TY - JOUR
AU - Nielsen, Dennis, Sandris
AB - ABSTRACT The viability of probiotics is affected by several factors during manufacturing, storage and gastrointestinal tract passage. Protecting the probiotics from harmful conditions is particularly critical for oxygen sensitive species like Akkermansia muciniphila, a bacterium which recently has been proposed as a next-generation probiotic candidate. Previously, we have developed a protocol for microencapsulating A. muciniphila in a xanthan/gellan gum matrix. Here, we report the enhanced survival during storage and in vitro gastric passage of microencapsulated A. muciniphila embedded in dark chocolate. Lactobacillus casei, as a representative species of traditional probiotics, was included in order to compare its behavior with that of A. muciniphila. For A. muciniphila we observed a 0.63 and 0.87 log CFU g−1 reduction during 60 days storage at 4°C or 15°C, respectively. The viability of L. casei remained stable during the same period. During simulated gastric transit (pH 3), microencapsulated A. muciniphila embedded in chocolate showed 1.80 log CFU mL−1 better survival than naked cells, while for L. casei survival was improved with 0.8 log CFU mL−1. In a hedonic sensory test, dark chocolate containing microcapsules were not significantly different from two commercially available chocolates. The developed protocol constitutes a promising approach for A. muciniphila dosage. next-generation probiotics, Akkermansia muciniphila, microencapsulation, probiotic chocolate INTRODUCTION The consumption of probiotics has been proposed as an active strategy in order to modulate the composition and activity of the gut microbiota (GM) and consequently for the therapeutic treatment of GM dysbiosis-associated diseases (El Hage, Hernandez-Sanabria and Van de Wiele 2017). Probiotics can be defined as ‘live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ (Hill et al.2014). However, classical probiotics have shown limited effects on human GM modulation and are—with the exception of human infants—not dominating commensal gut microbes (El Hage, Hernandez-Sanabria and Van de Wiele 2017; Martín et al.2017). Therefore, the necessity of developing next-generation probiotic formulations, consisting of indigenous, anaerobic and functional members of the GM present in healthy subjects, has been suggested (Walker and Lawley 2013). Akkermansia muciniphila relative abundance in the GM is inversely associated with several pathologies including inflammatory bowel disease, obesity and diabetes (Ottman et al.2017). Additionally, oral administration of this strict anaerobe can prevent and counteract the deleterious effects of a high-fat diet in mice (Derrien, Belzer and de Vos 2017). Based on these findings A. muciniphila is considered as a promising next-generation candidate for developing novel food or pharma supplements with beneficial effects (Cani and de Vos 2017). Probiotics must withstand adverse conditions such as osmotic stress and detrimental oxidation of proteins, lipids and DNA during drying processes and subsequent storage (França, Panek and Eleutherio 2007). Moreover, the passage through the upper gastrointestinal tract (GIT) represent an additional challenge to overcome, since acid stress in the stomach and exposure to bile in the small intestine results in bacterial cell membrane structural damage and macromolecule alterations (Mills et al.2011). To protect probiotics from environmental stresses, they can be coated with a physical barrier (Yao et al.2017) in order to improve the survival in food products, during both processing and storage, and during passage thorough the GIT (Ding and Shah 2009). This strategy may ensure the occurrence of relevant concentrations of viable cells at the time of consumption (Mortazavian et al.2007) and allows the delivery of live probiotic bacteria at the site of action. In this regard, microencapsulation of probiotics comprises a segregation of bacterial cells from the external environment by enclosing them in a covalently or ionically cross-linked polymer network (Cook et al.2012). Several studies report the microencapsulation