**2. The effects of probiotics on intestinal morphology and cell proliferation**

To investigate the effects of microorganisms on the development of digestive tract, generally animal models are used. Because, obtaining and examining intestinal mucosal samples of people are technically more difficult. The morphological parameters such as length of villi, depth of crypt, villi/crypt proportion, and surface area of villi are used to investigate the effects of microorganisms on intestinal morphology and cell proliferation. The height of villi and the depth of crypt are considered as the indicators of intestinal functions.

In the comparative studies conducted on germ free and conventional animals it has been determined that microorganisms located to digestive tract during the postnatal period caused to decreased villi length and increased crypt depth in conventional animals i.e. in pig (Willing & Van Kessel, 2007), in rat (Ishikawa et al., 1999) and in birds (Furuse & Okumura, 1994). However, it has been also reported that there is no difference between germ-free and conventional animals regarding development of villi (Sharma et al., 1995). It has been determined that length of villi is higher in gnotobiotic animal models as in germ-free animals compared to conventional animals (Herich et al., 2004). The effects of probiotics have been investigated by inoculating probiotics to germ free animals or supplementing conventional animals with probiotics.

#### **2.1 Villus height**

Willing & Van Kessel (2007) have reported that villus height was increased in gnotobiotic piglets inoculated *Lactobacillus fermentum* (monoassociated with *Lactobacillus fermentum*) and Di Giancamillo et al. (2008) also has reported increase in villus height in piglets supplemented with *Pediococcus acidilactici*. Yang et al. (2009) have investigated intestinal tract morphology of mice supplemented orally high, low and moderate doses of *Bifidobacterium adolescentis* BBMN23 and *Bifidobacterium adolescentis* BBMN68 after 2 and 4 weeks of application. Villus height was longer in low dose group compared to those of controls, but moderate and high doses did not affect it after two weeks. However after 4 weeks, villus height increased in all groups supplemented with probiotic. Similarly villus height increase has been reported in studies conducted on birds as an animal model (Samli et al., 2007; Awad et al., 2010).

Effects of the probiotic on villus height may change depending on the species of microorganism or probiotic. For example, villus height in duodenum and ileum increased but did not changed in jejunum of broiler chicks supplemented with *Pediococcus acidlactici* as probiotic (Taheri et al., 2010). On the other hand Günal et al. (2006) reported that villus height of jejunum and ileum increased in broiler chicks applied multi-microbe probiotic product. Segmental differences were also found in comparative studies conducted on germ free and conventional animals. Shirkey et al. (2006) have reported that villus height was the longest in jejunum of pigs supplemented with *Lactobacillus fermentum*. On the other hand Shurson et al. (1990) reported that germ-free pigs had longer ileal and duodenal villi, but shorter jejunal villi compared with their conventional counterparts. *Saccharomyces boulardii* is one of the yeast that has been using as probiotic. It has been determined that there was no significance change in villus height or crypt depth of intestinal biopsy samples obtained from volunteers who were supplemented with *Saccharomyces boulardii* for 14 days (Buts et al., 2002). However, another yeast species (*Saccharomyces cerevisiae*) was reported to increased villus height of ileum in birds (Zhang et al., 2005). Meslin and Sacquet (1984) who investigated microvilli on the surface of enterocytes reported that the microvilli were significantly shorter in all small intestinal regions when the micro flora was present. The decrease in microvillus length (due to the presence of micro flora) in germ free rat, was 5% in the duodenum, 9% in the jejunum and 18% in the ileum. Because increased villus height leads to increased surface area at the same time, digestion and absorption of disaccharides and dipeptides are promoted. In addition, it was indicated that longer villi are correlated with activation of cell mitosis (Samanya & Yamauchy, 2002).

#### **2.2 Crypt depth**

52 New Advances in the Basic and Clinical Gastroenterology

Probiotics are widely used for the promotion and improvement of health in humans and in animal species. Probiotics have been used as a biologically active substance in a large extend of pathologic conditions ranging from antibiotic-associated or travelers' diarrhea, irritable bowel syndrome (IBS), and lactose intolerance to dental caries, ulcers due to *Helicobacter pylori,* hepatic encephalopathy, intestinal motility disorders and neonatal necrotizing enterocolitis (Deshpande et al., 2011). It has been used as a growth, or production performance promoter in poultry species or farm animals. There are also numerous scientific reports about the interaction between probiotics and immune system. On the other hand, the effects of probiotics on digestive physiology and intestinal tract morphology have not been documented sufficiently. Therefore, the objective of this chapter is to assess the effects of probiotics on gastrointestinal physiology and morphology in human and animal models. The effects of probiotics on digestive and absorptive function of the intestine, expression of brush border enzymes and nutrient transport systems have been investigated in this chapter. The relationship between probiotics and gut motility or transit time of gastrointestinal content has also been highlighted. The effects of probiotics on morphological characteristics and the proliferation capacity of crypt and villus epithelium have been focused and in addition, the effects of probiotic on enteric nervous system have been evaluated. Finally, impact of the probiotics on the physical and functional barrier of

**2. The effects of probiotics on intestinal morphology and cell proliferation** 

and the depth of crypt are considered as the indicators of intestinal functions.

