A new protectant medium preserving bacterial viability

after freeze drying

Sara Bellali, Jacques Bou Khalil, Anthony Fontanini, Didier Raoult,
Jean-Christophe J.-C. Lagier

To cite this version:

Sara Bellali, Jacques Bou Khalil, Anthony Fontanini, Didier Raoult, Jean-Christophe J.-C. Lagier. A
new protectant medium preserving bacterial viability after freeze drying. Microbiological Research,
2020, 236, pp.126454. �10.1016/j.micres.2020.126454�. �hal-02517909�

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Version of Record: https://www.sciencedirect.com/science/article/pii/S0944501319306421
Manuscript_9eebb6a2395d6180ff63a1f8681dbc0e

1 A new protectant medium preserving bacterial viability after freeze drying

4 Sara Bellali1, Jacques Bou Khalil2, Anthony Fontanini2, Didier Raoult1 and Jean-Christophe

5 Lagier1 *.

1
7 Aix Marseille Univ., IRD, AP-HM, MEPHI, IHU Méditerranée Infection, Marseille, France.
2
8 IHU Méditerranée Infection, Marseille, France.

10 Corresponding author : Prof. Jean-Christophe LAGIER - Institut Hospitalo-Universitaire

11 11 Méditerranée Infection, 19-21 Boulevard Jean Moulin 13385 Marseille Cedex 05, France.

12 12 Phone: + 33 (0) 4 13 73 24 01. Fax: + 33 (0) 4 13 73 24 02. jclagier@yahoo.fr

13

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20 Keywords: freeze-drying, protectant medium, preservation, bacterial viability, survival, gut

21 microbiota.

22 Text word count: 4,339

23 Abstract word count: 246

1
© 2020 published by Elsevier. This manuscript is made available under the CC BY NC user license
https://creativecommons.org/licenses/by-nc/4.0/
24 Abstract

25 Freeze-drying technology has been widely considered for decades as a suitable technique to

26 preserve microorganisms. However, protective agents must be added prior to freeze drying to

27 improve the survival and storage stability of the bacteria.

28 The objective of our study was to evaluate the effect of a new protectant medium containing

29 sucrose (10%), trehalose (10%), skimmed milk (10%) and antioxidants on the viability of gut

30 bacteria under different storage conditions. Two strains were tested, Escherichia coli and

31 Akkermansia muciniphila, as examples of facultative aerobic and anaerobic bacteria,

32 respectively. We studied the cell viability and bacterial morphology in 5 fecal samples in the

33 presence and absence of this protectant medium using plating technique, flow cytometry and

34 scanning electron microscopy.

35 The results of bacterial viability assessed by plating method showed that the protectant

36 medium yielded higher survival rates for both strains whatever the storage conditions (85–

37 93%) compared to normal saline solution (0.36-37.50%). It also showed its effectiveness on

38 fecal samples, where bacterial viability after freeze-drying was 89.47±7.63% and

39 84.01±7.44%, as evidenced by flow cytometry analysis and plating method. However

40 unprotected samples showed the lowest cell viability at 19.01±12.88% and 13.23±9.56%, as

41 measured by flow cytometry and plating method. In addition, bacterial size and shape were

42 conserved in the protectant medium. In contrast, storage without protectant medium severely

43 damaged bacterial morphology.

44 In conclusion, our study is the first to use morphological features as well as culture-dependant

45 and culture-independent tests to evaluate the effectiveness of a new protectant medium.

2
46 Introduction

47 Freeze-drying has been the most commonly used technique to enhance the storage stability of

48 probiotics for decades. Probiotics are defined as living microorganisms that, once

49 administered in adequate amounts, confer a health benefit for the host (WHO, 2001). A

50 variety of probiotics such as yeast, Lactobacillus, and Bifidobacterium species have been

51 successfully preserved by freeze-drying (Biavati et al., 2000; Gomes and Malcata, 1999).

52 Recently, a novel group of obligate anaerobic bacteria was considered as the next generation

53 of probiotics in the treatment of inflammatory bowel disease (Everard et al., 2013; Van

54 Immerseel et al., 2010). Among them, Faecalibacterium prausnitzii is a strict anaerobic

55 bacterium that constitutes 3–5% of all fecal bacteria (Miquel et al., 2013) and is considered a

56 highly abundant butyrate producer (Breyner et al., 2017). Akkermansia muciniphila, a strict

57 anaerobic mucin-degrading bacterium, is also considered an abundant candidate of the healthy

58 human microbiota (1–5%) (Derrien et al., 2004). To our knowledge, only one recent study

59 reported the preservation of Akkermansia muciniphila by freeze-drying (Marcial-Coba et al.,

60 2018).

61 In addition, the human microbiota is known to play an important role in health and disease

62 (Wang et al., 2017). Several studies have already reported that fecal microbiota

63 transplantation (FMT) provided highly effective treatment of Clostridium difficile infection

64 (Brandt et al., 2012; Hocquart et al., 2018; Kassam et al., 2013; Surawicz et al., 2013; van

65 Nood et al., 2013). In recent years, FMT has emerged, evolving from the use of fresh fecal

66 microbiota to the cryopreservation of fecal microbiota (Hamilton et al., 2012). However,

67 colonoscopic administration remains invasive to patients and is therefore also complicated for

68 the healthcare units at the technical and organizational level. Other FMT modalities are

69 reported to be easier and less complicated. In fact, the orally-administered FMT via

70 encapsulated fresh microbiota (Louie et al., 2013) and the frozen/thawed capsules seem to be

3
71 the best choice (Youngster et al., 2014). In addition, oral FMT showed the same clinical

72 efficiencies as fresh FMT in the treatment of recurrent Clostridium difficile infection (Lee et

73 al., 2016). However, these FMT administrations are limited by storage and transport

74 conditions.

75 In order to overcome these complications and simplify these procedures, we suggested oral

76 freeze-dried FMT, a far gentler and esthetically pleasing format that could be self-

77 administrated (Staley et al., 2017). It would also be less expensive and time-consuming. To

78 date, few studies have addressed the issue of freeze-dried fecal samples ready to be used for

79 fecal transplantation (Hecker et al., 2016; Hirsch et al., 2015; Jiang et al., 2017; Staley et al.,

80 2017).

81 In fact, freeze-drying combines freezing and drying stresses that were known to be more

82 detrimental to sensitive bacteria than cryopreservation (Heylen et al., 2012). Before the

83 freeze-drying or lyophilization process, bacterial suspensions need to be frozen first and

84 subsequently dried under vacuum. However, without suitable protectant medium, freeze-

85 drying severely damages cell membranes and proteins (Carpenter et al., 1987; Carpenter and

86 Crowe, 1988; Crowe et al., 1990; Leslie et al., 1995), causing a decrease in viability (Panoff

87 et al., 1998; Wolfe and Bryant, 1999). Furthermore, the importance of adding protectants such

88 as disaccharides (e.g. trehalose, sucrose) (Leslie et al., 1995), polyols (e.g. mannitol, sorbitol)

89 (Ana S. Carvalho et al., 2003; Efiuvwevwere et al., 1999) and proteins (e.g. skimmed milk)

90 (Castro et al., 1997) prior to freezing or drying, played a major role in preserving cells and

91 improving their storage viability. Meanwhile, their protective mechanisms remained unclear

92 and not fully understood. Three major protective mechanisms were reported: (i) preventing

93 the intra and extracellular ice formation (Baumann, 1964; Fowler and Toner, 2006) (ii) water

94 replacement hypothesis by hydrogen bonds formation (Leslie et al., 1995) (ii) or glassy matrix

95 formation (Crowe et al., 1998).