of traditional probiotics in a wide spectrum of biopolymers such as alginate, chitosan, gelatin, xanthan gum, gellan gum, carrageenan, locust bean gum and pectin individually or in different combinations and show that, in comparison with free cells, the survival of microencapsulated bacteria is improved during storage and under simulated gastrointestinal conditions (Sun and Griffiths 2000; Kim et al.2008; Ding and Shah 2009; Fareez et al.2015; Yao et al.2017; Silva et al.2018). Recently, we have developed a protocol for encapsulating A. muciniphila in a xanthan/gellan gum matrix enhancing survival both during storage and during in vitro simulated transit of the upper GIT (Marcial-Coba et al.2018). If the microencapsulated probiotics are aimed to be administered via food intake, a proper food product must be used as a vehicle. In this regard, chocolate has been reported as an ideal carrier of probiotic Lactobacillus and Bifidobacterium based on its low water activity (aw), oxygen tension, low moisture permeability and high fat content, conferring good survival during storage as well as during in vitro simulated upper GIT passage (Possemiers et al.2010; Lalicic-Petronijevic et al.2015; Kemsawasd, Chaikham and Rattanasena 2016; Klindt-Toldam et al.2016). Furthermore, due to the content of flavonoid and phenolic substances, dark chocolate has been associated with potential health benefits (di Giuseppe et al.2008). This, together with the high consumption of this food product by people of all ages and all over the world (Konar et al.2016), makes chocolate an attractive alternative in order to dose high amounts of probiotics in small portions. The aim of this study was to evaluate the suitability of dark chocolate as a carrier of microencapsulated A. muciniphila in terms of survival during storage and in vitro simulated gastric passage as the first report regarding the addition of a next-generation probiotic in a food product. The same parameters were assessed for Lactobacillus casei, a robust and versatile oxygen-tolerant anaerobe as a representative of traditional probiotics, in order to contrast its performance with that of a strict anaerobic next-generation probiotic. MATERIALS AND METHODS Bacterial strains, microencapsulation and freeze-drying Both A. muciniphila and L. casei were propagated, microencapsulated and freeze-dried according to the protocol described by Marcial-Coba et al. (2018). Briefly, a 100 μL aliquot from a frozen stock culture of A. muciniphila DSM 22 959 was inoculated in 10 mL of brain heart infusion broth (BHI) and after incubation at 37°C during 48 h under strictly anaerobic conditions the propagation was scaled-up to a volume of 100 mL and consecutively to 510 mL, which were incubated under the same conditions during 48 h at each step. Similarly, 100 μL of frozen stock culture of L. casei Z11 (Larsen et al.2009; Marcial-Coba et al.2017) were consecutively propagated in 5, 50 and 255 mL of Lactobacilli MRS broth (Becton, Dickinson and Company, Sparks, USA). Each step consisted of 24 h incubations at 37 °C under aerobic conditions. Cells of both strains were harvested by centrifugation at 4500 × g during 15 min at 4°C and washed once with phosphate buffered saline (PBS, pH 7.4). Cell pellets were resuspended in a final volume of 4.5 mL of cryoprotectant solution (sucrose 5%, trehalose 5%). Suspended cells were carefully added to 45 ml of polymer mix (xanthan gum 1%, gellan gum 0.75%) previously described by Sun and Griffiths (2000), carefully homogenized and loaded (repetitively) in a 20 mL syringe. Microcapsules of both strains were formed by conventional extrusion, which consisted in dripping the former mixture, through a syringe needle (23 G × 1.25′, Sterican, B. Braun, Melsungen, Germany), into a constantly stirred (200 rpm) 0.2 M CaCl2 solution. Microcapsules were hardened during 30 min in the same solution, collected by filtration in sterile gauze, rinsed with sterile Milli-Q water and sorted in conic tubes containing 2 mL of cryoprotectant solution. Until this step, all the operations with A. muciniphila were done under anaerobic conditions in an anaerobic