**3.2. Methods for** *in vitro* **evaluation of effects of probiotics on intestinal microbiota**

The FAO/WHO refers to probiotics as live microorganisms that administered in adequate doses, benefit the health of the host [11]. The beneficial effects of ingesting probiotics

enhanced relief of the symptoms of lactose intolerance, treatment for diarrhea, reduction of serum cholesterol, enhanced immune response and anticarcinogenic effects [46].

Probiotics and Intestinal Microbiota: Implications in Colon Cancer Prevention 223

A major advance for *in vitro* fermentation systems was the development of continuous multi-stage models, which allow the simulation of horizontal processes. This type of system makes it easy to study the nutritional and physicochemical properties of intestinal microbiota, through the combination of three reactors connected in series, simulating the proximal, distal and transversal colon (see Figure 1). Later, Molly et al. [44] developed the human microbial ecosystem simulator (SHIME ®), which consists of a succession of five connected reactors, which represent the different parts of the human gastrointestinal tract with their respective values of pH, residence time and volumetric capacity (Figure 1). The five reactors are continually agitated and kept at a temperature of 37 ºC by means of a thermostat. The medium is kept in the anaerobic state, by daily injection of N2. The appropriate pH for each portion of the GI tract is controlled automatically by adding 1N

**Figure 1.** Computer controlled simulation of human microbial ecosystem (SHIME ®) housed in the

The adaptation, survival and proliferation of a human intestinal microbiota in continuous fermentation *in vitro* models are depended on environmental parameters such as pH, retention time, temperature, flow rate and oxygen depletion. The rigorous control of these factors allows steady established state in conditions the microbial composition and

The continuous cultivation model has been used in research on the metabolism and ecology of intestinal microbiota, with an emphasis on the use of probiotics [51, 54], prebiotics [55, 56] and the formation of fermentation products [57]. The *in vitro* modeling of host digestive functions in vitro coupled with multistage continuous fermentation, represents the most advanced attempt thus far at simulating interdependent physiological functions within the human gut, stomach lumen and small intestine. Human digestive functions that are

Probiotics Research Laboratory of FCF/UNESP-Brazil. Sivieri et al.[53]

metabolic activity, creating a reproducible system.

NaOH or concentrated HCl [44, 45].

The rising consumption of probiotic products by Europeans is mainly is in the form of dairy products containing generally *Lactobacillus* spp. and *Bifidobacterium* spp. However there are products in which the microorganisms used are strains of *Enterococcus* spp. or yeasts such as *Saccharomyces boulardii* [47]. Foods for human consumption containing lactic acid bacteria (LAB) include fermented milk, fruit juices, wine and sausages. Simple cultures or mixed microorganisms are used in probiotic preparations [48].

Several experimental observations have pointed to the potential protective effect of LAB against the development of tumors in the colon [49]. Within the intestinal microbiota, the LAB complex constitutes part of those bacteria able to promote a beneficial effect. They have an important role in retarding colon carcinogenesis by possibly of influencing metabolic, protective and immunological functions in the intestine [39]. The effect of intake of probiotics on intestinal native microbiota can be assessed through *in vivo* or *in vitro* models. *In vivo* models may involve healthy human volunteers, hospitalized patients or an animal model, but these models have some limitations such as high cost, delay in obtaining results and the type of food or drugs administered [50], whereas, *in vitro* models enable you to simplify the system and study separately the metabolism of native and added microbiota, in the presence of specific substrates [50].

*In vitro* fermentation models range from a simple batch system to more complex systems of continuous flow and multi-stage. *In vitro* gut fermentation models enable the stable cultivation of a complete intestinal microbiota for a defined and model-specific period of time. Selection of the appropriate model requires careful evaluation of the study objectives given the advantages and limitations exhibited by each type of system. Some existing systems are included in the batch, continuous culture, multi-stage continuous culture, continuous artificial digestive system and stationary systems [51].

 Batch fermentation is the growth of a pure or mixed bacterial suspension in a carefully selected medium without the further addition of nutrients. These models are generally closed systems is sealed bottles or reactors containing suspensions of fecal material which are maintained under anaerobic conditions. Several studies have already been carried out, using this type of model in research on the prebiotic potential of fructans. This template is particularly useful to investigate metabolic profiles of SCFAs arising from active metabolism of dietary compounds by intestinal microbiota [50].