4
 96 Each protectant medium impacts differently microorganisms and no universal protectant

97 medium has yet been developed (Champagne et al., 1991; Sanders et al., 1999). The glycerol

98 is one of the most commonly cryoprotectant used in preparation of frozen liquid suspensions

99 of microbiota (Costello et al., 2016; Hamilton et al., 2012; Kaito et al., 2018; Satokari et al.,

100 2015). However, this cryoprotectant is not recommended for freeze drying and is not useful

101 due to its viscosity which can lead to a sticky and an insufficient dried product not amenable

102 to encapsulation. For this, having a suitable protectant medium capable of preserving

103 anaerobic and aerobic bacteria during freeze drying process can be challenging (Font de

104 Valdez et al., 1983). Here, we investigate the effect of a protectant medium containing

105 sucrose (10%), trehalose (10%), skimmed milk (10%) and antioxidants to preserve

106 Akkermansia muciniphila and Escherichia coli during freezing, freeze-drying, and subsequent

107 storage for 30 days at different conditions. This protectant medium was also used in a specific

108 process to preserve fecal microbiota used in FMT. Thus, we evaluated the effectiveness and

109 preservation rate of this protectant medium by studying cell viability and bacterial

110 morphology in samples. To this end, we used plating technique, flow cytometry and scanning

111 electron microscopy.

112 Materials & methods

113 1. Preservation of individual bacteria

114 1.1.Protectant medium composition

115 In this work, we used a protectant medium selected on the basis of our previous study

116 patented in 2017 under the following number (N° WO/2018/234645). This medium contains

117 per g/L in phosphate buffered saline (PBS) (Life Technologies, Paiseley, United Kingdom)

118 the following elements: sucrose (10g); skimmed milk (10g); trehalose (5g); CaCl2 (0.1g);

119 MgCl2 (0.1g); KOH (0.3/0.6g) and tree antioxidants being: Ascorbic acid (1g); Uric acid

120 (0.4g) and Glutathione (0.1g). The pH of the medium was about 7.3 ± 0.2. Skimmed milk was
5
121 sterilized at 121°C for 15 min and the rest of the solution (antioxidants and sugar) was

122 sterilized by filtration through 0.22 μm filters.

123 1.2.Bacterial strains and growth conditions

124 In order to prove the effectiveness of the protectant medium, facultative aerobic and strict

125 anaerobic strains were tested. Escherichia coli (CSUR P1966) was grown in Columbia sheep

126 blood agar plates (BioMérieux, Marcy l'Etoile, France) at 37°C for 24 hours under aerobic

127 conditions. Akkermansia muciniphila strain (CSUR P6566) was grown in Columbia sheep

128 blood agar plates at 37°C for 48 hours under anaerobic conditions using a GasPak generator

129 (Becton Dickinson Microbiology Systems, Sparks, MD, USA). Both strains were isolated

130 from fecal materials of two healthy donors in our laboratory.

131 1.3.Freeze-drying procedures

132 Firstly, we chose the best time to harvest bacterial colonies with higher viable rates and less

133 dead bacteria based on flow cytometry measurements and microscopic observations at

134 different incubation times (12, 24 and 75 hours). Fresh Escherichia coli and Akkermansia

135 muciniphila were harvested directly from agar plates after 24 hours and 48 hours,

136 respectively, and mixed with two solutions: (a) normal saline solution (NaCl 0.9%) (Fresenius

137 Kabi, Sevres, France) as control, and (b) the protectant medium (milk 10% + sucrose 10% +

138 trehalose 5%+ antioxidants), with a final concentration of 2-3x1010 CFU/mL and 5-4x1010

139 CFU/mL, respectively, for Akkermansia muciniphila and Escherichia coli.

140 Bacterial suspensions in (a) normal saline solution and in (b) the protectant medium were

141 placed into three 2 mL, type I (Wheaton, Millville, NJ, USA) serum vials and frozen at -80°C

142 for 5 hours. Suspensions were desiccated in a DELTA 1-24 LSC-CHRIST freeze-dryer at a

143 condenser temperature of -80°C and at a chamber pressure of 0.63 mbar for 12 hours at 0°C,

144 followed by 3 hours at + 30°C. After freeze-drying, vials were sealed manually, and stored at

145 +4°C (Figure 1).

6
146 1.4.Storage conditions

147 Bacterial suspensions in presence of protectant medium or saline solution were stored for 48

148 hours and for 30 days under different conditions (Figure 1):

149 (i) Frozen at -80°C,

150 (ii) Frozen at -196°C by dipping the vials into liquid nitrogen (LN2),

151 (iii) Frozen at 80°C for 5 hours then freeze-dried,

152 (iiii) At +4°C.

153 After freeze-drying, samples were rehydrated and homogenized with phosphate buffered

154 saline (PBS) to return to their original volume (500 µL) at 25°C and incubated at room

155 temperature.

156 2. Preservation of fecal samples

157 2.1.Stool sample collection and preparation

158 Fresh fecal samples were obtained from 5 healthy donors (2 women, 3 men) from France,

159 with a normal body mass index. These volunteers were not subjected to any feeding trial,

160 specific diet, or antibiotic treatment for the last six months prior to sampling. Stool samples

161 were collected in a sterile stool container under anaerobic conditions by using a GasPak

162 generator (Becton, Dickinson and Sparks, USA) and were then immediately transported to the

163 laboratory. Donors were informed of the study and signed informed consents. The project

164 received the IHU Méditerranée Infection ethics committee agreement under number 2016-

165 011.

166 2.2.Stool sample preparation

167 Sample preparations were carried out under a sterile hood. Briefly, 50g of fecal matter was

168 blenderized (BOSCH Ultracompact 400W device) with 250 mL of normal saline solution

169 (NaCl 0.9%) (Fresenius Kabi, Sevres, France) for 5 min, then sieved using coffee strainers to

170 remove food debris. The slurry was centrifuged for 15 min at 6,000 x g. The pellet was
7
171 suspended in one half of the initial volume in 2 media: (a) the normal saline solution used as

172 control, and (b) the protectant medium (skimmed milk 10%+sucrose10% + trehalose 5% +

173 antioxidants). Then, bacterial suspensions were frozen at -80°C for 5 hours and directly

174 freeze-dried under the same conditions as described above, and finally stored at +4°C (Figure

175 1).

176 3. Cell viability determination

177 Bacterial viability of each storage condition, (a) in normal saline solution and (b) in protectant

178 medium was calculated before and after freeze-drying using viable counts and flow cytometry

179 method.

180 3.1.1. Enumeration of bacteria by plate count

181 Decimal dilutions were prepared with PBS. One hundred µL of the sample suspension was

182 mixed with 900 µL of PBS, vortexed for 10 seconds and serially diluted with PBS. The

183 Columbia agar plates were divided up into three or four lines into which 10µL of each

184 dilution were speared onto it. Each dilution was plated in triplicates. The plates were

185 incubated at 37°C for 48 h under aerobic and anaerobic (GasPak generator) conditions.

186 The number of colony-forming units per milliliter (CFU/mL) was determined and the viability

187 was defined as the percentage ratio of viable cells after freeze-drying and viable cells before

188 freeze-drying using the following equation:

⁄
189 Survival % = ⁄
x 100

190 3.1.2. LIVE/DEAD enumeration

191 The membrane integrity was determined using the LIVE/DEAD BacLight Bacterial Viability

192 Kit (Molecular Probes, Invitrogen, USA) as described previously (Bellali et al., 2019).

193 4. Scanning electron microscopy for bacterial morphology evolution

8
194 Fresh, frozen and freeze-dried samples were either directly smeared onto microscopy slides or

195 cyto-centrifuged on cytospin slides. Slides were then processed to image acquisition, after

196 staining with PTA (phosphotungstic acid 1 %) in order to check morphological appearance

197 changes and cell integrity.