chamber (Coy Laboratory products, USA). Gel beads were snap frozen in liquid nitrogen and subsequently freeze-dried during 24 h in a BOC Edwards Modulyo freeze-dryer (Buch & Holm, Herlev Denmark) with the condenser at a temperature of −40 °C at a chamber pressure of 0.2 mbar. Additionally, in order to determine the protective effect of the gel coating and the chocolate matrix against the conditions of a simulated gastric fluid, a batch of naked freeze-dried cells was produced as follows. Cells from a liquid culture (propagated as described above) in a final volume of 50 and 100 mL of L. casei and A. muciniphila, respectively, were collected by centrifugation (4500 × g, 15 min, 4°C), washed and resuspended in a final volume of 9 mL of cryoprotectant solution (sucrose 5%, trehalose 5%). Finally, freeze-drying was carried out under the same parameters as described above. Chocolate preparation and storage Commercially available 70% dark chocolate contained: 47 g of fat, 32 g of carbohydrates, 11 g of dietary fiber, 7.1 g of protein and 0.03 g of salt per 100 g (Marabou, Upplands Väsby, Sweden). It was properly tempered as lined out by Afoakwa et al. (2008), in four steps: melting (50°C), cooling to the point of crystallization (32°C), crystallization (27°C) and conversion of unstable crystals (31°C). After reaching the last temperature, freeze-dried microcapsules of each strain were added separately in a proportion of 2%, immediately after freeze-drying and gently dispersed. Portions of approximately 7.5 g were poured in sterile Petri dishes (50 mm × 15 mm) and packaged in laminated aluminum bags together with a container filled with approximately 30 g of silica gel. The atmosphere inside the package was composed of 90% N2 and 10% CO2 generated by using a sealing machine provided with vacuum and nitrogen purging system (Multivac A300, Germany). Samples were stored at 4°C or 15°C up to 60 days. Viability assessment during chocolate preparation and storage The number of Colony Forming Units (CFU) was determined at different time points: (i) microcapsules immediately after freeze-drying; (ii) chocolate samples immediately after solidification; (iii) chocolate samples after 15, 30 and 60 days of storage either at 4°C or 15°C. For the first time point, 100 mg microcapsules were dissolved in 10 mL of sodium citrate 1% (w/v) and serially diluted in PBS. Likewise, 5 g of chocolate samples containing microencapsulated bacteria were homogenized in 45 mL of preheated (37°C) sodium citrate 1% plus Tween 80 1% (w/v). Proper dilutions (10−2- 10−7 for A. muciniphila and 10−4- 10−8 for L. casei) of each replicate were plated twice on MRS or BHI agar depending on the species and incubated at 37°C during 48 h aerobically and 96 h anaerobically for L. casei and A. muciniphila, respectively. Water activity (aw) and microcapsules size determination The water activity of chocolate samples was measured shortly after solidification and after 15, 30 and 60 days of storage, using a water activity meter (AquaLab Series 3TE, DecagonDevices, Inc. USA) at 25°C. A picture of freeze-dried microcapsules was obtained with standardized distance and magnification settings employing a custom-tailored setup and the size of particles was determined by ImageJ software (Schneider, Rasband and Eliceiri 2012). For this purpose, the size of the images was calibrated with a millimeter scale. In vitro gastric passage Three different kinds of sample, previously stored anaerobically during 15 days at 4°C, were used to evaluate the survival of A. muciniphila and L. casei throughout in vitro gastric passage: (i) Chocolate containing microencapsulated bacteria 2% (w/w); (ii) microencapsulated bacteria and (iii) freeze-dried free cells. To that end, 5 g of chocolate were added into pre-warmed (37°C) Simulated Salivary Fluid (SSF), with pH adjusted to 7.0 as described by Minekus et al. (2014) and mixed for 1 min. Likewise, 100 mg of freeze-dried xanthan/gellan gum microcapsules, which is the approximately amount contained in 5 g of chocolate, and 100 mg of freeze-dried naked cells were added separately in 5 mL of SSF and mixed as previously