants),
    - condition n ° 3 = freezing in nitrogen / freeze-drying medium 14 (- Milk 10% + sucrosel0% + antioxidants), and
    - condition n ° 4 = freezing in nitrogen / freeze-drying medium 15 (Milk 10% + sucrose 10% + trehalose 5% + antioxidants).
    Lyophilization table.
    : Percentage of bacterial viability after
    % of terms viability% Anaerobes 186.20 294.52 392.48 494.07 Selle3 % Aerobic 95.53 97.56 96.89 98.92 % Anaerobes 91.27 93.55 94.64 95.24 Salt Ie4 % Aerobic 93.5 94.03 92.69 92.91
    The results of this study have shown that between (a) freezing at -80 ° C for 5 hours then lyophilization and (b) rapid freezing in liquid nitrogen then direct lyophilization, freezing in liquid nitrogen allows less loss of CFU.
    The viability results of aerobic and anaerobic bacteria were compared on two stools from two healthy donors from the same lyophilization medium (medium # 15 = 10% milk + 10% sucrose + 5% trehalose + antioxidants) frozen by rapid freezing in nitrogen (-195.79 ° C) for 1-2 min. in both cases but with two different lyophilization conditions in the DELTA 124 LSC-CHRIST lyophilizer. The two freeze-drying conditions were:
    1) pressure of 0.63 mbar, and plate temperature at 0 ° C for 12 hours; and
    2) 0.63 mbar pressure and plateau temperature at 0 ° C for 36 hours followed by 6 h at +30 ° C (according to the method of Staley et al. Am J Gastro 2017).
    The results do not show any difference between the two lyophilization methods.
    权利要求:
    Claims (15)
    [1" id="c-fr-0001]
    1. Method for preserving a sample of a set of aerobic and anaerobic bacteria other than lactic acid bacteria,
    5 in particular bacteria of the intestinal microbiota, comprising the following successive stages in which:
    a) a said sample of a set of said bacteria is prepared by dilution in an aqueous preservation medium comprising a combination of protective agents comprising (al) at least one
    10 antioxidant compound and (a2) at least one sugar, and (a3) milk proteins, and
    b) freezing of said sample recovered in step a) in liquid nitrogen at approximately -196 ° C for not more than 1 minute, preferably for 30 to 50 seconds, and
    C) lyophilization of said sample thus frozen is carried out to obtain a lyophilisate.
    [2" id="c-fr-0002]
    2. Method according to claim 1, characterized in that in step a), a said sample is prepared which is a clinical sample of stools by diluting said stools in an aqueous preservation medium, and
    20 the dispersion obtained is filtered to remove at least debris of sizes greater than 2mm.
    [3" id="c-fr-0003]
    3. Method according to claim 1 or 2, characterized in that in step a), said sample is diluted in an aqueous preservation medium comprising at least the combination of protective agents
    Comprising (al) at least one antioxidant compound and (a2) at least two different sugars, and (a3) milk proteins.
    [4" id="c-fr-0004]
    4. Method according to one of claims 1 to 3, characterized in that the milk proteins are in the form of whole milk, preferably at a concentration by weight in the said preservation medium of at least 5%, preferably 10 %.
    [5" id="c-fr-0005]
    5. Method according to one of claims 3 or 4, characterized in that said protective agents comprise a combination of antioxidant compounds chosen from ascorbic acid, uric acid and glutathione.
    [6" id="c-fr-0006]
    6. Method according to one of claims 1 to 5, characterized in that said protective agents comprise a combination of antioxidant compounds consisting of ascorbic acid at a concentration of Ig / L, uric acid at a concentration 0.4g / L and glutathione at a concentration of 0.1g / L.
    [7" id="c-fr-0007]
    7. Method according to one of claims 1 to 6, characterized in that the said protective agents comprise at least one sugar chosen from sucrose and trehalose.
    [8" id="c-fr-0008]
    8 Method according to one of Claims 1 to 7, characterized in that the said protective agents comprise at least one said sugar at a weight concentration in the said preservation medium of at least 5%, preferably 10%.
    [9" id="c-fr-0009]
    9. Method according to one of claims 1 to 8, characterized in that the said preservation medium is a buffer medium with a pH of 7 to 7.5 comprising in water the salts KCL, CaCi2, MgC12, KH2PO4, Na2HPO4, NaCL and KOH as well as at least one said protective agent.