198 We used a table top scanning electron microscope SEM (Hitachi TM4000 Plus) to evaluate

199 bacterial structures. The SEM has a capability of observing specimen in low vacuum pressure

200 (100 Pa to 101 Pa) to reduce charge-up on the specimen’s surface by the irradiated electrons.

201 Evacuation time after the loading of specimens into the SEM Chamber is shorter than 2

202 minutes, which is much quicker than conventional SEMs with high vacuum condition. The

203 imaging process for all samples in the presence and absence of protectant medium was

204 acquired at the same acquisition settings regarding magnification, intensity and voltage mode.

205 All settings are displayed on micrographs.

206 5. Statistical analysis

207 All experiments were carried out in triplicate. All viability data are expressed as the means

208 standard deviations (SDs). The difference between two means before and after freeze-drying

209 was calculated using the Mann-Whitney t-test (Prism v5.0, GraphPad). Means were

210 considered significantly different when P-value was less than 0.05. Differences between fresh

211 viable cells and all storage conditions were analyzed by one-way analysis of variance

212 followed by a Bonferroni’s multiple comparisons test (Prism v5.0, GraphPad).

213 Results

214 Evolution of the protectant medium efficacy by reporting the viability of individual

215 bacteria after freezing and freeze-drying

216 1. Viability of Escherichia coli and Akkermansia muciniphila after freezing and freeze

217 drying

9
218 The effect of protectant medium and saline water solution on the viability of Escherichia coli

219 and Akkermansia muciniphila under the different storage conditions (for 48 hours and 30 days

220 of storage) are summarized in Figure 2 and compared to fresh cultures suspended in

221 protectant medium and saline water solution.

222 (a) Freezing at -80°C

223 The viability of E. coli with protectant medium (85.00%) did not vary after 48 hours of

224 freezing and remained stable after 30 days of storage (84.00%). Similarly, the viability of A.

225 muciniphila did not greatly decreased after 48 hours of freezing (86.67%) and even after 30

226 days of storage (75.00%). However, the freezing in presence of normal saline had

227 significantly reduced the viability of E. coli and A. muciniphila to 25% and 0.67%

228 respectively. Thus, after 30 days of storage the viability remained reduced to 24.17% and

229 0.04% respectively.

230 (b) Freezing in liquid nitrogen (-196°C)

231 Similar results were obtained after fast freezing in liquid nitrogen, where frozen E. coli and A.

232 muciniphila with protectant medium exhibited high survival rates of 93.00% and 90.00%

233 respectively. The viability of both strains remained unchanged, even after 30 days of storage

234 (90.00% and 86.67%, respectively). However, unprotected E. coli and A. muciniphila showed

235 low viability of 37.50% and 25.56% respectively, compared to that observed in the presence

236 of a protectant medium. After 30 days of storage, the viability of E. coli and A. muciniphila

237 was 25.00% and 17.00% respectively.

238 (c) Freeze-drying

239 Results showed that freeze-dried E. coli and A. muciniphila in the presence of protectant

240 medium had the highest survival rates of 85.00% and 91.67% respectively after 48 hours. It

241 remained stable after 30 days of storage, 80.00% and 83.33% respectively. In the presence of

242 normal saline solution, the freeze-drying process severely damaged cell viability of E. coli
10
243 (2.67%) and A. muciniphila (0.36%) compared to freezing. The viability of E. coli and A.

244 muciniphila significantly decreased to 0.29% and 1x108 CFU/mL; 0.02%, respectively.

245 (d) Storage at +4°C

246 Bacterial suspensions of E. coli and A. muciniphila in the presence of protectant medium,

247 exhibited high survival rates of 80.00% and 90.00% respectively after 48 hours of storage at

248 4°C. Meanwhile, the viability of A. muciniphila and E. coli was declined to 60.00% and

249 41.65% respectively after 30 days of storage. However, both A. muciniphila and E. coli,

250 suspended in normal saline water, were affected within the 48 hours of storage, with a large

251 drop of viability (29.17% and 33.33%, respectively) after 48 hours. After 30 days of storage,

252 cell viability of E. coli and A. muciniphila (25.00%) and (16.67%), respectively were higher

253 than freezing or freeze drying.

254 2. Evaluation of bacterial morphology and shape integrity by scanning electron

255 microscopy after freezing and freeze-drying

256 Bacterial morphology and integrity of both Escherichia coli and Akkermansia muciniphila

257 treated with (a) normal saline solution or with (b) protectant medium in fresh, frozen and

258 freeze-dried state were evaluated for each conservation condition.

259 Micrographs of samples in the presence of normal saline solution showed damaged

260 Escherichia coli cells under all storage conditions tested (freezing or freeze-drying). The

261 degenerative aspect of the bacteria was clearly observed (Figure 3b1, 3b2, 3c1, 3c2, 3d1, 3d2).

262 Similar results were obtained in Akkermansia muciniphila with an irregularity in bacterial

263 shapes and an increase in cell size due to osmotic stress (2.59±1.45µm versus 0.78±0.24µm)

264 (Figure 4b1, 4b2, 4b3, 4b4). Nevertheless, morphology of both bacteria was preserved in the

265 presence of the protectant medium, which shape and size seemed to be better conserved.

266 Frozen or freeze-dried Akkermansia muciniphila were predominantly spherical, although

267 some were found to be irregular or elongated in the same way as fresh cells (Figure 4c1, 4c3,
11
268 4d1, 4d3). Interestingly, we detected small shapes of Escherichia coli that were osmotically-

269 dehydrated due to sucrose and trehalose contained in the protectant medium (Figures 3e1, 3e2,

270 3f1, 3f2, 3g1, 3h1).

271 Validation of the proof of concept by evaluating the effect of the protectant medium on

272 the viability of freeze-dried fecal samples

273 1. Enumeration of fecal material by plate method

274 Total bacterial counts (anaerobic and aerobic counts), and bacterial viability were presented in

275 Table 1. Total bacterial counts ranged from 3.42x109 CFU/mL to 6.92x109 CFU/mL, and

276 from 4.10x109 CFU/mL to 8.75x109 CFU/mL, respectively, for samples resuspended in

277 normal saline solution and in the protectant medium.

278 Before freeze-drying, we noticed differences between total cell counts in all fresh fecal

279 samples, whatever protected or not, so that the number of anaerobic bacteria was ten times

280 higher than that of aerobic bacteria in five samples. Moreover, anaerobic bacteria were much

281 more numerous for samples suspended in protectant medium (5.90x109 ±1.92 x109) than in

282 normal saline solution (4.25 x109±1.47 x109) (Table 1).

283 After freeze-drying, all five freeze-dried fecal samples with normal saline solution decreased

284 in viability and had a lower average survival rate of 13.23±9.56%, ranging from 1.69% to

285 26.27%, compared to samples dried in the presence of protectant medium, producing an

286 average survival rate of 84.01±7.44% ranging from 75.60% to 94.12% (Table 1).

287 As shown in Figure 5A, we found significant differences before and after freeze-drying when

288 the fecal sample was dried with normal saline solution (P=0.01). On the other hand, no

289 significant differences were found for samples freeze-dried in the presence of protectant

290 medium as compared to fresh fecal samples (Figure 5B).

12
291 2.1.Enumeration of fecal samples by flow cytometry

292 In addition to plate count methods, we used the flow cytometry method to assess the viability

293 of freeze-dried fecal samples. IP and SYTO9 were used simultaneously for viability

294 assessment before and after freeze-drying. Three bacterial populations were observed; live

295 (SYTO9-stained), dead (IP-stained) and injured, which were double stained (IP/SYTO9-

296 stained).

