**4. Effects of** *Enterococcus faecium* **CRL 183 on intestinal microbiota and colon cancer**

Probiotics and Intestinal Microbiota: Implications in Colon Cancer Prevention 231

immune system, positive changes in the intestinal microbiota, and reduction of colon cancer

Sivieri et al [123] studied the effect of daily ingestion of *Enterococcus faecium* CRL 183 (8 log CFU/mL) on the incidence of colorectal tumors induced by 1,2 dimethylhydrazine (DMH) in rats (20 mg/kg body weight, in a weekly dose, for 14 weeks). The experiment was conducted over 42 weeks and the rats were allocated to three groups: G1 - Control (not induced); G2 – Induced with DMH; G3 – Induced with DMH + *E. faecium* CRL 183. Thioglycollate-elicited peritoneal exudate cells (PECs) were harvested from animals in PBS and the adherent cells were obtained after incubation with LPS or RPMI-1640 (CO2 - 95:5, v/v). The cytokine levels (IL-4, IFN- γ and TNF- α*)* were determined in the supernatant of of the cell culture by ELISA. After euthanasia, colons were removed for histological analysis. The animals with induced colorectal cancer and that received the suspension of *Enterococcus faecium* CRL 183 (G3) showed a 50% reduction in average number of tumors compared to G2 (*P* < 0.001) (Figures 2 and 3). The total number of aberrant crypt foci (ACF), the total ACF/mm2, the number of crypts per ACF and the adenocarcinoma were also reduced in G3. In addition, G3

exhibited increased production of IL-4, IFN- γ and TNF- α by PECs compared to G2.

Anti-tumor activities of probiotic acid lactic bacteria have been attributed to an enhanced immune response [132]. The induction of TNF- α by probiotic bacteria would be necessary to initiate cross-talk between the immune cells associated with the lamina propria and the intestinal epithelial cells. IFN- γ is involved in the maturation of immune cells (dendritic cells), controls their cellular proliferation at the intestinal level and induce other cytokines, especially IL-4, IL-5 and IL-10. Because of its role in mediating macrophage and NK cell activation, IFN- γ is important in the host defense against intracellular pathogens, viruses and tumors [133]. According to Perdigón et al. [134] IL-4 exerts control over the inflammatory response induced by the carcinogen. In that study, the antitumor activity of *Enterococcus faecium* CRL 183 was attributed to its ability to modulate the immune response. It has been suggested that increasing the consumption of red meat and animal fat lead to an increased risk of developing cancer colon, in comparison with a vegetarian diet [23]. Several studies have demonstrated that the microbiota of the colon is involved in the etiology of the colon cancer and that the some strains of probiotic microorganism can have beneficial effects on the composition of the intestinal microbiota, stimulates the production of short chain fatty acids (SCFA) and inhibit the activity of enzymes that convert pro-carcinogens into

Based in these evidence, a study was carried out to determine if consumption of a soy product fermented with *E. faecium* CRL 183 was able to modify the fecal microbiota of rats fed a diet containing red meat [122]. The experiment was conducted during over days and the animals were randomly divided into six groups: GI - standard casein-based rodent feed; GII to GVI - beef-based feed. From the 30th day, G III–VI also ingested the following products: G III, *E. faecium*-fermented soy product; G IV, pure suspension of *E. faecium*; G V, sterilized fermented soy product; and G VI, unfermented soy product (3 mL kg−1 BW day−1). The feces of each animal were collected at the start (T0) and on the 30th (T30) and 60th (T60)

development [123, 125,128,130] .

carcinogens [39,49, 135, 136].

*Enterococcus* spp. are Gram-positive, non-sporulating, catalase and oxidase negative facultative anaerobes [72]. Species of this genus are natural constituents of the intestinal microbiota of humans and comprise the third-largest genus of lactic acid bacteria (LAB), after *Lactobacillus* spp. and *Streptococcus* spp. [117].

It is hard to determine the exact number of enterococci species, but from a microbiological and functional point of view, *Enterococcus faecalis* and *Enterococcus faecium* are considered the most important [117, 118].

Some strains of *Enterococcus* spp. exhibit antibiotic resistance, possess virulence factors (adhesions, invasins, pili and haemolysin) and may cause bacteremia, endocarditis and other infections [117]. However, commercial pharmaceutical preparations of enterococci include *Enterococcus faecium* SF68® (NCIMB 10415, produced by Cerbios-Pharma SA, Barbengo, Switzerland) and *Enterococcus faecalis* Symbioor 1 (SymbioPharm, Herborn, Germany), are on the market without reported health problems. Since 2008, *Enterococcus faecium* has been authorized for use in food and recognized as a probiotic microorganism in Brazil [119].

