To evaluate the generality of the use of this new cellulose sulphate based delivery method, five different strains of probiotic bacteria (L. acidophilus, L. johnsonii, L. casei, L. casei shirota and B. infantis) were encapsulated in CS (Fig.1A) and all survived the encapsulation process with good viability (60-70% for L. acidophilus and L. johnsonii, 90-100% for L. casei and B. infantis – results not shown). Good viability was also observed for other strains of probiotic bacteria obtained from the DSMZ including Lactobacillus plantarum subsp. plantarum (DSM 20174), Lactobacillus paracasei subsp. paracasei (DSM 20312), Bifidobacterium animalis subsp. lactis (DSM 10140) and Lactobacillus amylolyticus (DSM 11664) (data not shown), indicating that the CS is not toxic for all strains of bacteria and yeast analysed so far. Each CS capsule has a diameter of 0.7 mm and contains on average approximately 5 million L. casei, or 0.5 million L. acidophilus and B. infantis when full (after growth of bacteria within the capsule). The bacteria or yeast containing capsules (Fig 1B) are porous. Scanning Electron Microscopy of the capsules reveals a round shape with some indentations (Fig. 1C). Freeze-fracture of the capsules (Fig. 1D) reveals an outer gelated layer with thickness of about 5 µm, surrounding a space in which the cells are located [35]. The encapsulation process can also be adjusted so that capsules of a defined and reproducible size (with either increased or decreased diameter) can be produced (data not shown).
After encapsulation at fairly low bacterial density (2x106 CFU/ml), the CS capsules containing the bacteria (Fig. 2A) are incubated under standard bacterial growth conditions (appropriate medium and temperature with agitation) for 0,1 or 2 days) to allow the encapsulated bacteria to multiply. Experiments in which alarmarBlue®️ metabolic activity assays were carried out at various time points after encapsulation (Fig. 2A) revealed that the bacteria increased in number within the capsule within hours. As an example, the metabolic activity (expressed as Relative Light Units, RLU) was determined in capsules containing L. casei one and two days after encapsulation (Fig. 2B). Over this 24 hour period the metabolic activity in the capsules increased by 80% suggesting that the bacterial cell numbers had almost doubled. Similar results were obtained for all other bacteria or yeast encapsulated. As an example, Fig. 2C shows a similar increase in metabolic activity for E. coli K12. This was also visually evident when comparing the L. casei capsules immediately after encapsulation (Fig. 2D) with the capsules 24 hours later (Fig. 2E).
To evaluate whether the CS capsules could provide an effective protection against the killing of the encapsulated bacteria by stomach acid, L. casei were encapsulated and cultured for 1 to 2 days post encapsulation till the capsules were full (Fig. 3A). The capsules containing L. casei were then exposed to artificial gastric juice (AGJ) supplemented with pepsin and lysozyme (AGJ+P) for up to three hours or not exposed (0mins). After exposure of encapsulated L. casei for 3 hours to AGJ+P at pH 2, microscopic analysis clearly showed that the capsules remained intact with no deformation (Fig. 3B) even at high magnifications (Fig. 3C).
Similar results were obtained for all other bacteria or yeast encapsulated. As an example L. acidophilus (Fig. 3D and E) and B. infantis (Fig. 3F and G) containing capsules are shown after analogous AGJ+P exposure at low (Fig. 3D and F) and high (Fig. 3E and G) magnifications. These results show that acid exposure even for 3 hours did not affect the integrity of the capsules (compare with non-acid exposed L. casei containing capsules shown in Fig. 2D and E).
CS capsules containing L. casei were recovered immediately (0 mins) or after 1, 2 or 3 hours exposure to AGJ+P at pH 2 and the capsules dissolved using a decapsulation solution that releases the bacteria alive. After serial dilution in MRS medium and plating out on MRS agar plates (Fig. 3A), the growth of decapsulated bacteria exposed to AGJ+P at pH 2 for up to 3 hours (Fig. 3H blue diamonds) is no different to the growth of decapsulated bacteria cultured in MRS throughout and not exposed to AGJ+P (Fig. 3H orange squares).