Continuous culture fermentation models exist as either single- or multistage systems and are necessary to perform long-term studies, as substrate replenishment and toxic product removal are facilitated. Single-stage continuous fermentation models are often used to elucidate proximal colon function and metabolic activity as the mixing of digest from both the caecum and ascending colon is well simulated in these models [52].

These models have several advantages, such as: the ease of use of the system, the possibility of using radioactive substances and the low operation cost [28].

A major advance for *in vitro* fermentation systems was the development of continuous multi-stage models, which allow the simulation of horizontal processes. This type of system makes it easy to study the nutritional and physicochemical properties of intestinal microbiota, through the combination of three reactors connected in series, simulating the proximal, distal and transversal colon (see Figure 1). Later, Molly et al. [44] developed the human microbial ecosystem simulator (SHIME ®), which consists of a succession of five connected reactors, which represent the different parts of the human gastrointestinal tract with their respective values of pH, residence time and volumetric capacity (Figure 1). The five reactors are continually agitated and kept at a temperature of 37 ºC by means of a thermostat. The medium is kept in the anaerobic state, by daily injection of N2. The appropriate pH for each portion of the GI tract is controlled automatically by adding 1N NaOH or concentrated HCl [44, 45].

222 Lactic Acid Bacteria – R & D for Food, Health and Livestock Purposes

microorganisms are used in probiotic preparations [48].

continuous artificial digestive system and stationary systems [51].

the caecum and ascending colon is well simulated in these models [52].

of using radioactive substances and the low operation cost [28].

of dietary compounds by intestinal microbiota [50].

the presence of specific substrates [50].

enhanced relief of the symptoms of lactose intolerance, treatment for diarrhea, reduction of

The rising consumption of probiotic products by Europeans is mainly is in the form of dairy products containing generally *Lactobacillus* spp. and *Bifidobacterium* spp. However there are products in which the microorganisms used are strains of *Enterococcus* spp. or yeasts such as *Saccharomyces boulardii* [47]. Foods for human consumption containing lactic acid bacteria (LAB) include fermented milk, fruit juices, wine and sausages. Simple cultures or mixed

Several experimental observations have pointed to the potential protective effect of LAB against the development of tumors in the colon [49]. Within the intestinal microbiota, the LAB complex constitutes part of those bacteria able to promote a beneficial effect. They have an important role in retarding colon carcinogenesis by possibly of influencing metabolic, protective and immunological functions in the intestine [39]. The effect of intake of probiotics on intestinal native microbiota can be assessed through *in vivo* or *in vitro* models. *In vivo* models may involve healthy human volunteers, hospitalized patients or an animal model, but these models have some limitations such as high cost, delay in obtaining results and the type of food or drugs administered [50], whereas, *in vitro* models enable you to simplify the system and study separately the metabolism of native and added microbiota, in

*In vitro* fermentation models range from a simple batch system to more complex systems of continuous flow and multi-stage. *In vitro* gut fermentation models enable the stable cultivation of a complete intestinal microbiota for a defined and model-specific period of time. Selection of the appropriate model requires careful evaluation of the study objectives given the advantages and limitations exhibited by each type of system. Some existing systems are included in the batch, continuous culture, multi-stage continuous culture,

 Batch fermentation is the growth of a pure or mixed bacterial suspension in a carefully selected medium without the further addition of nutrients. These models are generally closed systems is sealed bottles or reactors containing suspensions of fecal material which are maintained under anaerobic conditions. Several studies have already been carried out, using this type of model in research on the prebiotic potential of fructans. This template is particularly useful to investigate metabolic profiles of SCFAs arising from active metabolism

Continuous culture fermentation models exist as either single- or multistage systems and are necessary to perform long-term studies, as substrate replenishment and toxic product removal are facilitated. Single-stage continuous fermentation models are often used to elucidate proximal colon function and metabolic activity as the mixing of digest from both

These models have several advantages, such as: the ease of use of the system, the possibility

serum cholesterol, enhanced immune response and anticarcinogenic effects [46].

**Figure 1.** Computer controlled simulation of human microbial ecosystem (SHIME ®) housed in the Probiotics Research Laboratory of FCF/UNESP-Brazil. Sivieri et al.[53]

The adaptation, survival and proliferation of a human intestinal microbiota in continuous fermentation *in vitro* models are depended on environmental parameters such as pH, retention time, temperature, flow rate and oxygen depletion. The rigorous control of these factors allows steady established state in conditions the microbial composition and metabolic activity, creating a reproducible system.