297 The relative percentages of live, dead and injured bacterial populations obtained before and

298 after freeze-drying for the five fecal samples are presented in Figure 6. Before freeze-drying,

299 the relative percentage of viable fecal bacteria in the presence of normal saline solution were:

300 84.01% (sample 1), 66.65% (sample 2), 61.93% (sample 3), 68.90% (sample 4), and

301 65.98%(sample 5).Therefore, after freeze-drying it decreased to 16.67% (sample 1), 23.48%

302 (sample 2), 5.85% (sample 3), 18.67% (sample 4) and 2.27% (sample 5) (Figure 6).

303 Simultaneously, dead and injured bacterial populations increased, whereas, in the presence of

304 the protectant medium, we did not observe any significant changes before (79.87%, 64.52%,

305 68.38%, 73.13%, and 70.73%) and after (73.10%, 56.77%, 55.54%, 74.12%, 60.31%) freeze-

306 drying (Figure 6). Overall, the presence of protectant medium during freeze-drying showed a

307 higher protection of bacteria (89.47±7.63%), while normal saline solution damaged most of

308 them (19.01±12.88%). These results were similar to those found by plate counting method.

309 Furthermore, using statistical analysis, we found a significant difference (P= 0.0079) between

310 live bacterial counts before and after freeze-drying of samples dried with saline solution

311 (Figure 5C). Meanwhile, viable count of samples dried with protectant medium were not

312 statistically significant (Figure 5D).

313 3. Electron microscopy of fecal material before and after freeze drying

13
314 The processed fecal samples showed significant differences of their bacterial components at

315 the level of shape and size. Before freeze-drying, stool samples in the presence of normal

316 saline solution showed a bigger bacterial size (1.06±0.15µm) (Figure 7a1-a2) compared to

317 those suspended in protectant medium (0.70±0.14µm) (Figure 7b1-b2). Furthermore, after

318 freeze-drying, the protectant medium conferred a more stable state to the bacteria, where size

319 and shape remained conserved and smaller (0.61±0.17µm) (Figure 7d1-d2) in contrast to the

320 normal saline solution (1.05±0.16µm) (Figure 7c1-c2).

321 Discussion

322 Freeze-drying in the presence of a protective medium to ensure optimal bacterial viability

323 plays a key role in microbiology nowadays. This quality of preservation is extremely needed

324 in FMT and probiotics production.

325 In this study, we evaluated the impact of freeze drying on the bacterial viability using the

326 normal saline solution as a control typically used in the majority (62%) of clinical studies of

327 FMT (Gough et al., 2011; Van Nood et al., 2013), and a new protectant medium containing

328 sucrose (10%), trehalose (10%), skimmed milk (10%) and three antioxidants (uric acid,

329 ascorbic acid and glutathione). Those compounds were found in several studies, in

330 combination or individually, to be effective in protecting bacteria against the injuries during

331 freezing or freeze drying and improved storage stability. Several investigators have reported

332 the positive effect of trehalose on bacterial survival during freeze drying (Jain and Roy, 2010;

333 Mensink et al., 2017). Trehalose is also known as an antioxidant protecting membranes

334 against oxidative stress (Herdeiro et al., 2006). In addition, trehalose and sucrose were the

335 most disaccharides commonly used as protectants during the freezing-drying process

336 (Broeckx et al., 2016; Carpenter and Crowe, 1988; A. S. Carvalho et al., 2003, 2002; Crowe

337 et al., 1998, 1988; Leslie et al., 1995; Linders et al., 1997; Paiva and Panek, 1996; Zayed and

338 Roos, 2004). These two sugars were capable of protecting cell membranes by water

14
339 replacement involved hydrogen bonding between sugars and polar-head groups of the

340 phospholipids (Crowe et al., 1990; Leslie et al., 1995; Rudolph and Crowe, 1985). They also

341 were capable of reducing the ice formation by increasing the shrinkage of cells before

342 freezing (Fowler and Toner, 2006). Remarkably, this dehydration mechanism was observed in

343 our results carried out by scanning electron microscopy, where cells of E. coli were found

344 dehydrated before and after freezing and drying due to the presence of sugars that induced

345 osmosis-derived dehydration. In contrast, in the absence of sugars, cells of E. coli and

346 Akkermansia muciniphila were found injured and severally damaged after freezing and drying

347 due to ice crystal formation, as described previously in several studies (Champagne et al.,

348 1991; Sanders et al., 1999). Such a mechanism still remains unclear.

349 Proteins also provided an additional protective effect by covering the cells and balancing the

350 cell membranes during freeze-drying and storage (Buitink et al., 2000). Indeed, skimmed milk

351 was selected as an efficient drying medium (Bevilacqua et al., 2012; Hubálek, 2003), it

352 contains proteins providing an additional protective layer for the cells (Abadias et al., 2001;

353 Carvalho et al., 2004) and stabilizing membrane components (Castro et al., 1996; Selmer-

354 Olsen et al., 1999). Another feature of skimmed milk is its ability to dry easier and provide a

355 higher yield of dry matter. According to the study reported by Zayed and Roos (Zayed and

356 Roos, 2004), the addition of skimmed milk to the mixture of trehalose and sucrose gave a

357 higher survival rate during subsequent storage (Abadias et al., 2001).

358 Furthermore, previous studies reported that the mixture of many components of protectant

359 medium (e.g. sucrose + trehalose, sucrose + trehalose + skimmed milk…etc.) could result in a

360 better protection of microorganisms than single component due to additive or synergic

361 protective effects (Berner and Viernstein, 2006; Celik and O’Sullivan, 2013; Hubálek, 2003;

362 Jalali et al., 2012; Keivani Nahr et al., 2015; Ming et al., 2009; Sharma et al., 2014; Yang et

363 al., 2007; Yu et al., 2017; Zhang et al., 2014).

15
364 Regarding total anaerobic bacteria in fecal samples prior to freeze drying, we found that

365 samples suspended with our protectant medium had more anaerobes than samples without

366 protectant. The higher number of anaerobes was explained in our previous study (N°

367 WO/2018/234645) by the presence of antioxidants in our protectant medium preserving

368 sensitive anaerobic bacteria from oxygen. In addition to Akkermansia muciniphila, our

369 protectant medium showed superior efficacy to preserve other fastidious anaerobes such as

370 Treponema denticola and Treponema pectinovorum (Data unpublished) and also extremely

371 oxygen-sensitive (EOS) bacteria such as Methanobrevibacter smithii and Faecalibacterium

372 prausnitzii (Data unpublished).

373 Creating a medium protecting the majority of the gut bacteria was challenging. We chose

374 Escherichia coli as an example of aerobic bacteria and Akkermansia muciniphila as an

375 example of anaerobic bacteria in addition to its beneficial effects. We recommend the freeze

376 drying of anaerobic and aerobic bacteria using the protectant medium comprising sucrose

377 (10%), trehalose (10%), skimmed milk (10%) and antioxidants. We believe that this

378 protectant medium holds great promises for therapeutic purposes such as fecal microbiota

379 transplant and probiotics.

380 The idea of creating this protectant medium comes from many conservation strategies and for

381 the first time we used morphological, culture-dependent and culture-independent tests to

382 evaluate its effectiveness. Further studies will be conducted to test freeze-dried microbiota

383 transplantation in oral capsules using this protectant medium.

384 Acknowledgments

385 We sincerely thank Takashi Irie, Kyoko Imai, Shigeki Matsubara, Taku Sakazume, Yusuke

386 Ominami, Hishada Akiko and the Hitachi team of Japan (Hitachi High-Technologies

387 Corporation, Science & Medical Systems Business Group 24-14, Nishi-shimbashi 1-chome,

16
388 Minato-ku, Tokyo 105-8717 Japan) for the collaborative study conducted together with the

389 IHU Méditerranée Infection, and for the installation of a TM4000 microscope at IHU

390 Méditerranée Infection facility. The authors also thank Magdalen Lardière for English

391 reviewing.

392 Funding source

393 This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée

394 Infection, the National Research Agency under the program « Investissements d’avenir »,

395 reference ANR-10-IAHU-03.

