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Probiotic, Prebiotic Research: Taking It Outside (In Vitro)

Probiotic, Prebiotic Research: Taking It Outside (In Vitro)

The health benefits of food products and ingredients can only be fully understood and evaluated if their digestive and fermentative profiles have been characterized under standardized protocols. In general, in vivo approaches (including animal experiments and human clinical trials), despite their recognized scientific relevance, have a number of disadvantages that hinder high-throughput screening. These include labor- and time-consuming setups resulting in high costs. Experimental variability can also confound results because the composition of a person’s gut microbiota can be as specific as his or her fingerprint. Sampling difficulties also occur with in vivo trials due to the inherent complexity of the gastrointestinal tract. Lastly, with in vivo models, ethical constraints must be taken into consideration; any study conducted on humans/animals must be a prioriapproved by a designated panel of experts to determine whether or not the research should be done. Given these in vivo difficulties, high-throughput in vitro technologies that simulate the human gastrointestinal tract have become increasingly important in evaluating the putative benefits of food compounds. Moreover, the mechanistic results obtained using an in vitro approach may subsequently indicate whether further, costlier in vivo studies are worth pursuing.

Well-designed in vitro simulation technologies are essential to assessing the effects of pro- and prebiotics on modulating the intestinal microbiome towards improved health.1 In this respect, 20 years ago the University of Ghent (Belgium) developed the first prototype of the SHIME (Simulator of the Human Intestinal Microbial Ecosystem) technology.2 The SHIME is a scientifically validated and internationally accepted simulator of the gastrointestinal tract and a valuable tool for innovative product development. Gastrointestinal research specialists at the company ProDigest (Ghent, Belgium) have developed a full technology platform around this system to provide customized solutions for gastrointestinal research in the food, functional food, and pharmaceutical industries.

 

SHIME Applications

The SHIME is a dynamic and controlled model of the gastrointestinal tract that simulates sequentially the stomach, small intestine digestions, and the three colonic regions (ascending, transverse, and descending), with its complex and stable microbial communities representative of the human colonic microbiota.3,4

This technology has been used to investigate the activity of different prebiotics.5-8 For example, using the SHIME, inulin fermentation was shown to result in a two-fold increase in butyrate and propionate,5,7 two of the main short-chain fatty acids produced upon carbohydrate fermentation and known to promote health benefits.9 Moreover, inulin was shown to specifically modulate the population of Bifidobacteria, both quantitatively and qualitatively.5,7

Using the SHIME, fermentation of arabinoxylan-oligosaccharides (AXOS), another class of prebiotics, resulted in increased propionate production and modulated the microbial community towards a more beneficial composition,6 particularly regarding mucosa-associated bacteria.10 This last study made use of an upgraded version of the SHIME—the Mucosal-SHIME, or M-SHIME. This new platform allows researchers to study the bacteria that adheres to the mucus in the gut—thereby more accurately mimicking the human gut11—where intestinal epithelial cells are protected by a layer of mucus secreted by goblet cells. This mucus layer provides a habitat for commensal bacteria, preventing the adherence and penetration of pathogens.12

The beneficial effects of these prebiotics have been further validated in vivo13-15 using both animal studies and human clinical trials, thereby confirming the relevance and predictive value of the different SHIME platforms.1 There are many other examples of how the SHIME can accurately simulate what occurs in vivo, including the metabolism of polyphenols, the survivability of probiotics, and relative cholesterol-lowering effects.

SHIME has been used to study EpiCor, a dried fermentate derived from the baker’s yeast Saccharomyces cerevisiae. EpiCor is a proprietary ingredient of Embria Health Sciences (Ankeny, IA), which employs two of this article’s authors. EpiCor has been shown to have immunomodulatory properties both in vivo and in vitro.16-19 Placebo-controlled human trials show that regular consumption of EpiCor is able to reduce cold/flu-like symptoms,16 reduce allergic rhinitis-induced nasal congestion,17 activate NK cells, and enhance erythrocyte health and mucosal immune protection in healthy individuals.18 Moreover, EpiCor has been reported to induce direct activation of human natural killer cells and B lymphocytes and to increase their chemotactic potential in vitro.19 However, the mechanism behind these immune-protective effects remains largely unknown and is likely mediated by EpiCor’s fermentation by-products and subsequently induced alterations in the gastrointestinal tract’s microbial community.

Where EpiCor is concerned, SHIME technology, coupled with the mucus-adhesion assay, studied the intestinal fate of EpiCor. (An article based on this study is in preparation.) A first, short-term screening study of increasing doses of EpiCor indicated that this product is well-fermented in a saccharolytic manner and in a linear dose-response fashion. When compared to the poorly fermented cellulose and even to the prebiotic fiber inulin, EpiCor showed the highest butyrate production 48 hours after incubation. Moreover, the potential prebiotic properties of EpiCor were demonstrated by a dose-dependent increase in Lactobacilli and an increase in Bifidobacteria at an intermediate dose. (Article in preparation.) The effect of EpiCor in the different colonic compartments was also evaluated in long-term experiments using the SHIME. This allowed us to investigate the dynamic changes taking place within the different microbial communities over a long period of time due to daily intake of a predetermined dose of EpiCor. EpiCor administration led to a decrease in coliforms and Staphylococci, two groups of bacteria typically associated with the presence of potential pathogens. In addition, a significant increase in Lactobacilli was observed in the three colonic compartments (ascending, transverse, and descending colon). (Article in preparation.)