To investigate the effects of microorganisms on the development of digestive tract, generally animal models are used. Because, obtaining and examining intestinal mucosal samples of people are technically more difficult. The morphological parameters such as length of villi, depth of crypt, villi/crypt proportion, and surface area of villi are used to investigate the effects of microorganisms on intestinal morphology and cell proliferation. The height of villi

In the comparative studies conducted on germ free and conventional animals it has been determined that microorganisms located to digestive tract during the postnatal period caused to decreased villi length and increased crypt depth in conventional animals i.e. in pig (Willing & Van Kessel, 2007), in rat (Ishikawa et al., 1999) and in birds (Furuse & Okumura, 1994). However, it has been also reported that there is no difference between germ-free and conventional animals regarding development of villi (Sharma et al., 1995). It has been determined that length of villi is higher in gnotobiotic animal models as in germ-free animals compared to conventional animals (Herich et al., 2004). The effects of probiotics have been investigated by inoculating probiotics to germ free animals or supplementing

Willing & Van Kessel (2007) have reported that villus height was increased in gnotobiotic piglets inoculated *Lactobacillus fermentum* (monoassociated with *Lactobacillus fermentum*) and Di Giancamillo et al. (2008) also has reported increase in villus height in piglets supplemented with *Pediococcus acidilactici*. Yang et al. (2009) have investigated intestinal tract morphology of mice supplemented orally high, low and moderate doses of *Bifidobacterium adolescentis* BBMN23 and *Bifidobacterium adolescentis* BBMN68 after 2 and 4

gastrointestinal tract has been evaluated in this chapter.

conventional animals with probiotics.

**2.1 Villus height** 

The data related to effects of probiotics on crypt depth are inconsistent. Probably there are variations depending on the species, the dose or the application of used probiotic. Yang et al. (2009) have reported that crypt depth decreased in mice supplemented with moderate and high doses of probiotic (*Bifidobacterium adolescentis* BBMN23) for 2 weeks, but on the contrary it was increased in low dose probiotic supplemented group compared to controls. However after 4 weeks of application, increased crypt depth has been reported in moderate and high doses groups. Willing & Van Kessel (2007) have reported that crypt depth was increased in piglets inoculated with *Lactobacillus fermentum* (monoassociated with *Lactobacillus fermentum*) compared to conventional animals. Similarly, increased crypt depth in duodenum, jejunum and ileum of chicks supplemented with *Bacillus subtilis* has been found (Pelicano et al., 2005).

Scharek et al. (2005) have reported that there was no significant change in the crypt depth in proximal jejunum of pigs supplemented with *Enterococcus faecium* 68. Similarly, it has been stated that crypt depth didn't change in duodenum, but decreased in ileum of broiler chicks supplemented with *Lactobacillus sp* (Awad et al., 2009). In addition, it has been reported that crypt depth was not changed in broiler chicks supplemented with *Saccharomyces cerevisiae*

The Impact of Probiotics on the Gastrointestinal Physiology 55

fatty acids (SCFA) (Sakata et al., 1999). Investigators have suggested that proliferative effects of probiotics on intestinal epithelial cells are based on the probiotic induced increased SCFA production. In the same way, Di Giancamillo et al. (2008) have reported that enterocyte

Mogilner et al. (2007) have reported that there was a decrease in enterocyte death via

It has been suggested that bacteria used as probiotic affect functions and counts of the goblet cells in intestinal mucosa other than enterocytes. Because the mucus secreted by goblet cells is one of the factors composing intestinal barrier, it has significance on the prevention of

Gauffin Cano et al. (2002) reported an increase in the number of goblet cells after the administration of the probiotic strain *Lactobacillus casei* CRL 431 in a malnourished mouse model. In addition there is ample evidence that intestinal microbiota affect goblet cell dynamics, including mucus secretion and composition either directly by the secretion of bioactive factors or indirectly by the activation of host immune cells (Sharma et al., 1995).