Currently, several strains of *Enterococcus faecium* are considered safe for human consumption, being used as starter cultures in cheese making and other fermented products and recognized as probiotic microorganisms [120]. The use of *Enterococcus faecium* as a starter culture in various fermented foods can be explained by its resistance to high concentrations of NaCl and low pH, and its ability to produce different aromas.

The strain of *Enterococcus faecium* CRL 183 was isolated by researchers from at the Reference Center for Lactobacillus (Cerela-Argentina), from cheese samples of Tafí – a traditional homemade cheese from the highland province of Tucuman, Argentina [121]. *In vitro* and *in vivo* studies showed that *Enterococcus faecium* CRL 183 is able to adhere to the intestinal cells, resists the gastrointestinal environment and colonizes the large intestine of rats, thus satisfying the requirement for a probiotic microorganism [122, 123]. Furthermore, this strain has no antibiotic resistance and no virulence factors, ensuring its safe use as a starter culture [121].

*Enterococcus faecium* CRL 183 has been investigated by our research group for about 20 years, with the objective of defining its functional properties in the free form or associated with food products [122,123,124,125,126, 127, 128, 129, 130].

The best functional effects of *Enterococcus faecium* CRL 183 were obtained when this microorganism was used as a starter culture of a yogurt-like fermented soy product (soy yogurt) [129]. This product has sensorial and technological properties similar to fermentedmilk yogurt drinks and has exhibited functional properties in animal tests and clinical trials. Among the beneficial effects of the soy product fermented with *Enterococcus faecium* CRL 183, the following deserve special attention: improved of lipid profile, modulation of the immune system, positive changes in the intestinal microbiota, and reduction of colon cancer development [123, 125,128,130] .

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

after *Lactobacillus* spp. and *Streptococcus* spp. [117].

**colon cancer** 

Brazil [119].

[121].

most important [117, 118].

**4. Effects of** *Enterococcus faecium* **CRL 183 on intestinal microbiota and** 

*Enterococcus* spp. are Gram-positive, non-sporulating, catalase and oxidase negative facultative anaerobes [72]. Species of this genus are natural constituents of the intestinal microbiota of humans and comprise the third-largest genus of lactic acid bacteria (LAB),

It is hard to determine the exact number of enterococci species, but from a microbiological and functional point of view, *Enterococcus faecalis* and *Enterococcus faecium* are considered the

Some strains of *Enterococcus* spp. exhibit antibiotic resistance, possess virulence factors (adhesions, invasins, pili and haemolysin) and may cause bacteremia, endocarditis and other infections [117]. However, commercial pharmaceutical preparations of enterococci include *Enterococcus faecium* SF68® (NCIMB 10415, produced by Cerbios-Pharma SA, Barbengo, Switzerland) and *Enterococcus faecalis* Symbioor 1 (SymbioPharm, Herborn, Germany), are on the market without reported health problems. Since 2008, *Enterococcus faecium* has been authorized for use in food and recognized as a probiotic microorganism in

Currently, several strains of *Enterococcus faecium* are considered safe for human consumption, being used as starter cultures in cheese making and other fermented products and recognized as probiotic microorganisms [120]. The use of *Enterococcus faecium* as a starter culture in various fermented foods can be explained by its resistance to high

The strain of *Enterococcus faecium* CRL 183 was isolated by researchers from at the Reference Center for Lactobacillus (Cerela-Argentina), from cheese samples of Tafí – a traditional homemade cheese from the highland province of Tucuman, Argentina [121]. *In vitro* and *in vivo* studies showed that *Enterococcus faecium* CRL 183 is able to adhere to the intestinal cells, resists the gastrointestinal environment and colonizes the large intestine of rats, thus satisfying the requirement for a probiotic microorganism [122, 123]. Furthermore, this strain has no antibiotic resistance and no virulence factors, ensuring its safe use as a starter culture

*Enterococcus faecium* CRL 183 has been investigated by our research group for about 20 years, with the objective of defining its functional properties in the free form or associated with

The best functional effects of *Enterococcus faecium* CRL 183 were obtained when this microorganism was used as a starter culture of a yogurt-like fermented soy product (soy yogurt) [129]. This product has sensorial and technological properties similar to fermentedmilk yogurt drinks and has exhibited functional properties in animal tests and clinical trials. Among the beneficial effects of the soy product fermented with *Enterococcus faecium* CRL 183, the following deserve special attention: improved of lipid profile, modulation of the

concentrations of NaCl and low pH, and its ability to produce different aromas.

food products [122,123,124,125,126, 127, 128, 129, 130].