We mention these studies done on EpiCor to demonstrate how the SHIME can be a useful and relevant model to study the effects of food components and ingredients on human health. Altogether, for EpiCor, the results obtained by using the SHIME indicate a gradual change over time to both an improved microbial community composition and an improved fermentative profile. These findings, together with the increased butyrate production observed, are likely to help explain the immunomodulatory mechanisms of EpiCor.

 

Extension of the SHIME: Cross-Talk with the Host Compartment

The original SHIME system, as with all other in vitro simulators, lacks a real host biological compartment. In other words, it simulates the bacteria living in the gastrointestinal tract but the tissues of the host are not simulated. Since these gut wall tissues are an important part of the immune response, the lack of a real host biological compartment and associated tissues makes it impossible to test the ultimate effects of food products on the human intestinal epithelia and immune system. Several approaches have been taken to overcome this limitation.

ProDigest has developed a number of cell culture assays to be used in combination with the SHIME, thereby increasing the predictive value of the in-house technology platform. These cells (a combination of enterocytes and macrophages), after proper differentiation and confluent growth, simulate a simplified gut wall, thereby allowing investigation of the potential effects of a test product on the host. Samples taken from specific colonic compartments of the SHIME can be directly introduced in these cell models, and changes in biological parameters investigated.11 The unique aspect of this model relates to the fact that it is not just the intact test product that is tested but rather the complete matrix containing both the product and its metabolites produced by the microbial community. Enabling these experiments using the complete gut matrix is certainly more relevant to gastrointestinal health research, considering that the ultimate goal is to evaluate the final net health effects of the tested food product.

In order to further assess the specific effect of EpiCor, we made use of a co-culture in vitro model—exposing the cells to the SHIME colonic content—where human intestinal epithelial-like cells are co-cultured in the presence of immune cells (described in Marzorati M et al., 2012),11 based on the model described by Satsu and colleagues (2006).20 By mimicking the intestinal mucosal environment, this model allows testing for putative beneficial effects both on the intestinal epithelia and on the immune system. We took a small sample from the SHIME (representative of what is inside our colon when ingesting EpiCor), brought it in contact with a simplified structure (co-culture of epithelial-like and immune cells) representative of our gut wall, and measured specific parameters to understand the response of the host to EpiCor. Moreover, the morphological characteristics and activated mechanisms of this co-culture model simulate the inflammatory status observed in inflammatorybowel disease (IBD), such as Crohn’s disease and ulcerative colitis; therefore, it represents a suitable in vitro model with which to screen for test compounds that can be used to treat or prevent IBD-like symptoms.20

Finally, the attempt to integrate the host with the SHIME has been taken a step further. ProDigest, in collaboration with the University of Ghent, recently developed the Host-Microbiota Interaction (HMI) module21 to be connected with the SHIME system. This is a two-compartment reactor that allows the flow of the metabolites formed within the colonic vessels of the SHIME (upper microbiota compartment) over an integrated lower compartment containing enterocyte-like cells separated by a semi-permeable membrane and a mucus layer.11, 21 The advantage of this device over the co-culture model just described is that it allows testing the effects of the newly formed metabolites on the host-derived enterocyte-like cells in real-time and for up to 48 hours (the approximate time that an enterocyte, during its turnover, is usually exposed to the luminal content of the colon) rather than at a single time point.

Using EpiCor, the integration of the HMI module in the SHIME platform was validated by testing the effects of an anti-inflammatory compound administered to the SHIME for one week. The secretion of pro-inflammatory cytokines by Caco-2 cells grown on the HMI lower compartment was followed up for 48 hours in comparison to the control. The HMI module connected with the SHIME and treated with EpiCor showed lower production of the pro-inflammatory cytokines IL-8 and IL-1-beta. (Article submitted by Marzorati M et al., 2012.)

 

A Time to SHIME

In conclusion, this integrated approach allows the testing for potential pro- and prebiotic effects of food compounds—on the one hand, by evaluating their metabolic fate upon digestion and colonic fermentation, and on the other hand, by assessing the net effect of the formed metabolites on intestinal epithelial function and immune parameters.

This innovative technology platform is designed to be used in the food, functional food, and pharmaceutical industries. Moreover, recent R&D conducted by ProDigest led to the development of a similar platform dedicated to the pig gastrointestinal tract (SPIME), creating new opportunities for the feed industry. Due to its high flexibility, the SHIME technology platform is not limited to pre- and probiotics. For instance, it is also used to evaluate the fate in the gastrointestinal tract of contaminants, the bioavailability of active ingredients, the dissolution of capsules, and the relative targeted delivery of various products (e.g., peptides, enzymes, APIs).