In a quantitative evaluation of metabolic activity as a surrogate for bacterial number, comparing four different bacteria to demonstrate the generality of the observations, bacteria were either encapsulated and then allowed to grow to fill the capsules over two days, or left non-encapsulated. The relative viability of encapsulated or non-encapsulated bacteria was determined using the indirect metabolic alarmarBlue®️ Assay and initial metabolic activities normalized and set to 100% (Fig 4A). The non-encapsulated and encapsulated bacteria were then exposed to AGJ+P or AGJ for different times before the relative viability was again determined using the alarmarBlue®️ Assay (Fig. 4A). Free, non-encapsulated ( green lines) or encapsulated (¢ red lines) L. acidophilus (Fig. 4B), L. johnsonii (Fig. 4C), B. infantis (Fig. 4D) and L. casei shirota (Fig. 4E) were exposed to AGJ+P at pH 2 for 3 mins, 0.5 hour, 1 hour and 2 hours and the viability after AGJ+P exposure plotted as a percentage of the initial viability (before exposure). The viability of the bacteria in AGJ without pepsin or acid was also measured (¿ blue lines). The results showed that all four strains of encapsulated probiotic bacteria (red lines) survived AGJ+P at pH 2 better than non-encapsulated bacteria (green lines), where viability was reduced to undetectable levels after 30 minutes for all four bacteria (Fig. 4A, B, C and D).
In a second set of experiments, L. casei as an exemplar bacteria and Saccharomyces boulardii as an exemplar yeast were used. The resistance of non-encapsulated freeze dried bacteria or yeast, or bacteria or yeast encapsulated in CS, allowed to grow to fill the capsules, and then freeze dried to mimic the normal formulation of a commercial bacteria or yeast preparation as a freeze dried powder, was evaluated over a 4 hour period in AGJ+P at pH 2, the mean fasting retention time in the stomach [36]. This was followed by one hour exposure to bile. Normalized CFU of freeze dried encapsulated (¢ red lines) or non-encapsulated ( green lines) L. casei (Fig. 5B) or Saccharomyces boulardii (Fig. 5C) were exposed to AGJ+P at pH 2 for four hours, followed by exposure for 1 hour to bile and the number of surviving bacteria or yeast was determined after decapsulation, serial dilution and titration on agar plates. Results were plotted as the change in Relative Viability over time based on an initial Relative Viability set as 1. The viability of the free, non-encapsulated bacteria or yeast in AGJ without pepsin or acid was also measured ( orange lines), as was the viability of encapsulated bacteria or yeast exposed to AGJ at pH 7 (¿ blue lines) and showed no overall changes in viability over the course of the experiment. The viability of non-encapsulated L. casei was reduced ~8 logs within 1 hour exposure to AGJ+P (Fig. 5B. green line 1 hour point) whereas encapsulated L. casei exposed to AGJ+P at pH 2 for 4 hours, followed by 1 hour bile exposure showed no significant effect (Fig. 5B. ¢ red line average of 5 and 6 hours points). Similarly, the viability of non-encapsulated S. boulardii was reduced ~5 logs within 1 hours exposure to AGJ+P (Fig. 5C. green line 1 hour point) whereas encapsulated S. boulardii exposed to AGJ+P at pH 2 for 4 hours, followed by 1 hour bile exposure showed no significant effect (Fig. 5C. ¢ red line average of 5 and 6 hours points). In both cases the addition of bile juice to the encapsulated microbiota caused a transient reduction in cell number followed by recovery within the next hour.
To evaluate whether encapsulated bacteria were released after transit through the stomach and intestine as a result of a combination of the presence of low amounts of active cellulase produced by representatives of Bacillus genus in the human gastrointestinal tract [37], and peristaltic movement causing breakage or bursting of the capsules, both in vitro and in vivo experiments were carried out.
To demonstrate release under these conditions in vitro, CS capsules were incubated at room temperature with gentle shaking in various concentrations of cellulose chosen to reflect those produced by commensal bacillus species in the human gastrointestinal tract [37]. Fig. 6 shows visually the effects of overnight incubation and shaking without cellulase (Control), and with increasing amounts of cellulase (1U/ml, 5U/ml and 10U/ml). Incubation with10 U/ml cellulase and overnight shaking caused the capsules to visually disintegrate (Fig. 6). Table 1 shows the results of the complete experiment in which cellulase concentrations between 0.01U/ml and 10U/ml were tested with or without touch and after incubation for between 1 hour and overnight. Cellulase concentrations of 0.05 U/ml were sufficient to cause capsule disruption (+) on touch after 8 hours (Table 1), whilst even concentrations as low as 0.01 U/ml caused capsule disruption (+) on touch after overnight incubation.