explained. Conditions of the human stomach were simulated in a static in vitro model according with Minekus et al. (2014). At the beginning of this phase, the whole volume of the former oral bolus was added into 10 mL of pre-warmed (37 °C) Simulated Gastric Fluid (SGF) with 2000 U mL−1 of pepsin from porcine gastric mucosa (EC 232–629-3) (Sigma-Aldrich, St. Louis, USA) in the final digestion mixture and pH was adjusted to 3.0 by the addition of 1 M HCl. The digestion vessel was incubated aerobically at 37 °C in a shaking incubator (225 rpm) for 30 min. After 15 min of incubation, the pH of the simulated gastric chyme was measured and adjusted to 3.0 if necessary. At the end of the incubation period, pH was measured again and adjusted to 6.0 in order to stop the reaction. To evaluate the survival of both strains after exposure to gastric conditions, 10 mL of the in vitro digested chyme were diluted in 90 mL of sodium citrate 1% plus Tween 80 1%; sodium citrate 1% or PBS for the chocolate, microcapsules or free cells samples, respectively. Proper dilutions were plated on BHI or MRS agar and incubated at 37°C for 96 h under anaerobic conditions and for 48 h under aerobic conditions for A. muciniphila and L. casei, respectively. In order to determine the viability loss suffered during the in vitro assay, these results were compared with the concentration of viable cells present in the above-mentioned SSF suspension, which was diluted, plated and incubated, as described above, as a function of the type of sample and bacterial strain. Sensory analysis To preliminarily assess the potential consumer acceptance of dark chocolate enclosing xanthan/gellan gum microcapsules, 17 untrained panelists, from different nationalities between the ages of 25 and 47, evaluated the chocolate samples using a Hedonic scale ranging from 1 to 9, where 1 corresponded to ‘extremely dislike’ and 9 to ‘extremely like’. Besides dark chocolate containing freeze-dried placebo microcapsules without bacterial cells, samples of dark chocolate without beads and dark chocolate with small particles of dried orange (Marabou, Upplands Väsby, Sweden) were also included. All three kinds of samples were tempered as described above and exhibited the same shape. Panelists were asked to rank the general appreciation as well as their perception regarding taste and mouth feel of each of the 3 kinds of samples. They were also asked to indicate how willing they are about consuming chocolate containing perceptible microcapsules as a therapeutic treatment. Statistical analysis Quantification of viable cells during storage and in vitro gastric passage was performed in duplicate and triplicate, respectively, and the corresponding results were expressed as means ± standard deviation. In order to compare the concentration of viable cells occurring in chocolate samples over storage and the viability loss among three different treatments (chocolate + microcapsules, only microcapsules or freeze-dried free cells) during in vitro simulated gastric passage, one-way ANOVA was applied. Tukey's post hoc test was used to determine differences between the mean values. A confidence level of 95% was set for both tests. RESULTS AND DISCUSSION Viability during chocolate preparation and storage Xanthan/gellan gum microcapsules right after freeze-drying had a particle size of 1.88 ± 0.32 mm and contained 9.66 ± 0.02 or 11.05 ± 0.03 log CFU g−1 of A. muciniphila and L. casei, respectively. Addition of microcapsules to melted dark chocolate, in a proportion of 2%, resulted in a concentration of 7.87 ± 0.03 and 9.23 ± 0.01 log CFU g−1 (Fig. 1). Both of these values were close to the theoretical concentrations, 7.96 log CFU g−1 for A. muciniphila and 9.35 log CFU g−1 for L. casei, as a function of the proportion used for the chocolate mixture. This suggests that the addition to melted dark chocolate did not result in an additional death of previously microencapsulated cells in the polymer matrix used in this study. Figure 1. Open in new tabDownload slide Viability (log CFU g−1) of microencapsulated A. muciniphila and L. casei embedded in dark chocolate 70% right after