396 SB, JCL and DR are co-inventors of a patent (N° WO/2018/234645).

397 Competing Interests

398 The authors declare no competing interests.

399

17
400 References

401 Abadias, M., Benabarre, A., Teixidó, N., Usall, J., Viñas, I., 2001. Effect of freeze drying and
402 protectants on viability of the biocontrol yeast Candida sake. Int. J. Food Microbiol.
403 65, 173–182.
404 https://doi.org/10.1016/S0168-1605(00)00513-4.

405 Abadias, M., Teixidó, N., Usall, J., Benabarre, A., Vinas, I., 2001. Viability, efficacy, and
406 storage stability of freeze-dried biocontrol agent Candida sake using different
407 protective and rehydration media. J. Food Prot. 64, 856–861.
408 https://doi.org/10.4315/0362-028X-64.6.856.

409 Baumann, D.P., 1964. Preservation of lactic cultures. J Dairy Sci 47:674–676.
410 https://doi.org/10.31274/rtd-180813-1582.

411 Bellali, S., Lagier, J.-C., Raoult, D., Khalil, B., Yaacoub, J., 2019. Among live and dead
412 bacteria, the optimization of sample collection and processing remains essential in
413 recovering gut microbiota components. Front. Microbiol. 10, 1606.
414 https://doi.org/10.3389/fmicb.2019.01606
415 Berner, D., Viernstein, H., 2006. Effect of protective agents on the viability of Lactococcus
416 lactis subjected to freeze-thawing and freeze-drying. Sci. Pharm. 74, 137–149.
417 https://doi.org/10.3797/scipharm.2006.74.137.

418 Bevilacqua, A., Cagnazzo, M.T., Caldarola, C., Ciuffreda, E., Dragano, A.R., Franchino, S.,
419 Lauriola, R., Pacifico, A., Corbo, M.R., Sinigaglia, M., 2012. Bifidobacteria as
420 Potential Functional Starter Cultures: A Case Study by MSc Students in Food Science
421 and Technology (University of Foggia, Southern Italy). Food Nutr. Sci. 03, 55–63.
422 https://doi.org/10.4236/fns.2012.31010.

423 Biavati, B., Vescovo, M., Torriani, S., Bottazzi, V., 2000. Bifidobacteria: history, ecology,
424 physiology and applications. Ann. Microbiol. 50, 117–132.

425 Brandt, L.J., Aroniadis, O.C., Mellow, M., Kanatzar, A., Kelly, C., Park, T., Stollman, N.,
426 Rohlke, F., Surawicz, C., 2012. Long-term follow-up of colonoscopic fecal microbiota
427 transplant for recurrent Clostridium difficile infection. Am. J. Gastroenterol. 107,
428 1079–1087.
429 https://doi.org/10.1038/ajg.2012.60.

430 Breyner, N.M., Michon, C., de Sousa, C.S., Vilas Boas, P.B., Chain, F., Azevedo, V.A.,
431 Langella, P., Chatel, J.M., 2017. Microbial Anti-Inflammatory Molecule (MAM) from
432 Faecalibacterium prausnitzii shows a protective effect on DNBS and DSS-Induced
433 colitis model in mice through Inhibition of NF-κB pathway. Front. Microbiol. 8, 114.
434 https://doi.org/10.3389/fmicb.2017.00114.

435 Broeckx, G., Vandenheuvel, D., Claes, I.J.J., Lebeer, S., Kiekens, F., 2016. Drying techniques
436 of probiotic bacteria as an important step towards the development of novel
437 pharmabiotics. Int. J. Pharm. 505, 303–318.
438 https://doi.org/10.1016/j.ijpharm.2016.04.002.

439 Buitink, J., van den Dries, I.J., Hoekstra, F.A., Alberda, M., Hemminga, M.A., 2000. High
440 Critical Temperature above Tg May Contribute to the Stability of Biological Systems.

18
441 Biophys. J. 79, 1119–1128.
442 https://doi.org/10.1016/S0006-3495(00)76365-X.

443 Carpenter, J.F., Crowe, J.H., 1988. Modes of stabilization of a protein by organic solutes
444 during desiccation. Cryobiology 25, 459–470.
445 https://doi.org/10.1016/0011-2240(88)90054-5.

446 Carpenter, J.F., Crowe, L.M., Crowe, J.H., 1987. Stabilization of phosphofructokinase with
447 sugars during freeze-drying: characterization of enhanced protection in the presence of
448 divalent cations. Biochim. Biophys. Acta BBA - Gen. Subj. 923, 109–115.
449 https://doi.org/10.1016/0304-4165(87)90133-4.

450 Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X., Gibbs, P., 2002. Survival of
451 freeze-dried Lactobacillus plantarum and Lactobacillus rhamnosus during storage in
452 the presence of protectants. Biotechnol. Lett. 24, 1587–1591.
453 https://doi.org/10.1023/a:1020301614728.

454 Carvalho, A.S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X., Gibbs, P., 2004. Relevant factors
455 for the preparation of freeze-dried lactic acid bacteria. Int. Dairy J. 14, 835–847.
456 https://doi.org/10.1016/j.idairyj.2004.02.001.

457 Carvalho, Ana S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X., Gibbs, P., 2003. Protective
458 effect of sorbitol and monosodium glutamate during storage of freeze-dried lactic acid
459 bacteria. Le Lait 83, 203–210.
460 https://doi.org/10.1051/lait:2003010.

461 Carvalho, A. S., Silva, J., Ho, P., Teixeira, P., Malcata, F.X., Gibbs, P., 2003. Effects of
462 Addition of Sucrose and Salt, and of Starvation upon Thermotolerance and Survival
463 During Storage of Freeze-dried Lactobacillus delbrueckii ssp bulgaricus. J. Food Sci.
464 68, 2538–2541.
465 https://doi.org/10.1111/j.1365-2621.2003.tb07057.x.

466 Castro, H.P., Teixeira, P.M., Kirby, R., 1997. Evidence of membrane damage in Lactobacillus
467 bulgaricus following freeze drying. J. Appl. Microbiol. 82, 87–94.
468 https://doi.org/10.1111/j.1365-2672.1997.tb03301.x.

469 Castro, H.P., Teixeira, P.M., Kirby, R., 1996. Changes in the cell membrane of Lactobacillus
470 bulgaricus during storage following freeze-drying. Biotechnol. Lett. 18, 99–104.
471 https://doi.org/10.1007/BF00137819.

472 Celik, O.F., O’Sullivan, D.J., 2013. Factors influencing the stability of freeze-dried stress-
473 resilient and stress-sensitive strains of bifidobacteria. J. Dairy Sci. 96, 3506–3516.
474 https://doi.org/10.3168/jds.2012-6327.

475 Champagne, C.P., Gardner, N., Brochu, E., Beaulieu, Y., 1991. The Freeze-Drying of Lactic
476 Acid Bacteria. A Review. Can. Inst. Food Sci. Technol. J. 24, 118–128.
477 https://doi.org/10.1016/S0315-5463(91)70034-5.

478 Costello, S.P., Tucker, E.C., La Brooy, J., Schoeman, M.N., Andrews, J.M., 2016.
479 Establishing a Fecal Microbiota Transplant Service for the Treatment of Clostridium
480 difficile Infection. Clin. Infect. Dis. 62, 908–914. https://doi.org/10.1093/cid/civ994.

19
481 Crowe, J.H., Carpenter, J.F., Crowe, L.M., 1998. The role of vitrification in anhydrobiosis.
482 Annu. Rev. Physiol. 60, 73–103. https://doi.org/10.1146/annurev.physiol.60.1.73.