To confirm the in vitro observations that encapsulated bacteria are protected from acid and bile exposure and can be released by the action of cellulases in the lower intestine, two different concentration of non-encapsulated E. coli-LUX or encapsulated E. coli-LUX were administrated to mice by the gavage technique (Fig. 7A). Briefly, freeze dried capsules containing E. coli-LUX (Fig. 7B and C) were rehydrated and decapsulated before being subjected to serial dilution and plating out (Fig. 7D). The number of bacteria per capsule was determined, and the number of capsules calculated that contained either 2.7x109 CFU or 5.3 x 109 CFU. In parallel free non-encapsulated E. coli-LUX that had also been freeze dried and rehydrated were titrated and the volume containing either 2.7x109 CFU or 5.3 x 109 CFU calculated. E. coli-LUX have previously been shown to colonize the mouse gastrointestinal (GI) tract to high levels [31], carry the luxCDABE operon and constitutively auto-luminesce in the absence of exogenous substrate [32]. E. coli-LUX were chosen to allow clear identification and differentiation of the encapsulated bacteria compared to commensal bacteria already present in the mouse which are needed to enable the testing of commensal bacteria cellulase- mediated release of the encapsulated bacteria. Either 2.7x109 CFU or 5.3 x 109 CFU E. coli-LUX were administered to nude mice, either as free bacteria, or in capsules, by oral gavage. There was no lethality and no untoward observations of toxicity during the duration of the study. After 24 hours, mice were euthanized. No significant observations were recorded at necropsy. Organs and feces were collected and placed individually in wells of multi-well plates (Fig. 7A).
Fig. 7E shows the intensity of the bioluminescent signal from colon (upper left well), stomach (upper center well), cecum (upper right well), feces 2 hours post gavage (lower left well), feces 4 hours post gavage (lower center well), feces 24 hours post gavage (lower right well) in mice fed 2.7x109 CFU free E. coli-LUX (left most plate, M1), 5.3x109 CFU free E. coli-LUX (second from left plate, M2), 2.7x109 CFU encapsulated E. coli-LUX (third from left plate, M3) and 5.3x109 CFU encapsulated E. coli-LUX (right most plate, M4). The intensity of bioluminescent signal was not detectable in the tissue samples collected from mice treated with non-encapsulated E. coli-LUX (top rows of two left most plates), and only in the 2 hours feces from non-encapsulated E. coli-LUX (left most well on bottom row of two left most plates). In contrast, a clear bioluminescent signal was seen in the colon of mice treated with encapsulated E. coli-LUX (top left wells of the two rightmost plates). Similarly, the collected feces after 2, 4 and 24 hours showed detectable bioluminescent signal in the mice treated with encapsulated E. coli-LUX (bottom wells of the two rightmost plates).
The bioluminescent signal was quantitated after various timepoints of exposure and the quantitative analysis is shown in Fig. 7F. The signal was detectable mostly in the colon and feces of mice treated with encapsulated E. coli-LUX. Fig. 7F shows the similar amounts of bacteria were found to have remained in the stomach 24 hours after gavage of marked bacteria regardless of whether they were encapsulated or not (Fig. 7F), however more bacteria were found in the cecum in those mice receiving encapsulated rather than non-encapsulated bacteria and this difference was even more marked and more than 1 log higher in the large intestine (colon). Similar differences in amounts of living bacteria were also seen in fecal pellets 2 and 4 hours post-gavage as well as 24 hours after gavage (Fig. 7F). GI transit in a mouse is around 4-6 hrs [38] [39] [40]. Thus, the data suggests that not only are the encapsulated bacteria protected from acid destruction during passage through the stomach, but additionally there is release and colonization of the intestine as evidenced by the continued presence of marked bacteria in the feces at a constant level even after 24 hours.