The possibilities are endless. 

 

References

1. Marzorati M et al. “The use of the SHIME-related technology platform to assess the efficacy of pre- and probiotics.” AgroFOOD Industry Hi-Tech, vol. 20, no. 3 (May/June 2009): 50-53.

2. Molly K et al. “Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem.” Applied Microbiology and Biotechnology, vol. 39, no. 2 (May 1993): 254-258.

3. Molly K et al. “Validation of the simulator of the human intestinal microbial ecosystem (SHIME) reactor using microorganism-associated activities.” Microbial Ecology in Health and Disease, vol. 7, no. 4 (April 1994): 191-200.

4. Macfarlane GT et al. “Validation of a three-stage compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon.” Microbial Ecology, vol. 35, no. 2 (March 1998): 180-187.

5. Van de Wiele T et al. “Prebiotic effects of chicory inulin in the simulator of the human intestinal microbial ecosystem.” FEMS Microbiology Ecology, vol. 51, no. 1 (December 2004): 143-153.

6. Sanchez JI et al. “Arabinoxylan-oligosaccharides (AXOS) affect the protein/carbohydrate fermentation balance and microbial population dynamics of the Simulator of Human Intestinal Microbial Ecosystem.” Microbial Biotechnology, vol. 2, no. 1 (January 2009): 101-113.

7. Van de Wiele T et al. “Inulin-type fructans of longer degree of polymerization exert more pronounced in vitro prebiotic effects.” Journal of Applied Microbiology, vol. 102, no. 2 (February 2007): 452-460.

8. Van den Abbeele P et al.“In vitro model to study the modulation of the mucin-adhered bacterial community.” Applied Microbiology and Biotechnology, vol. 83, no. 2 (May 2009): 349-359.

9. Hijova E et al. “Short chain fatty acids and colonic health.” Bratislava Medical Journal, vol. 108, no. 8 (2007): 354-358.

10. Van den Abbeele P et al. “Arabinoxylans and inulin modulate the luminal and mucosa associated bacteria in vitro and in vivo” in Dietary Fibre - New frontiers for food and health. (eds. J.J. McCleary B, Topping D, & e. van der Kamp JW) (Wageningen Academic Publishers, 2009): 233-249.

11. Marzorati M et al. “An in vitro technology platform to assess host-microbiota interactions in the gastrointestinal tract.” AgroFOOD Industry Hi-Tech, vol. 23, no. 6 (November/December 2012): 8-11.

12. Hansson GC. “Role of mucus layers in gut infection and inflammation.” Current Opinion in Microbiology, vol. 15, no. 1 (February 2012): 57-62.

13. Van Craeyveld V et al. “Structurally different wheat-derived arabinoxylooligosaccharides have different prebiotic and fermentation properties in rats.”Journal of Nutrition, vol. 138, no. 12 (December 2008): 2348-2355.

14. Kolida S et al. “Prebiotic capacity of inulin-type fructans.” Journal of Nutrition, vol. 137, no. 11S (November 2007): 2503S-2506S.

15. Neyrinck AM et al. “Prebiotic effects of wheat arabinoxylan related to the increase in Bifidobacteria, Roseburia and Bacteroides/Prevotellain diet-induced obese mice.” PLoS One, vol. 6, no. 6 (September 2011): e20944.

16. Moyad MA et al. “Immunogenic yeast-based fermentate for cold/flu-like symptoms in nonvaccinated individuals.” Journal of Alternative and Complementary Medicine, vol. 16, no. 2 (February 2010): 213-218.

17. Moyad MA et al. “Immunogenic yeast-based fermentation product reduces allergic rhinitis-induced nasal congestion: a randomized, double-blind, placebo-controlled trial.” Advances in Therapy, vol. 26, no. 8 (August 2009): 795-804.

18. Jensen GS et al. “A double-blind placebo-controlled, randomized pilot study: Consumption of a high-metabolite immunogen from yeast culture has beneficial effects on erythrocyte health and mucosal immune protection in healthy subjects.” Open Nutrition Journal, vol. 2, no. 8 (September 2008): 68-75.

19. Jensen GS et al. “An anti-inflammatory immunogen from yeast culture induces activation and alters chemokine receptor expression on human natural killer cells and B lymphocytes in vitro.” Nutrition Research, vol. 27, no. 6 (June 2007): 327-335.

20. Satsu H et al. “Induction by activated macrophage-like THP-1 cells of apoptotic and necrotic cell death in intestinal epithelial Caco-2 monolayers via tumor necrosis factor-alpha.” Experimental Cell Research, vol. 312, no. 19 (November 2006): 3909-3919.

21. Marzorati M et al. “Patent application (WO2010/118857A2)” (University of Ghent) Belgium (2010).

 
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