    [10" id="c-fr-0010]
    10. Method according to one of claims 1 to 9, characterized in that said storage medium comprises:
    (al) a combination of antioxidant compounds consisting of ascorbic acid at a concentration of lg / L, uric acid at a concentration of 0.4 g / L and glutathione at a concentration of O, lg / L, and (a2) sucrose at a concentration by weight in the said storage medium of 5%, and trehalose at a concentration by weight in the said storage medium 10%, and (a3) milk proteins at a concentration by weight in the says 10% conservation medium.
    [11" id="c-fr-0011]
    11. Method according to one of claims 1 to 10, characterized in that said sample comprises at least the following aerobic bacteria and anaerobic bacteria: Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli, Klebsiella oxytoca, Streptococcus agalactiae, Serratia marcescens, Proteus mirabilis, Staphylococcus epidermidis, BaciUus circulans, Streptococcus pneumoniae, Staphylococcus hominis and Acinetobacter baumanii.
    [12" id="c-fr-0012]
    12. Method one of claims 1 to 10, characterized in that the said sample comprises at least the following strict anaerobic bacteria: Bacteroides fragitis, Bacteroides nordii, Bacteroides thetaiotaomicron, Clostidium butyricum, Clostridium massilioamazoniensis, Clostridium beijerinckii, Clostridium irregular Firtegia , Propionlbacterium acnes, Propionibacterlum avidum, Haemophitus influenzae.
    [13" id="c-fr-0013]
    13. Method one of claims 1 to 12, characterized in that in step c) the frozen sample is deposited in a lyophilizer for at least 12 hours at a temperature of at least 0 ° C and at a pressure of vacuum emptied by no more than lmbar.
    [14" id="c-fr-0014]
    14. Sample lyophilisate of a set of aerobic and anaerobic bacteria of the intestinal microbiota other than lactic acid bacteria, obtained by a process according to one of claims 1 to 13, characterized in that it comprises:
    - at least one protective agent consisting in at least the combination of protective agents consisting in (a1) at least one antioxidant compound and (a2) at least one sugar, and (a3) milk proteins, and
    - an amount of live bacteria, preferably the number of:
    5 - at least 10 th CFU / mL bacteria of each of the categories of aerobic bacteria and anaerobic bacteria respectively cultured after 48 hours of storage at 4 ° C., and
    - at least 10 8 CFU / mL bacteria from each of the categories of aerobic bacteria and anaerobic bacteria respectively
    10 culture after 6 weeks of storage at 4 ° C.
    [15" id="c-fr-0015]
    15. Lyophilisate according to claim 14 characterized in that said sample is a clinical sample of human stool.
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    同族专利:
    公开号 | 公开日
    WO2018234645A1|2018-12-27|
    EP3642323A1|2020-04-29|
    FR3067928B1|2020-02-28|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2015159004A1|2014-04-16|2015-10-22|Fondation Mediterranee Infection|Method and liquid medium for transporting and preserving bacteria|
    WO2017103225A1|2015-12-18|2017-06-22|Institut National De La Recherche Agronomique|Lyophilized composition for preserving microbiota in its ecosystem|
    FR2829147B1|2001-08-30|2003-12-12|Agronomique Inst Nat Rech|PROCESS FOR THE PREPARATION OF A LYOPHILIZED COMPOSITION CONTAINING LACTIC BACTERIA HAVING IMPROVED BACTERIAL VIABILITY AND ACTIVITY DURING AMBIENT TEMPERATURE STORAGE AND COMPOSITION OBTAINED|DK3436054T3|2016-09-13|2019-11-11|Allergan Inc|STABILIZED NON-PROTEIN CLOSTRID TOXIN COMPOSITIONS|
    AU2020267139A1|2019-05-01|2021-11-25|The Procter & Gamble Company|Probiotic bacterial strains that produce short chain fatty acids and compositions comprising same|
    TW202136493A|2019-12-20|2021-10-01|英商4D製藥研究有限公司|Lyophilisation process|
    法律状态:
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1755796|2017-06-23|
    FR1755796A|FR3067928B1|2017-06-23|2017-06-23|METHOD FOR PRESERVING A SAMPLE OF BACTERIA|FR1755796A| FR3067928B1|2017-06-23|2017-06-23|METHOD FOR PRESERVING A SAMPLE OF BACTERIA|
    PCT/FR2018/051036| WO2018234645A1|2017-06-23|2018-04-25|Method for preserving a sample of bacteria|
    EP18725288.7A| EP3642323A1|2017-06-23|2018-04-25|Method for preserving a sample of bacteria|
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