The continuous cultivation model has been used in research on the metabolism and ecology of intestinal microbiota, with an emphasis on the use of probiotics [51, 54], prebiotics [55, 56] and the formation of fermentation products [57]. The *in vitro* modeling of host digestive functions in vitro coupled with multistage continuous fermentation, represents the most advanced attempt thus far at simulating interdependent physiological functions within the human gut, stomach lumen and small intestine. Human digestive functions that are

reproduced in the TIM-1 small intestine model include bile secretion, motility, pH and absorption capacity of the upper intestine. Proximal colon simulator models such as TIM-2 include other host functions such as peristaltic mixing and water and metabolite absorption. The combination of TIM-1 and TIM-2 models led to the creation of an artificial digestive system which has been used to investigate pharmaceutical drug delivery and advanced nutritional studies [58, 59].

Probiotics and Intestinal Microbiota: Implications in Colon Cancer Prevention 225

Bacterial glucuronidase appears to have an important role in the initiation of colon cancer, due to its ability to hydrolyze several glucuronides and carcinogenic aglycones in the intestinal lumen [65,68]. The nitroreductase and azoreductase take past in to the formation

Both harmful and beneficial bacteria are commonly found in the intestines and differ in their enzymatic activity [70]. In general, bacteria from the genera *Bifidobacterium* and *Lactobacillus* produced a very little activity of enzymes that convert pro-carcinogens into carcinogens, compared with bacteria from the genera *Bacteroides* and *Clostridium* [71]. Therefore, the activities of these enzymes in the lumen might be correlated with the number of lactic acid bacteria (LAB) in the intestine [72]. This suggests that increasing the proportion of LAB in the gut could diminish the levels of xenobiotic metabolizing enzymes [71]. Thus, the effect of probiotic microorganisms on fecal enzyme activities might be explained by this mechanism.

In a preliminary study, on feces of small animal, the animal supplementation of a high cholesterol diet with a mixture of probiotic strains of *L. johnsonii* and *L. reuteri* for 5 weeks

Gorbach and Goldin [65] studied, in humans, the effect of ingestion of *L. acidophilus* NCFM strains about the activity of-glucuronidase, nitroreductase and azoreductase. Both strains had a similar effect and caused a significant decline in the activity of these three enzymes. A reverse effect was found 10 to 30 days after the end of the intake of these bacteria, suggesting that continuous consumption of *L. acidophilus* is necessary for maintaining.

Benno and Mitsuoka [73] and Spanhaak et al. [66] also found in humans, a significant reduction in the activity glucuronidase after intake of *Bifidobacterium longum* and *L casei* Shirota, respectively. On the other hand, Marteau et al. [74] verified in healthy volunteers that the regular consumption of a fermented dairy product (100 g three times per day) containing *L. acidophilus*, *B. bifidum*, *Streptococcus thermophilus* and *S. cremoris* for 3 weeks decreased the feces nitroreductase activity from baseline but not that of β-glucuronidase or

Feces metabolites are also indicators of bacterial activity. Changes in enzyme activities and the concentration of ammonia, phenol and cresol have been detected in volunteers who consumed Lactobacilli [65]. Other metabolites with possible adverse effects are N-nitroso

A wide variety of microrganisms can produce ammonia, for example, enterobacteria, bacteroides and clostridia. Ammonia is considered a potential promoter of tumor in the colon and it can increase the rate of neoplastic transformation in the intestine. According to Benno and Mitsuoka [73], reducing the proportion of clostridia and bacteroides could explain the decrease in the concentration of ammonia in individuals who consumed fecal *B.* 

Epidemiological studies indicate an association between the risk of developing colon cancer and the consumption of high fat diets [7,75, 76]. For the digestion of fats, bile acids

compounds, diacylglycerol and secondary bile acids [49].

significantly decreased the activity of fecal-glucuronidase and azoreductase [67].

of aromatic amines harmful to the body [69].

azoreductase.

*longum.*

The use of a multidisciplinary biological systems approach, in combination with '-omics' platforms as outlined will facilitate the most advanced system for unraveling the complex microbial and host factors governing human gut microbiota functionality [60].

In vitro fermentation models are an innovative technological platform where the greatest advantages are exhibited by the virtually limitless experimental capacity as experimentation is not restricted by ethical concerns. Host intestinal function is only partially simulated in some model designs (e.g. TIM-1 and TIM-2) and together with microbial population balancing remains a major challenge of in vitro gut fermentation modeling.