483 Crowe, J.H., Carpenter, J.F., Crowe, L.M., Anchordoguy, T.J., 1990. Are freezing and
484 dehydration similar stress vectors? A comparison of modes of interaction of stabilizing
485 solutes with biomolecules. Cryobiology 27, 219–231.
486 https://doi.org/10.1016/0011-2240(90)90023-W.

487 Crowe, J.H., Crowe, L.M., Carpenter, J.F., Rudolph, A.S., Wistrom, C.A., Spargo, B.J.,
488 Anchordoguy, T.J., 1988. Interactions of sugars with membranes. Biochim. Biophys.
489 Acta BBA - Rev. Biomembr. 947, 367–384.
490 https://doi.org/10.1016/0304-4157(88)90015-9.

491 Derrien, M., Vaughan, E.E., Plugge, C.M., de Vos, W.M., 2004. Akkermansia muciniphila
492 gen. nov., sp. nov., a human intestinal mucin-degrading bacterium. Int. J. Syst. Evol.
493 Microbiol. 54, 1469–1476.
494 https://doi.org/10.1099/ijs.0.02873-0.

495 Efiuvwevwere, B.J.O., Gorris, L.G.M., Smid, E.J., Kets, E.P.W., 1999. Mannitol-enhanced
496 survival of Lactococcus lactis subjected to drying. Appl. Microbiol. Biotechnol. 51,
497 100–104.
498 https://doi.org/10.1007/s002530051369.

499 Everard, A., Belzer, C., Geurts, L., Ouwerkerk, J.P., Druart, C., Bindels, L.B., Guiot, Y.,
500 Derrien, M., Muccioli, G.G., Delzenne, N.M., 2013. Cross-talk between Akkermansia
501 muciniphila and intestinal epithelium controls diet-induced obesity. Proc. Natl. Acad.
502 Sci. 110, 9066–9071.
503 https://doi.org/10.1073/pnas.1219451110.

504 Font de Valdez, G., Savoy de Giori, G., Pesce de Ruiz Holgado, A., Oliver, G., 1983.
505 Comparative study of the efficiency of some additives in protecting lactic acid bacteria
506 against freeze-drying. Cryobiology 20, 560–566.
507 https://doi.org/10.1016/0011-2240(83)90044-5.

508 Fowler, A., Toner, M., 2006. Cryo-Injury and Biopreservation. Ann. N. Y. Acad. Sci. 1066,
509 119–135.
510 https://doi.org/10.1196/annals.1363.010.

511 Gomes, A.M., Malcata, F.X., 1999. Bifidobacterium spp. and Lactobacillus acidophilus:
512 biological, biochemical, technological and therapeutical properties relevant for use as
513 probiotics. Trends Food Sci. Technol. 10, 139–157.
514 https://doi.org/10.1016/S0924-2244(99)00033-3.

515 Gough, E., Shaikh, H., Manges, A.R., 2011. Systematic review of intestinal microbiota
516 transplantation (fecal bacteriotherapy) for recurrent Clostridium difficile infection.
517 Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 53, 994–1002.
518 https://doi.org/10.1093/cid/cir632.

519 Hamilton, M.J., Weingarden, A.R., Sadowsky, M.J., Khoruts, A., 2012. Standardized frozen
520 preparation for transplantation of fecal microbiota for recurrent Clostridium difficile
521 infection. Am. J. Gastroenterol. 107, 761–767.
522 https://doi.org/10.1038/ajg.2011.482.
20
523 Hecker, M.T., Obrenovich, M.E., Cadnum, J.L., Jencson, A.L., Jain, A.K., Ho, E., Donskey,
524 C.J., 2016. Fecal Microbiota Transplantation by Freeze-Dried Oral Capsules for
525 Recurrent Clostridium difficile Infection. Open Forum Infect. Dis. 3, ofw091.
526 https://doi.org/10.1093/ofid/ofw091.

527 Herdeiro, R.S., Pereira, M.D., Panek, A.D., Eleutherio, E.C.A., 2006. Trehalose protects
528 Saccharomyces cerevisiae from lipid peroxidation during oxidative stress. Biochim.
529 Biophys. Acta BBA - Gen. Subj. 1760, 340–346.
530 https://doi.org/10.1016/j.bbagen.2006.01.010.

531 Heylen, K., Hoefman, S., Vekeman, B., Peiren, J., De Vos, P., 2012. Safeguarding bacterial
532 resources promotes biotechnological innovation. Appl. Microbiol. Biotechnol. 94,
533 565–574.
534 https://doi.org/10.1007/s00253-011-3797-y.

535 Hirsch, B.E., Saraiya, N., Poeth, K., Schwartz, R.M., Epstein, M.E., Honig, G., 2015.
536 Effectiveness of fecal-derived microbiota transfer using orally administered capsules
537 for recurrent Clostridium difficile infection. BMC Infect. Dis. 15, 191.
538 https://doi.org/10.1186/s12879-015-0930-z.

539 Hocquart, M., Lagier, J.-C., Cassir, N., Saidani, N., Eldin, C., Kerbaj, J., Delord, M., Valles,
540 C., Brouqui, P., Raoult, D., Million, M., 2018. Early Fecal Microbiota Transplantation
541 Improves Survival in Severe Clostridium difficile Infections. Clin. Infect. Dis. 66,
542 645–650.
543 https://doi.org/10.1093/cid/cix762.

544 Hubálek, Z., 2003. Protectants used in the cryopreservation of microorganisms. Cryobiology
545 46, 205–229.
546 https://doi.org/10.1016/S0011-2240(03)00046-4.

547 Jain, N.K., Roy, I., 2010. Trehalose and protein stability. Curr. Protoc. Protein Sci. Chapter 4,
548 Unit 4.9.
549 https://doi.org/10.1002/0471140864.ps0409s59.

550 Jalali, M., Abedi, D., Varshosaz, J., Najjarzadeh, M., Mirlohi, M., Tavakoli, N., 2012.
551 Stability evaluation of freeze-dried Lactobacillus paracasei subsp. tolerance and
552 Lactobacillus delbrueckii subsp. bulgaricus in oral capsules. Res. Pharm. Sci. 7, 31–
553 36.

554 Jiang, Z.D., Ajami, N.J., Petrosino, J.F., Jun, G., Hanis, C.L., Shah, M., Hochman, L.,
555 Ankoma-Sey, V., DuPont, A.W., Wong, M.C., Alexander, A., Ke, S., DuPont, H.L.,
556 2017. Randomised clinical trial: faecal microbiota transplantation for recurrent
557 Clostridum difficile infection - fresh, or frozen, or lyophilised microbiota from a small
558 pool of healthy donors delivered by colonoscopy. Aliment. Pharmacol. Ther. 45, 899–
559 908.
560 https://doi.org/10.1111/apt.13969.

561 Kaito, S., Toya, T., Yoshifuji, K., Kurosawa, S., Inamoto, K., Takeshita, K., Suda, W.,
562 Kakihana, K., Honda, K., Hattori, M., Ohashi, K., 2018. Fecal microbiota
563 transplantation with frozen capsules for a patient with refractory acute gut graft-

21
564 versus-host disease. Blood Adv. 2, 3097–3101.
565 https://doi.org/10.1182/bloodadvances.2018024968.

566 Kassam, Z., Lee, C.H., Yuan, Y., Hunt, R.H., 2013. Fecal microbiota transplantation for
567 Clostridium difficile infection: systematic review and meta-analysis. Am. J.
568 Gastroenterol. 108, 500–508.
569 https://doi.org/10.1038/ajg.2013.59.

570 Keivani Nahr, F., Mokarram, R.R., Hejazi, M.A., Ghanbarzadeh, B., Sowti Khiyabani, M.,
571 Zoroufchi Benis, K., 2015. Optimization of the nanocellulose based cryoprotective
572 medium to enhance the viability of freeze dried Lactobacillus plantarum using
573 response surface methodology. LWT - Food Sci. Technol. 64, 326–332.
574 https://doi.org/10.1016/j.lwt.2015.06.004.