Either gastrointestinal motility or the kinetic of its content is one of the most important variations providing gastrointestinal tract's comfort. The changes in motility can make symptoms varying from constipation to diarrhea come into being (Ohashi & Ushida, 2009). The interest to probiotics, due to their motility regulatory effects, is rising in functional

The influence of microorganisms on intestinal motility was reported for the first time by Abraham and Bishop. These researchers observed that both small and large intestinal transit time and gastric emptying decreased in germ-free animals (Abraham & Bishop, 1967). Husebye et al. (1994) detected that phase-3 intervals in migrating motor complex (MMC) in germ-free animals were extended. In addition, they noticed that the motility became normal when specific pathogen free bacteria were inoculated in these animals' intestines. They also noticed that the motility of intestines became normal when probiotics were inoculated

Actually gastrointestinal motility and microorganisms in the tracts are mutually influencing each other. The presence or the absence of the motility affects microorganisms' colonization and also the motility is being altered in case the microorganisms are lacking. Both migrating motor complex in stomach and the one way peristaltic movements in small intestine influence the colonization in the area (Quigley, 2011). Thus the decrease of intestinal motility

In addition to commensal bacteria in gastrointestinal tract, those used as probiotic were also detected to be influencing the motility (Williams et al., 2010). Diverse researches related to the subject in both human and animal models were conducted. Massi et al. (2006), observed *in vitro* the influence of probiotics on motility in ileum and proximal colon segments isolated from guinea pigs. They realized that *Lactobacillus* and cytoplasmic extract obtained from *Bifidobacterium* caused a contraction in ileum and a relaxation in proximal colon. They claimed that the extract mentioned above does not exert its effect via muscarinic receptors

proliferation increased in piglets supplemented with *Pediococcus acidilactici*.

pathogen invasion in digestive tract.

gastrointestinal disorders.

instead of doing so with commensal bacteria.

causes small intestinal bacterial overgrowth (SIBO).

apoptosis in rats with short bowel syndrome supplemented with *Lactobacillus* GG.

**3. The effect of probiotics on the motility of the gastrointestinal tract** 

yeast (Zhang et al., 2005). Increased crypt depth indicates that both mucosal secretion (Chiou et al., 1996) and cell turnover are high (Yason et al., 1987).

#### **2.3 Villus height/crypt depth ratio**

In studies conducted on germ free animals it has been determined that the villus height/crypt depth ratio is higher in germ free animals than conventional animals (Heneghan et al., 1984). Awad et al. (2010) have reported that villus height to crypt depth ratio increased in duodenum and ileum of chicks supplemented with *Lactobacillus* sp. Similarly, supplementation of multi-microbe probiotic product has been reported to cause increased villus height to crypt depth ratio in duodenum and ileum (Kim et al., 2011). It has been indicated that, increased villus height to crypt depth ratio are directly correlated with an increased epithelial turnover (Fan et al., 1997). Therefore, it may be concluded that bacteria used as probiotic positively affect development of intestinal epithelia.

#### **2.4 Villus surface area**

The effects of probiotics on villus surface area may change depending on the segment which bacteria colonized. For example, jejunum villus surface area increased, but duodenum or ileum surface area did not affected in chicks supplemented with probiotic containing *Lactobacillus acidophilus*, *Lactobacillus casei*, *Bifidobacterium bifidum*, and *Enterococcus faecium* species (Smirnov et al., 2005). Similarly Samanya and Yamauchy (2002) have reported that *Bacillus subtilis natto* increased villus surface dose dependently but this increase varied between different segments. Increased surface area allows for increased intestinal absorptive area. Increased absorptive area is useful because digested nutrients pass into the villi through diffusion, so effectiveness of diffusion increases.

#### **2.5 Cell proliferation, migration and turnover**

Proliferation, migration, number and apoptosis of cells in intestinal tract are affected by secreted molecules or fermented products of microorganisms in digestive tract.

It has been determined that cell count and mitotic index in intestinal villus and crypt was higher in gnotobiotic rats mono-associated with *Lactobacillus rhamnousus* GG compared to germ-free or conventional animals (Banasaz et al., 2002). Probiotics both increase cells in intestinal mucosa and affect the migration of cells in crypt to the tip of villus. Canonici et al. (2011) have revealed by *in vitro* and *in vivo* studies that *Saccharomyces boulardii* accelerates migration of intestinal enterocytes through crypt-villus axis by activating α2β1 integrin collagen receptor. Because of this effect, *Saccharomyces boulardii* accelerates the repair of intestinal epithelium damage.