preparation and over 15, 30 and 60 days of storage at 4°C or 15°C. Each value is the mean ± standard deviation of two separate experiments. a Statistically significant from the initial concentration at the 95% of confidence level. Figure 1. Open in new tabDownload slide Viability (log CFU g−1) of microencapsulated A. muciniphila and L. casei embedded in dark chocolate 70% right after preparation and over 15, 30 and 60 days of storage at 4°C or 15°C. Each value is the mean ± standard deviation of two separate experiments. a Statistically significant from the initial concentration at the 95% of confidence level. As shown in Fig. 1, L. casei remained highly stable during 60 days of storage, without suffering a significant viability loss neither at 4°C nor at 15°C. Similar results were observed by Nebesny et al. (2007) for L. casei and L. paracasei embedded in dark chocolate, showing no variation on its viability after 60 days of storage at 4°C, but an approximately 0.5 log CFU g−1at 18°C. A. muciniphila, on the other hand, was stable for 15 days and exhibited a non-significant reduction during the first 30 days of storage at both temperatures, but a significant 0.63 ± 0.05 and 0.87 ± 0.05 log CFU g−1 reduction was observed after 60 days of storage at 4°C and 15°C, respectively. In a previous study, we observed that microencapsulated A. muciniphila after 30 days of storage at 4°C suffered a significant 0.57 log CFU g−1 reduction (Marcial-Coba et al.2018). This suggests that chocolate provided an additional protective effect during storage. Moreover, the combined use of both, biopolymer and chocolate matrices and the metabolically inactive condition of A. muciniphila seems to be a promising strategy in order to maintain its viability during a relatively long period, since, as reported by van der Ark et al. (2017), metabolically active A. muciniphila, encapsulated in a double emulsion suffered a dramatic viability loss of 2 log CFU g−1 after only 72 h of storage at 4°C. Additionally, in accordance with the physical properties of chocolate, no significant variation regarding aw measurement was observed during the above mentioned time of storage. It ranged between 0.20 and 0.27 for both strains (Table 1). Table 1. Water activity (aw) of chocolate samples containing microencapsulated A. muciniphila DSM22959 or L. casei Z11 stored during 15, 30 and 60 days at 4°C or 15°C. Time 0 15 days 4°C 15 days 15°C 30 days 4°C 30 days 15°C 60 days 4°C 60 days 15°C A. muciniphila 0.26 ± 0.02 0.21 ± 0.03 0.23 ± 0.02 0.24 ± 0.02 0.24 ± 0.03 0.23 ± 0.04 0.24 ± 0.02 L. casei 0.27 ± 0.02 0.25 ± 0.02 0.23 ± 0.03 0.22 ± 0.02 0.20 ± 0.02 0.24 ± 0.02 0.23 ± 0.01 Time 0 15 days 4°C 15 days 15°C 30 days 4°C 30 days 15°C 60 days 4°C 60 days 15°C A. muciniphila 0.26 ± 0.02 0.21 ± 0.03 0.23 ± 0.02 0.24 ± 0.02 0.24 ± 0.03 0.23 ± 0.04 0.24 ± 0.02 L. casei 0.27 ± 0.02 0.25 ± 0.02 0.23 ± 0.03 0.22 ± 0.02 0.20 ± 0.02 0.24 ± 0.02 0.23 ± 0.01 Dark chocolate samples contained freeze-dried xanthan/gellan gum microcapsules 2% (w/w) with A. muciniphila or L. casei were stored under anaerobic conditions over 60 days at 4°C or 15°C. Each value is the mean ± the standard deviation of two separate measurements. Open in new tab Table 1. Water activity (aw) of chocolate samples containing microencapsulated A. muciniphila DSM22959 or L. casei Z11 stored during 15, 30 and 60 days at 4°C or 15°C. Time 0 15 days 4°C 15 days 15°C 30 days 4°C 30 days 15°C 60 days 4°C 60 days 15°C A. muciniphila 0.26 ± 0.02 0.21 ± 0.03 0.23 ± 0.02 0.24 ± 0.02 0.24 ± 0.03 0.23 ± 0.04 0.24 ± 0.02 L. casei 0.27 ± 0.02 0.25 ± 0.02 0.23 ± 0.03 0.22 ± 0.02 0.20 ± 0.02 0.24 ± 0.02 0.23 ± 0.01 Time 0 15 days 4°C 15 days 15°C 30 days 4°C 30 days 15°C 60 days 4°C 60 days 15°C A. muciniphila 0.26 ± 0.02 0.21 ± 0.03 0.23 ± 0.02 0.24 ± 0.02 0.24 ± 0.03 0.23 ± 0.04 0.24 ± 0.02 L. casei 0.27 ± 0.02 0.25 ± 0.02 0.23 ± 0.03 0.22 ± 0.02 0.20 ± 0.02 0.24 ± 0.02 0.23 ± 0.01 Dark chocolate samples contained freeze-dried xanthan/gellan gum microcapsules 2% (w/w) with A. muciniphila or L. casei were stored under anaerobic conditions over 60 days at 4°C or 15°C. Each value is the mean ± the