Sivieri et al [123] studied the effect of daily ingestion of *Enterococcus faecium* CRL 183 (8 log CFU/mL) on the incidence of colorectal tumors induced by 1,2 dimethylhydrazine (DMH) in rats (20 mg/kg body weight, in a weekly dose, for 14 weeks). The experiment was conducted over 42 weeks and the rats were allocated to three groups: G1 - Control (not induced); G2 – Induced with DMH; G3 – Induced with DMH + *E. faecium* CRL 183. Thioglycollate-elicited peritoneal exudate cells (PECs) were harvested from animals in PBS and the adherent cells were obtained after incubation with LPS or RPMI-1640 (CO2 - 95:5, v/v). The cytokine levels (IL-4, IFN- γ and TNF- α*)* were determined in the supernatant of of the cell culture by ELISA. After euthanasia, colons were removed for histological analysis. The animals with induced colorectal cancer and that received the suspension of *Enterococcus faecium* CRL 183 (G3) showed a 50% reduction in average number of tumors compared to G2 (*P* < 0.001) (Figures 2 and 3). The total number of aberrant crypt foci (ACF), the total ACF/mm2, the number of crypts per ACF and the adenocarcinoma were also reduced in G3. In addition, G3 exhibited increased production of IL-4, IFN- γ and TNF- α by PECs compared to G2.

Anti-tumor activities of probiotic acid lactic bacteria have been attributed to an enhanced immune response [132]. The induction of TNF- α by probiotic bacteria would be necessary to initiate cross-talk between the immune cells associated with the lamina propria and the intestinal epithelial cells. IFN- γ is involved in the maturation of immune cells (dendritic cells), controls their cellular proliferation at the intestinal level and induce other cytokines, especially IL-4, IL-5 and IL-10. Because of its role in mediating macrophage and NK cell activation, IFN- γ is important in the host defense against intracellular pathogens, viruses and tumors [133]. According to Perdigón et al. [134] IL-4 exerts control over the inflammatory response induced by the carcinogen. In that study, the antitumor activity of *Enterococcus faecium* CRL 183 was attributed to its ability to modulate the immune response.

It has been suggested that increasing the consumption of red meat and animal fat lead to an increased risk of developing cancer colon, in comparison with a vegetarian diet [23]. Several studies have demonstrated that the microbiota of the colon is involved in the etiology of the colon cancer and that the some strains of probiotic microorganism can have beneficial effects on the composition of the intestinal microbiota, stimulates the production of short chain fatty acids (SCFA) and inhibit the activity of enzymes that convert pro-carcinogens into carcinogens [39,49, 135, 136].

Based in these evidence, a study was carried out to determine if consumption of a soy product fermented with *E. faecium* CRL 183 was able to modify the fecal microbiota of rats fed a diet containing red meat [122]. The experiment was conducted during over days and the animals were randomly divided into six groups: GI - standard casein-based rodent feed; GII to GVI - beef-based feed. From the 30th day, G III–VI also ingested the following products: G III, *E. faecium*-fermented soy product; G IV, pure suspension of *E. faecium*; G V, sterilized fermented soy product; and G VI, unfermented soy product (3 mL kg−1 BW day−1). The feces of each animal were collected at the start (T0) and on the 30th (T30) and 60th (T60)

days of the experiment, to determine the viable cell counts of total aerobic and anaerobic bacteria, *Enterococcus* spp., Enterobacteria, *Lactobacillus* spp., *Clostridium* spp., *Bacteroides*  spp. and *Bifidobacterium* spp.