575 Lee, C.H., Steiner, T., Petrof, E.O., Smieja, M., Roscoe, D., Nematallah, A., Weese, J.S.,
576 Collins, S., Moayyedi, P., Crowther, M., Ropeleski, M.J., Jayaratne, P., Higgins, D.,
577 Li, Y., Rau, N.V., Kim, P.T., 2016. Frozen vs Fresh Fecal Microbiota Transplantation
578 and Clinical Resolution of Diarrhea in Patients With Recurrent Clostridium difficile
579 Infection: A Randomized Clinical Trial. JAMA 315, 142–149.
580 https://doi.org/10.1001/jama.2015.18098.

581 Leslie, S.B., Israeli, E., Lighthart, B., Crowe, J.H., Crowe, L.M., 1995. Trehalose and sucrose
582 protect both membranes and proteins in intact bacteria during drying. Appl. Environ.
583 Microbiol. 61, 3592–3597.

584 Linders, L.J.M., Wolkers, W.F., Hoekstra, F.A., van ’t Riet, K., 1997. Effect of Added
585 Carbohydrates on Membrane Phase Behavior and Survival of DriedLactobacillus
586 plantarum. Cryobiology 35, 31–40. https://doi.org/10.1006/cryo.1997.2021.

587 Louie, T., Cannon, K., O’grady, H., Wu, K., & Ward, L., 2013. Fecal microbiome
588 transplantation (FMT) via oral fecal microbial capsules for recurrent Clostridium
589 difficile infection (rCDI). ID week, 201389.
590 Marcial-Coba, M.S., Cieplak, T., Cahú, T.B., Blennow, A., Knøchel, S., Nielsen, D.S., 2018.
591 Viability of microencapsulated Akkermansia muciniphila and Lactobacillus plantarum
592 during freeze-drying, storage and in vitro simulated upper gastrointestinal tract
593 passage. Food Funct. 9, 5868–5879.
594 https://doi.org/10.1039/c8fo01331d.

595 Mensink, M.A., Frijlink, H.W., van der Voort Maarschalk, K., Hinrichs, W.L.J., 2017. How
596 sugars protect proteins in the solid state and during drying (review): Mechanisms of
597 stabilization in relation to stress conditions. Eur. J. Pharm. Biopharm. Off. J.
598 Arbeitsgemeinschaft Pharm. Verfahrenstechnik EV 114, 288–295.
599 https://doi.org/10.1016/j.ejpb.2017.01.024.

600 Ming, L.C., Rahim, R.A., Wan, H.Y., Ariff, A.B., 2009. Formulation of Protective Agents for
601 Improvement of Lactobacillus salivarius I 24 Survival Rate Subjected to Freeze
602 Drying for Production of Live Cells in Powderized Form. Food Bioprocess Technol.
603 2, 431–436.
604 https://doi.org/10.1007/s11947-009-0184-0.

22
605 Miquel, S., Martín, R., Rossi, O., Bermúdez-Humarán, L.G., Chatel, J.M., Sokol, H., Thomas,
606 M., Wells, J.M., Langella, P., 2013. Faecalibacterium prausnitzii and human intestinal
607 health. Curr. Opin. Microbiol. 16, 255–261.
608 https://doi.org/10.1016/j.mib.2013.06.003.

609 Paiva, C.L.A., Panek, A.D., 1996. Biotechnological Applications of the Disaccharide
610 Trehalose, in: Biotechnology Annual Review. Elsevier, pp. 293–314.
611 https://doi.org/10.1016/S1387-2656(08)70015-2.

612 Panoff, J.M., Thammavongs, B., Guéguen, M., Boutibonnes, P., 1998. Cold stress responses
613 in mesophilic bacteria. Cryobiology 36, 75–83.
614 https://doi.org/10.1006/cryo.1997.2069.

615 Rudolph, A.S., Crowe, J.H., 1985. Membrane stabilization during freezing: The role of two
616 natural cryoprotectants, trehalose and proline. Cryobiology 22, 367–377.
617 https://doi.org/10.1016/0011-2240(85)90184-1.

618 Sanders, J.W., Venema, G., Kok, J., 1999. Environmental stress responses in Lactococcus
619 lactis. FEMS Microbiol. Rev. 23, 483–501.
620 https://doi.org/10.1111/j.1574-6976.1999.tb00409.x.

621 Satokari, R., Mattila, E., Kainulainen, V., Arkkila, P.E.T., 2015. Simple faecal preparation
622 and efficacy of frozen inoculum in faecal microbiota transplantation for recurrent
623 Clostridium difficile infection – an observational cohort study. Aliment. Pharmacol.
624 Ther. 41, 46–53. https://doi.org/10.1111/apt.13009.

625 Selmer‐Olsen, E., Birkeland, S.-E., Sørhaug, T., 1999. Effect of protective solutes on leakage
626 from and survival of immobilized Lactobacillus subjected to drying, storage and
627 rehydration. J. Appl. Microbiol. 87, 429–437.
628 https://doi.org/10.1046/j.1365-2672.1999.00839.x.

629 Sharma, R., Sanodiya, B.S., Thakur, G.S., Jaiswal, P., Sharma, A., Bisen, P.S., 2014.
630 Standardization of lyophilization medium for Streptococcus thermophilus subjected to
631 viability escalation on freeze drying. Microbiol. Res. 5.
632 https://doi.org/10.4081/mr.2014.5402.

633 Staley, C., Hamilton, M.J., Vaughn, B.P., Graiziger, C.T., Newman, K.M., Kabage, A.J.,
634 Sadowsky, M.J., Khoruts, A., 2017. Successful Resolution of Recurrent Clostridium
635 difficile Infection using Freeze-Dried, Encapsulated Fecal Microbiota; Pragmatic
636 Cohort Study. Am. J. Gastroenterol. 112, 940–947.
637 https://doi.org/10.1038/ajg.2017.6.

638 Surawicz, C.M., Brandt, L.J., Binion, D.G., Ananthakrishnan, A.N., Curry, S.R., Gilligan,
639 P.H., McFarland, L.V., Mellow, M., Zuckerbraun, B.S., 2013. Guidelines for
640 diagnosis, treatment, and prevention of Clostridium difficile infections. Am. J.
641 Gastroenterol. 108, 478–498; quiz 499.
642 https://doi.org/10.1038/ajg.2013.4.

643 Van Immerseel, F., Ducatelle, R., De Vos, M., Boon, N., Van De Wiele, T., Verbeke, K.,
644 Rutgeerts, P., Sas, B., Louis, P., Flint, H.J., 2010. Butyric acid-producing anaerobic
645 bacteria as a novel probiotic treatment approach for inflammatory bowel disease. J.

23
646 Med. Microbiol. 59, 141–143.
647 https://doi.org/10.1099/jmm.0.017541-0.

648 van Nood, E., Vrieze, A., Nieuwdorp, M., Fuentes, S., Zoetendal, E.G., de Vos, W.M., Visser,
649 C.E., Kuijper, E.J., Bartelsman, J.F.W.M., Tijssen, J.G.P., Speelman, P., Dijkgraaf,
650 M.G.W., Keller, J.J., 2013. Duodenal infusion of donor feces for recurrent
651 Clostridium difficile. N. Engl. J. Med. 368, 407–415.
652 https://doi.org/10.1056/NEJMoa1205037.

653 Wang, B., Yao, M., Lv, L., Ling, Z., Li, L., 2017. The Human Microbiota in Health and
654 Disease. Engineering 3, 71–82.
655 https://doi.org/10.1016/J.ENG.2017.01.008

656 WHO, F., 2001. Evaluation of health and nutritional properties of powder milk and live lactic
657 acid bacteria. Food Agric. Organ. U. N. World Health Organ. Expert Consult. Rep. 1–
658 34.