standard deviation of two separate measurements. Open in new tab In vitro gastric simulation After in vitro stomach passage at pH 3, microencapsulated strains enclosed in dark chocolate exhibited the lowest viability loss corresponding to 1.04 ± 0.2 and 0.19 ± 0.12 log CFU mL−1 for A. muciniphila and L. casei, respectively. This value was not significantly different from the log CFU mL−1 reduction resulting from the simulated gastric transit of microcapsules (non-embedded in chocolate). However, the log CFU mL−1 reduction of both former treatments was significantly lower than a loss of 3.07 ± 0.06 and 1.43 ± 0.11 log CFU mL−1 suffered by free cells of A. muciniphila and L. casei, respectively (Table 2). Table 2. Viability (log CFU mL−1) of microencapsulated and subsequently enclosed in chocolate; only microencapsulated; or free cells of A. muciniphila DSM22959 and L. casei Z11 during in vitro gastric passage. Akkermansia muciniphila After oral phase After stomach Chocolate + microcapsules 7.47 ± 0.06 6.44 ± 0.20 Microcapsules 7.77 ± 0.06 6.13 ± 0.10 Free cells 7.72 ± 0.13 4.64 ± 0.06a Lactobacillus casei After oral phase After stomach Chocolate + microcapsules 8.74 ± 0.02 8.55 ± 0.12 Microcapsules 9.16 ± 0.04 8.73 ± 0.07 Free cells 9.14 ± 0.16 7.71 ± 0.11a Akkermansia muciniphila After oral phase After stomach Chocolate + microcapsules 7.47 ± 0.06 6.44 ± 0.20 Microcapsules 7.77 ± 0.06 6.13 ± 0.10 Free cells 7.72 ± 0.13 4.64 ± 0.06a Lactobacillus casei After oral phase After stomach Chocolate + microcapsules 8.74 ± 0.02 8.55 ± 0.12 Microcapsules 9.16 ± 0.04 8.73 ± 0.07 Free cells 9.14 ± 0.16 7.71 ± 0.11a In vitro gastric simulation lasted for 30 min at approximately pH 3 and was preceded by simulated oral phase during 1 min at pH 7 for each of the experimental treatments applied for both strains. Dark chocolate enclosing microcapsules 2% possessed a concentration of 7.75 ± 0.03 log CFU/g for A. muciniphila and 9.03 ± 0.1 for L. casei. Xanthan/gellan gum microcapsules (1:0.75) originally contained 9.52 ± 0.06 log CFU/g for A. muciniphila and 10.82 ± 0.07 for L. casei. Freeze-dried free cells exhibited an original concentration of 8.85 ± 0.05 log CFU/g for A. muciniphila and 10.77 ± 0.11 for L. casei. pH values during digestion process: dark chocolate + microcapsules with A. muciniphila: pH0 min = 2.95 ± 0.1, pH15 min = 4.2 ± 0.16, pH30 min = 3.37 ± 0.12; dark chocolate + microcapsules with L. casei: pH0 min = 3.0 ± 0.08, pH15 min = 4.13 ± 0.12, pH30 min = 3.21 ± 0.06; microcapsules with A. muciniphila: pH0 min = 2.93 ± 0.12, pH15 min = 3.42 ± 0.08, pH30 min = 3.18 ± 0.14; microcapsules with L. casei: pH0 min = 3.03 ± 0.08, pH15 min = 3.4 ± 0.12, pH30 min = 3.15 ± 0.13; free cells of A. muciniphila: pH0 min = 2.93 ± 0.12, pH15 min = 3.1 ± 0.07, pH30 min = 3.01 ± 0.11; Free cells of L. casei: pH0 min = 2.96 ± 0.81, pH15 min = 3.16 ± 0.06, pH30 min = 3.03 ± 0.12. Each value is the mean ± the standard deviation of three separate experiments. a log reduction significantly higher than that of the chocolate + microcapsules and only microcapsules treatment. Open in new tab Table 2. Viability (log CFU mL−1) of microencapsulated and subsequently enclosed in chocolate; only microencapsulated; or free cells of A. muciniphila DSM22959 and L. casei Z11 during in vitro gastric passage. Akkermansia muciniphila After oral phase After stomach Chocolate + microcapsules 7.47 ± 0.06 6.44 ± 0.20 Microcapsules 7.77 ± 0.06 6.13 ± 0.10 Free cells 7.72 ± 0.13 4.64 ± 0.06a Lactobacillus casei After oral phase After stomach Chocolate + microcapsules 8.74 ± 0.02 8.55 ± 0.12 Microcapsules 9.16 ± 0.04 8.73 ± 0.07 Free cells 9.14 ± 0.16 7.71 ± 0.11a Akkermansia muciniphila After oral phase After stomach Chocolate + microcapsules 7.47 ± 0.06 6.44 ± 0.20 Microcapsules 7.77 ± 0.06 6.13 ± 0.10 Free cells 7.72 ± 0.13 4.64 ± 0.06a Lactobacillus casei After oral phase After stomach Chocolate + microcapsules 8.74 ± 0.02 8.55 ± 0.12 Microcapsules 9.16 ± 0.04 8.73 ± 0.07 Free cells 9.14 ± 0.16 7.71 ± 0.11a In vitro gastric simulation lasted for 30 min at approximately pH 3 and was preceded by simulated oral phase during 1 min at