Probiotics and Intestinal Microbiota: Implications in Colon Cancer Prevention 233

Another study, using New Zealand rabbits with induced hypercholesterolemia as an animal model, was conducted to investigate the possible correlation between fecal microbiota, serum lipid parameters and atherosclerotic lesion development. It was show that, after 60 days of the experiment, intake of the probiotic soy product (with or without isoflavones) was correlated with significant increases (P<0.05) in *Lactobacillus* spp.*, Bifidobacterium* spp. and *Enterococcus* spp. and a decrease in the enterobacteria population (Cavallini et al., 2011). The studies conducted by Bedani et al (2010) and Cavallini et al. (2011) suggest that daily ingestion of the soy product fermented with *Enterococcus faecium* CRL 183, or the pure culture of this probiotic microorganism, may contribute to a beneficial balance of the fecal

Currently, other studies, using animal models and an *in vitro* simulator of human intestinal microbial ecosystem (SHIME), are being conducted by our research group in order to elucidate the possible mechanisms involved in the protective effect of *Enterococcus faecium* CRL 183 against colon cancer and the importance of the modulation of fecal microbiota and

From the above discussion, it is evident that probiotics have the capacity to modulate the intestinal microbiota and the immune system, to the benefit of the host organism, reducing the risk of many chronic degenerative diseases, among them colon cancer. It appears also that the actions performed by probiotics are species-strain-dependent, so that several effects or actions can occur with the same bacterial genus. However, the results of several experiments reported in the literature, highlight a degree of controversy concerning the effects observed, especially regarding the various types of cancers and it is difficult to compare these studies. Such controversies are due mainly to large variations in the time of the experiment - usually prevailing those of short duration the experimental models, bacterial strains and the doses and frequencies of administration of probiotics. In this sense, it is important that further studies be done to define and standardize these variables mentioned,

and especially to elucidate the mechanisms involved in each of the observed effects.

acting as adjuvants in specific treatments for existing diseases.

It showed also be mentioned that, according there is also in the literature, that probiotics studied are taken almost exclusively in milk as can be observed in the products available on the market. This condition often makes them inappropriate for certain lactose intolerant population groups on those and allergic to milk proteins. Thus, alternative vehicles for probiotics, free of lactose and of β-lactoglobulin, such as the aqueous extract of soybeans, for example, deserve special attention from researchers seeking to develop products with a good nutritional profile and suitable to transport the probiotic specified for the purpose desire. It is expected that in the near future, as results of the interaction of various fields of study such as food science and technology, nutrition, microbiology, genetic engineering and molecular biology the market can offer consumers products that are more accessible and effective, reducing the risk of certain diseases, particularly certain types of cancer, and

stimulation of the immune system in the disease pathogenesis [53].

microbiota.

**5. Conclusions** 

**Figure 2.** Topographic view of macroscopic growths by G2 – Induced with DMH. Sivieri et al [123]

**Figure 3.** Topographic view of macroscopic growths by G3 – Induced with DMH + *E. faecium* CRL 183. Sivieri et al [123]

By day T30 days of experiment, rats on a red meat-based diet exhibited an increase in the population of total anaerobes, enterobacteria and enterococci and a decrease in the numbers of lactobacilli and bifidobacteria. From T30 to T60, the obtained results showed that fermented soy product and pure *Enterococcus faecium* CRL 183 suspension promoted an increase in the numbers of lactobacilli (0.45 log CFU g−1 and 1.83 log CFU g−1, respectively). During the same period, only the animals treated with pure *Enterococcus faecium* CRL 183 suspension showed a rise in the fecal bifidobacterium population. The fermented soy product promoted a slight fall in the *Bacteroides* spp. population (2*.*80 ± 0*.*20 to 2*.*34 ± 0*.*07 log CFU g−1), but the counts of *clostridia*. and enterobacteria were unchanged.

Another study, using New Zealand rabbits with induced hypercholesterolemia as an animal model, was conducted to investigate the possible correlation between fecal microbiota, serum lipid parameters and atherosclerotic lesion development. It was show that, after 60 days of the experiment, intake of the probiotic soy product (with or without isoflavones) was correlated with significant increases (P<0.05) in *Lactobacillus* spp.*, Bifidobacterium* spp. and *Enterococcus* spp. and a decrease in the enterobacteria population (Cavallini et al., 2011).

The studies conducted by Bedani et al (2010) and Cavallini et al. (2011) suggest that daily ingestion of the soy product fermented with *Enterococcus faecium* CRL 183, or the pure culture of this probiotic microorganism, may contribute to a beneficial balance of the fecal microbiota.

Currently, other studies, using animal models and an *in vitro* simulator of human intestinal microbial ecosystem (SHIME), are being conducted by our research group in order to elucidate the possible mechanisms involved in the protective effect of *Enterococcus faecium* CRL 183 against colon cancer and the importance of the modulation of fecal microbiota and stimulation of the immune system in the disease pathogenesis [53].