659 Wolfe, J., Bryant, G., 1999. Freezing, drying, and/or vitrification of membrane- solute-water
660 systems. Cryobiology 39, 103–129.
661 https://doi.org/10.1006/cryo.1999.2195

662 Yang, L., Ma, Y., Zhang, Y., 2007. Freeze-drying of live attenuated Vibrio anguillarum
663 mutant for vaccine preparation. Biologicals 35, 265–269.
664 https://doi.org/10.1016/j.biologicals.2007.03.001.

665 Youngster, I., Russell, G.H., Pindar, C., Ziv-Baran, T., Sauk, J., Hohmann, E.L., 2014. Oral,
666 capsulized, frozen fecal microbiota transplantation for relapsing Clostridium difficile
667 infection. JAMA 312, 1772–1778.
668 https://doi.org/10.1001/jama.2014.13875.

669 Yu, Y., Zhang, Z., Wang, Y., Liao, M., Li, B., Xue, L., 2017. Optimization of protectant,
670 salinity and freezing condition for freeze-drying preservation of Edwardsiella tarda. J.
671 Ocean Univ. China 16, 831–839.
672 https://doi.org/10.1007/s11802-017-3331-7.

673 Zayed, G., Roos, Y.H., 2004. Influence of trehalose and moisture content on survival of
674 Lactobacillus salivarius subjected to freeze-drying and storage. Process Biochem. 39,
675 1081–1086.
676 https://doi.org/10.1016/S0032-9592(03)00222-X.

677 Zhang, Y., Ng, I.-S., Yao, C., Lu, Y., 2014. Orthogonal array deciphering MRS medium
678 requirements for isolated Lactobacillus rhamnosus ZY with cell properties
679 characterization. J. Biosci. Bioeng. 118, 298–304.
680 https://doi.org/10.1016/j.jbiosc.2014.02.018.
681

682 Tables and Figures

683 Table 1: Total bacterial count (CFU/mL) before and after freeze drying, in the presence of

684 protectant medium and normal saline solution and survival rates (%) after freeze drying.

24
Samples Medium of Culture Before freeze- drying After freeze-drying % of viability

conservation conditions (CFU/mL) (CFU/mL)

Normal saline Anaerobe 3,00E+09 3,10E+08 10,33

Aerobe 4,22E+08 7,82E+07 18,53

Total cells 3,42E+09 3,88E+08 11,34

Sample 1

Protectant Anaerobe 4,60E+09 4,10E+09 67,39

medium
Aerobe 2,22E+08 1,82E+08 81,98

Total cells 4,82E+09 4,28E+09 88,80

Normal saline Anaerobe 6,33E+09 1,74E+09 27,49

Aerobe 5,92E+08 7,82E+07 13,21

Total cells 6,92E+09 1,82E+09 26,27

Sample 2

Protectant Anaerobe 7,60E+09 6,00E+09 78,95

medium
Aerobe 2,00E+08 1,82E+08 91,00

Total cells 7,80E+09 6,18E+09 79,26

Normal saline Anaerobe 3,33E+09 2,44E+08 7,33

Aerobe 5,70E+08 6,82E+07 11,96

Total cells 3,90E+09 3,12E+08 8,01

Sample 3

Protectant Anaerobe 4,00E+09 3,00E+09 75,00

medium
Aerobe 1,00E+08 1,00E+08 100,00

Total cells 4,10E+09 3,10E+09 75,61

Normal saline Anaerobe 3,30E+09 6,44E+08 19,52

Sample 4

Aerobe 5,70E+08 8,60E+07 15,09

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 Total cells 3,87E+09 7,30E+08 18,86

Protectant Anaerobe 5,00E+09 4,70E+09 94,00

medium
Aerobe 1,00E+08 1,00E+08 100,00

Total cells 5,10E+09 4,80E+09 94,12

Normal saline Anaerobe 5,28E+09 1,00E+08 1,89

Aerobe 8,00E+08 3,00E+06 0,38

Total cells 6,08E+09 1,03E+08 1,69

Sample 5

Protectant Anaerobe 8,30E+09 7,00E+09 84,34

medium
Aerobe 4,50E+08 2,00E+08 44,44

Total cells 8,75E+09 7,20E+09 82,29

Normal saline Anaerobe 13,31

(4,25±1.47)×109 (6,08±6.64)×108
(±10.18)

Aerobe (5,91±1.35)×108 (6,27±3.40)×107 11,83 (±6.87)

Total cells (4,84±1.56)×109 (6,70±6.80)×108 13,23 (±9.56)

Mean

Protectant Anaerobe (5,90±1.92)×109 (4,96±1.76)×109 84,28 (±7.63)

medium
Aerobe 83,49
(2,14±1.43)×108 (1,53±4.88)×108
(±23.07)

Total cells (6,11±2.03)×109 (5,11±1.79)×109 84,01 (±7.44)

685

686 Figure 1: Schematic figure illustrating the design of the protocols established to evaluate the

687 effect of (a) saline water and (b) the protectant medium on the viability of bacteria after

688 freezing and freeze-drying. (I) On individual bacteria, Akkermansia muciniphila and

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689 Escherichia coli. (II) On fecal samples. Bacterial viability and cell integrity were evaluated

690 using the plating method, flow cytometry and scanning electron microscopy.

691 Figure 2: Effect of protectant medium and saline water solution on the viability of (A)

692 Akkermansia muciniphila expressed in log CFU/mL, (B) Escherichia coli expressed in log

693 CFU/mL (C) A. muciniphila expressed the % of viability and (D) E. coli expressed the % of

694 viability, during freezing (at 80°C and in liquid nitrogen), freeze drying, and at 4°C after 48

695 hours and 30 days of storage. Freeze-dried cells were stored at 4°C. Viability was measured

696 before and after all storage conditions using the plate counting method. Statistically

697 significant differences between fresh cells and all storage conditions were analyzed by one-

698 way analysis of variance followed by Bonferroni‘s multiple comparisons test. ∗P < 0.01 ∗∗P

699 < 0.001 ∗∗∗P < 0.0001, ns: not significant.

700 Figure 3: Scanning electron micrographs of Escherichia coli conserved in normal saline

701 solution and with protectant medium in fresh state (a1- a2, e1- e2, respectively), after freeze

702 drying (b1- b2, f1- f2, respectively), freezing at -80°C (c1- c2, g1- g2, respectively) and in liquid

703 nitrogen (d1- d2, h1- h2, respectively). Blue asterisk: dehydrated bacteria, yellow asterisk:

704 non-dehydrated bacteria, yellow arrows: skimmed milk particles.

705 Figure 4: Scanning electron micrographs of Akkermansia muciniphila conserved in normal

706 saline solution and with protectant medium in fresh state (a1- a4, c1- c4, respectively) and after

707 freeze-drying (b1- b4, d1- d2, respectively). Yellow asterisk: Akkermansia muciniphila

708 bacterium, yellow arrows: skimmed milk particles.

709 Figure 5: Statistical differences of viable count of 5 fecal samples before and after freeze

710 drying in presence of protectant medium and normal saline solution. Viable counts were

711 performed by plating count methods (log CFU/mL) (A, B) and flow cytometry technique (log

712 cells/ mL) (C, D). ∗P < 0.01, ns: not significant.

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713 Figure 6: Relative abundance and flow cytometry measurements (Cells/mL) of Live/Dead

714 and Injured bacterial population of 5 fecal samples before and after freezing drying using

715 normal saline solution and protectant medium.

716 Figure 7: Scanning electron micrographs of stool sample conserved in normal saline

717 solution and with protectant medium before (a1- a2, b1- b2, respectively) and after freeze

718 drying (c1- c2, d1- d2, respectively).

719

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