Results related to the effects of probiotics on the proliferation of intestinal epithelium cells are controversial. Mogilner et al. (2007) have reported that *Lactobacillus* GG' did not affect proliferation of enterocytes in rats with short bowel syndrome. Similarly, it has been reported that *Saccharomyces boulardii* did not affect proliferation of enterocytes (Canonici et al., 2011). However Ichikawa et al. (1999) have suggested that *Clostridium butyricum* and *Lactobacillus casei* had tropic effect on digestive tract by enhancing proliferation of intestinal epithelial cells. It has been reported that probiotics increased the production of short-chain

yeast (Zhang et al., 2005). Increased crypt depth indicates that both mucosal secretion

In studies conducted on germ free animals it has been determined that the villus height/crypt depth ratio is higher in germ free animals than conventional animals (Heneghan et al., 1984). Awad et al. (2010) have reported that villus height to crypt depth ratio increased in duodenum and ileum of chicks supplemented with *Lactobacillus* sp. Similarly, supplementation of multi-microbe probiotic product has been reported to cause increased villus height to crypt depth ratio in duodenum and ileum (Kim et al., 2011). It has been indicated that, increased villus height to crypt depth ratio are directly correlated with an increased epithelial turnover (Fan et al., 1997). Therefore, it may be concluded that

The effects of probiotics on villus surface area may change depending on the segment which bacteria colonized. For example, jejunum villus surface area increased, but duodenum or ileum surface area did not affected in chicks supplemented with probiotic containing *Lactobacillus acidophilus*, *Lactobacillus casei*, *Bifidobacterium bifidum*, and *Enterococcus faecium* species (Smirnov et al., 2005). Similarly Samanya and Yamauchy (2002) have reported that *Bacillus subtilis natto* increased villus surface dose dependently but this increase varied between different segments. Increased surface area allows for increased intestinal absorptive area. Increased absorptive area is useful because digested nutrients pass into the villi

Proliferation, migration, number and apoptosis of cells in intestinal tract are affected by

It has been determined that cell count and mitotic index in intestinal villus and crypt was higher in gnotobiotic rats mono-associated with *Lactobacillus rhamnousus* GG compared to germ-free or conventional animals (Banasaz et al., 2002). Probiotics both increase cells in intestinal mucosa and affect the migration of cells in crypt to the tip of villus. Canonici et al. (2011) have revealed by *in vitro* and *in vivo* studies that *Saccharomyces boulardii* accelerates migration of intestinal enterocytes through crypt-villus axis by activating α2β1 integrin collagen receptor. Because of this effect, *Saccharomyces boulardii* accelerates the repair of

Results related to the effects of probiotics on the proliferation of intestinal epithelium cells are controversial. Mogilner et al. (2007) have reported that *Lactobacillus* GG' did not affect proliferation of enterocytes in rats with short bowel syndrome. Similarly, it has been reported that *Saccharomyces boulardii* did not affect proliferation of enterocytes (Canonici et al., 2011). However Ichikawa et al. (1999) have suggested that *Clostridium butyricum* and *Lactobacillus casei* had tropic effect on digestive tract by enhancing proliferation of intestinal epithelial cells. It has been reported that probiotics increased the production of short-chain

secreted molecules or fermented products of microorganisms in digestive tract.

bacteria used as probiotic positively affect development of intestinal epithelia.

through diffusion, so effectiveness of diffusion increases.

**2.5 Cell proliferation, migration and turnover** 

intestinal epithelium damage.

(Chiou et al., 1996) and cell turnover are high (Yason et al., 1987).

**2.3 Villus height/crypt depth ratio** 

**2.4 Villus surface area** 

fatty acids (SCFA) (Sakata et al., 1999). Investigators have suggested that proliferative effects of probiotics on intestinal epithelial cells are based on the probiotic induced increased SCFA production. In the same way, Di Giancamillo et al. (2008) have reported that enterocyte proliferation increased in piglets supplemented with *Pediococcus acidilactici*.

Mogilner et al. (2007) have reported that there was a decrease in enterocyte death via apoptosis in rats with short bowel syndrome supplemented with *Lactobacillus* GG.

It has been suggested that bacteria used as probiotic affect functions and counts of the goblet cells in intestinal mucosa other than enterocytes. Because the mucus secreted by goblet cells is one of the factors composing intestinal barrier, it has significance on the prevention of pathogen invasion in digestive tract.

Gauffin Cano et al. (2002) reported an increase in the number of goblet cells after the administration of the probiotic strain *Lactobacillus casei* CRL 431 in a malnourished mouse model. In addition there is ample evidence that intestinal microbiota affect goblet cell dynamics, including mucus secretion and composition either directly by the secretion of bioactive factors or indirectly by the activation of host immune cells (Sharma et al., 1995).