pH 7 for each of the experimental treatments applied for both strains. Dark chocolate enclosing microcapsules 2% possessed a concentration of 7.75 ± 0.03 log CFU/g for A. muciniphila and 9.03 ± 0.1 for L. casei. Xanthan/gellan gum microcapsules (1:0.75) originally contained 9.52 ± 0.06 log CFU/g for A. muciniphila and 10.82 ± 0.07 for L. casei. Freeze-dried free cells exhibited an original concentration of 8.85 ± 0.05 log CFU/g for A. muciniphila and 10.77 ± 0.11 for L. casei. pH values during digestion process: dark chocolate + microcapsules with A. muciniphila: pH0 min = 2.95 ± 0.1, pH15 min = 4.2 ± 0.16, pH30 min = 3.37 ± 0.12; dark chocolate + microcapsules with L. casei: pH0 min = 3.0 ± 0.08, pH15 min = 4.13 ± 0.12, pH30 min = 3.21 ± 0.06; microcapsules with A. muciniphila: pH0 min = 2.93 ± 0.12, pH15 min = 3.42 ± 0.08, pH30 min = 3.18 ± 0.14; microcapsules with L. casei: pH0 min = 3.03 ± 0.08, pH15 min = 3.4 ± 0.12, pH30 min = 3.15 ± 0.13; free cells of A. muciniphila: pH0 min = 2.93 ± 0.12, pH15 min = 3.1 ± 0.07, pH30 min = 3.01 ± 0.11; Free cells of L. casei: pH0 min = 2.96 ± 0.81, pH15 min = 3.16 ± 0.06, pH30 min = 3.03 ± 0.12. Each value is the mean ± the standard deviation of three separate experiments. a log reduction significantly higher than that of the chocolate + microcapsules and only microcapsules treatment. Open in new tab The high survival of microencapsulated strains enclosed in dark chocolate after in vitro gastric passage might be associated not only to the additional coating of cells by the food matrix, but also to a buffering effect of chocolate in the SGF, since pH after 15 min of incubation was spontaneously increased to 4.2 ± 0.16 for A. muciniphila and 4.13 ± 0.12 for L. casei. Such shift during the incubation was not observed on the other two treatments. Although, there are no other studies evaluating the combined effect of both biopolymer coating and dark chocolate matrix as in the present investigation, Possemiers et al. (2010) reported that L. helveticus encased in a dark chocolate matrix showed a significantly higher survival rate (80%) than that of free cells (17%) after in vitro gastric transit at pH 2, which is comparable with our results. Likewise, L. acidophilus NCFM embedded in 72% dark chocolate, with an initial concentration of 9 log CFU g−1, was reduced by 1.9 log CFU g−1 during exposure to in vitro static gastric conditions for 35 min (Klindt-Toldam et al.2016). Consistent with the present outcomes, we observed in a previous study (Marcial-Coba et al.2018) that A. muciniphila, microencapsulated as in this investigation, exhibited a survival rate of approximately 1% and 49.8% after fasted (pH 2) and fed (pH 4) in vitro gastric simulation, respectively. Likewise, van der Ark et al. (2017) developed an encapsulation protocol of fresh metabolically active A. muciniphila in a water–in-oil-in–water double emulsion. They showed a relative viability of encapsulated cells corresponding to 6.6% after 2 hours of in vitro gastric passage at pH 3 (van der Ark et al.2017) based on the same protocol as used in this study (Minekus et al.2014). This survival rate might be comparable with a 2.4% and 9.9% of remaining viable A. muciniphila microencapsulated in xanthan/gellan gum (1:0.75) or microencapsulated in the same polymer mixture and additionally embedded in dark chocolate 70% after in vitro stomach passage (pH 3), but higher than 0.08% corresponding to the survival rate of freeze-dried free cells exposed to the same conditions. Sensory analysis Although the dark chocolate samples enclosing microcapsules obtained the lowest mean score in the three categories evaluated by the panelists, it was not significantly lower than the score of the other two types of samples (Table 3). It might reflect that the slightly salty taste, due to CaCl2 residues, and crunchiness of freeze-dried microcapsules do not strongly influence the sensory acceptance of chocolate prepared as in the present study. Table 3. Sensory analysis of dark chocolate and dark chocolate with the addition of chopped dried orange or xanthan/gellan gum microcapsules. Dark chocolate Dark chocolate + dried