Efficient Protection of Probiotics for Delivery to the gastric tract by Cellulose Sulphate Encapsulation


 Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota can restore health. This can be achieved by oral delivery of members of the microbiome (including probiotics) or by fecal microbiome transplantation. In order to provide their health promoting effects, microbiota must survive (i) transport and storage (i.e. shelf life) and (ii) transit through the highly acid conditions in the stomach and bile salts in the small intestine. We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS) that protects members of the microbiota from stomach acid and bile.


Viability in acid and testing
1 Artificial gastric juice (AGJ) was produced by mixing HCl (pH2), 2 pepsin (10 g/L), NaCl (2.79 g/L), KCl (8.74 g/L), CaCl2 (0.24 3 g/L), glucose (77 g/L), glucosamine (33 g/L), lysozyme (1.52 4 g/L). Control gastric juice (CGJ) had the same composition as 5 AGJ, except that the HCl, pepsin and lysozyme were not 6 added.  The released bacteria were diluted in 10 fold dilution steps in 16 Testing of encapsulated bacteria in mice 1 A genetically modified strain of E. coli K12 MG1655 kindly 2 provided by Mark Tangney and colleagues [23] was used that 3 colonises the mouse gastrointestinal (GI) tract to high levels 4 [24]. It carries the luxCDABE operon so that it constitutively 5 auto-bioluminesces in the absence of exogenous substrate [25]. 6 Two groups of male nude mice (Charles River/ Nu-FOXn1 nu ) 7 received two different concentrations (2.7x10 9 CFU (dose 1) or 8 5.4x10 9 CFU (dose 2)) of non-encapsulated E. coli-Lux or 9 encapsulated E. coli-Lux [26] [27] administered in 600µl of saline 10 which was orally dosed by gavage. Fecal pellets were collected 11 2 hours, 4 hours and 24 hours post gavage. At 24 hours after 12 gavage of test articles, animals were euthanized. After the 13 necropsy, the stomach, cecum and colon were harvested. The 14 organs and fecal pellets were subjected to bioluminescence 15 imaging using an IVIS 200 spectrum (Perkin Elmer) imaging 16 system. The luminescent exposure time was optimized and the 17 samples were exposed to the emission spectrum of luciferase for 5, 1, and 0.5 seconds. The tissue samples and feces were 1 exposed to the emission spectrum of luciferase for 10 seconds, 2 1, and 2 minutes. The bioluminescence was measured with an 3 open filter. The signal was visualized as pseudocolor images 4 indicating light intensity (red being the most intense and blue and dry ice. They can be stored at -80 o C at this step. The 1 capsules were freeze dried using a commercially available 2 freeze drying machine. When the collecting chamber 3 temperature of the freeze dryer reached -80 o C, the vacuum 4 pump was started and frozen vials with half-opened caps were 5 placed into the freeze drying machine quickly, the door closed 6 and the vacuum pump immediately re-started. Once freeze 7 drying was completed, the freeze-dryer door was opened, the 8 caps quickly closed and sealed with parafilm to ensure the 9 vacuum and airtightness of vials. The freeze-dried vials were 10 stored at room temperature. within the capsule). The encapsulation process is such that 9 beads of a defined and reproducible size can be produced with 10 either increased or decreased diameter as might be needed.  To evaluate whether the capsules could provide an effective 6 protection against stomach acid, artificial gastric juice (AGJ) 7 supplemented with pepsin and lysozyme (AGJ+P) was used.

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Exposure times up to 4 hours at pH2 [28] were tested, the mean 9 fasting retention time in the stomach. plating out on MRS agar plates, the growth of encapsulated bacteria exposed to AGJ+P at pH 2.0 for 3 hours (Fig. 1E) is no 1 different to the growth of encapsulated bacteria cultured in 2 MRS throughout and not exposed to AGJ+P or acid (Fig. 1F). In a quantitative evaluation, free, non-encapsulated ( green 5 lines) or encapsulated (¢ red lines) L. acidophilus ( Fig. 2A), L. Saccharomyces boulardi (Fig. 3B) were exposed to AGJ+P at 3 pH2 for four hours, followed by exposure for 1 hour to bile. The 4 viability of the free, non-encapsulated bacteria or yeast in AGJ 5 without pepsin or acid was also measured ( orange lines), as 6 was the viability of encapsulated bacteria or yeast exposed to 7 AGJ at pH 7.0 (¿ blue lines) and showed no changes in 8 viability. The viability of non-encapsulated L. casei was reduced 9 ~8 logs within 1 hours exposure to AGJ+P (A. green line 1 10 hour point) whereas encapsulated L. casei exposed to AGJ+P 11 at pH 2 for 4 hours, followed by 1 hour bile exposure showed 12 no significant effect (A. ¢ red line 5 and 6 hours points).