orange Dark chocolate + microcapsules General appreciation 7.71 ± 1.36 7.12 ± 1.45 6.41 ± 2.03 Taste 7.82 ± 1.38 7.12 ± 1.87 6.29 ± 2.27 Mouth feel 7.47 ± 1.29 7.24 ± 1.35 6.29 ± 2.27 Dark chocolate Dark chocolate + dried orange Dark chocolate + microcapsules General appreciation 7.71 ± 1.36 7.12 ± 1.45 6.41 ± 2.03 Taste 7.82 ± 1.38 7.12 ± 1.87 6.29 ± 2.27 Mouth feel 7.47 ± 1.29 7.24 ± 1.35 6.29 ± 2.27 Each value is the mean ± the standard deviation of 17 individual interviews using a 9-point Hedonic scale. Open in new tab Table 3. Sensory analysis of dark chocolate and dark chocolate with the addition of chopped dried orange or xanthan/gellan gum microcapsules. Dark chocolate Dark chocolate + dried orange Dark chocolate + microcapsules General appreciation 7.71 ± 1.36 7.12 ± 1.45 6.41 ± 2.03 Taste 7.82 ± 1.38 7.12 ± 1.87 6.29 ± 2.27 Mouth feel 7.47 ± 1.29 7.24 ± 1.35 6.29 ± 2.27 Dark chocolate Dark chocolate + dried orange Dark chocolate + microcapsules General appreciation 7.71 ± 1.36 7.12 ± 1.45 6.41 ± 2.03 Taste 7.82 ± 1.38 7.12 ± 1.87 6.29 ± 2.27 Mouth feel 7.47 ± 1.29 7.24 ± 1.35 6.29 ± 2.27 Each value is the mean ± the standard deviation of 17 individual interviews using a 9-point Hedonic scale. Open in new tab Furthermore, the general willingness of panelists to consume dark chocolate containing perceptible microcapsules as a therapeutic treatment of a hypothetical pathology, obtained an average score of 8.41 ± 0.91 on a 9-point Hedonic scale indicating a great willingness to use chocolate as a vehicle for probiotics. The present outcomes only provide a preliminary notion of the potential sensory acceptance of dark chocolate with embedded freeze-dried microcapsules, as an alternative vehicle for the therapeutic administration of a next-generation probiotic, since the development of a commercial product containing live cells of A. muciniphila still requires further studies to be performed and the approval for human consumption. CONCLUSION In this study we show, as a proof of concept, that microencapsulation in a xanthan/gellan gum gel matrix and further embedding in dark chocolate conferred an efficient protection to A. muciniphila, a strict anaerobic next-generation probiotic candidate. This was reflected, not only, in a viability loss <1 log CFU g−1 and a final concentration ≥7 log CFU g−1 after 60 days of storage, but also in a high survival rate after in vitro gastric transit at pH 3. This protective effect, probably based on the stable physical properties of both matrices, was also reproduced in L. casei, a robust representative species of traditional probiotics. Moreover, chocolate enables and makes the administration of microcapsules easier than just particles. At the same time, its consumption appears more attractive than a medication in a capsule or tablet format. As a future perspective, additional studies must be performed in order to determine the influence of chocolate composition on the survival of a next-generation probiotic candidate such as A. muciniphila. Based on our results, we conclude that the methodology described in this study might be a promising dosage protocol, after further studies that demonstrate the therapeutic efficiency and safety of A. muciniphila when administered to human subjects. Acknowledgements M. S. Marcial-Coba was supported by a grant from Ecuadorian Secretariat of Higher Education, Science, Technology and Innovation—SENESCYT (open call - 2014). Confilcts of interest. None declared. REFERENCES Afoakwa EO , Paterson A , Fowler M et al. . Modelling tempering behaviour of dark chocolates from varying particle size distribution and fat content using response surface methodology . Innov Food Sci Emerg 2008 ; 9 : 527 – 33 . 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TI - Dark chocolate as a stable carrier of microencapsulated Akkermansia muciniphila and Lactobacillus casei
JF - FEMS Microbiology Letters
DO - 10.1093/femsle/fny290s
DA - 2019-10-01
UR - https://www.deepdyve.com/lp/oxford-university-press/dark-chocolate-as-a-stable-carrier-of-microencapsulated-akkermansia-aFZeSPpRaO
SP - i24
VL - 366
IS - Supplement_1
DP - DeepDyve
ER -