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Similarly the viability of non-encapsulated S. boulardi was 14 reduced ~5 logs within 1 hours exposure to AGJ+P (B. green 15 line 1 hour point) whereas encapsulated S. boulardi exposed to 16 AGJ+P at pH 2 for 4 hours, followed by 1 hour bile exposure 17 showed no significant effect (B. ¢ red line 5 and 6 hours points). In both cases the addition of bile juice to the 1 encapsulated microbiota caused a transient reduction in cell 2 number followed by recovery within the next hour. gastrointestinal tract [29,30]. Fig. 4 shows visually the effects of overnight incubation and shaking without cellulase (Control), 1 and with increasing amounts of cellulase (1U/ml, 5U/ml and 2 10U/ml). Incubation with10 U/ml cellulase and overnight 3 shaking caused the capsules to visually disintegrate (Fig. 4).
4 Table 1 shows the results of the complete experiment in which 5 cellulase concentrations between 0.01U/ml and 10U/ml were 6 tested with or without touch and after incubation for between 1 7 hour and overnight. Cellulase concentrations of 0.05 U/ml were 8 sufficient to cause capsule disruption (+) on touch after 8 hours 9 (Table 1) (Table 2).  shows the similar amounts of bacteria were found to have 1 remained in the stomach 24 hours after gavage of marked 2 bacteria regardless of whether they were encapsulated or not 3 (Fig. 5B), however more bacteria were found in the cecum in 4 those mice receiving encapsulated rather than non-5 encapsulated bacteria and this difference was even more 6 marked and more than 1 log higher in the large intestine 7 (colon). Similar differences in amounts of living bacteria were 8 also seen in fecal pellets 2 and 4 hours post-gavage as well as 9 24 hours after gavage (Fig. 5B). GI transit in a mouse is around Use of poly-L-lysine (PLL) to coat the alginate encapsulated L. 14 acidophilus or L. casei has less of a protective effect after 15 exposure to AGJ at pH 1.55 for two hours with losses in viability 16 of 4-5 logs and of 5-6 logs respectively [19]. In another study 17 losses of viability of around 3 logs have been shown for alginate capsules coated with palm oil and PLL exposed to AGJ 1 at pH 2 for two hours for a wide variety of bacteria (L. Most recently, a study has shown that a layer-by-layer 7 approach using chitosan, followed by alginate and repeated 8 (LbL -(CHI/ALG)2) and even a multi-layered Chitosan capsule 9 alone (LbL-(CHI/L100)2) can afford effective protection against 10 pH2 over two hours with only loss of 1 log in viability in AGJ [38].

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However, this study was conducted in the absence of pepsin.

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Thus, there is still a need to find simple methods to protect with 13 high efficiency bacteria delivered by the oral route, such as in 14 FMT from gastric conditions including enzymatic destruction by 15 pepsin and lysozyme.
We have shown here, for a number of commonly used probiotic 1 strains, the ability of cellulose sulphate encapsulation to protect 2 from low pH in artificial gastric juice containing pepsin, followed 3 by treatment with bile. Cellulose sulphate encapsulation offers 4 exceptional protection (Fig. 2) also for strains thought 5 previously to be acid resistant such as L. casei shirota and L.   We were able to 2 mimic this effect in vitro using equivalent concentration ranges 3 of cellulase and gentle agitation overnight (Fig. 4 and Table 1). 4 We also observed a slow release of bacteria through pores of 5 the cellulose beads in vitro that presumably also occurs in vivo 6 (see Table 2). The use of FMT is complicated by the high heterogeneity of 10 fecal samples since no two samples from different individual 11 donors will ever be the same [48]. In this light, the ability to    harvested. These organs, as well as the feces were placed in E. coli-LUC (M2) (orange bars), 2.7x10 9 CFU of encapsulated 1 E. coli-LUX (M3) (grey bars) or 5.3x10 9 CFU of encapsulated E.