**2.2.2 Amount of mutans streptococci**

If oral infection by mutans streptococci is not sufficient, dental caries is not induced in the experimental animal despite feeding with a caries-inducing diet. The amount of mutans streptococci adhered on teeth also influences the development of caries. Accordingly, the amount of mutans streptococci in the oral cavity should be measured periodically until the end of the experimental schedule. According to several studies, dental caries is definitely induced if the amount of mutans streptococci is >105 colony-forming units (CFU)/mL (Ooshima et al, 1993; Tsunehiro et al, 1997).

#### **2.2.3 Amount of sucrose intake and texture of diets**

Dental caries is positively correlated with the amount of sucrose intake (Sreebny, 1982). Therefore, the amount of diet that the animals ingest needs to be equal among feeding groups. Furthermore, the Vipeholm Dental Caries Study clarified that the texture of food containing sucrose influences the occurrence of dental caries (Gustafsson et al, 1954). In that study, subjects ate several foods (e.g., bread, chocolate, caramel) containing sucrose. The incidence of caries was higher in the group consuming "gooey" foods between meals than in the control group. Namely, the ingestion of sucrose that causes the adhesion to the teeth surface becomes a high risk of dental caries induction. Therefore, the texture and configuration of test materials containing phytochemicals added to the animal experimental diet are important to get significance. If the texture and taste of test substances are unique and likely to influence intake and adhesion, the method to reduce these factors should be implemented.

### **2.2.4 The indirect effect of anticariogenic substances on body except for tooth**

The test substance might have multiple functions apart from anticariogenic effects. Test substances such as ELM, ES and Alg53, which have been used in our experiments, have inhibitory effect on α-glucosidase. Therefore, if experimental animals are given an αglucosidase inhibitor and sucrose, the latter is not digested by intestinal disaccharidases and reaches the large intestine, where it is fermented by microbiota. These intestinal microbiota produce short-chain fatty acids, CO2, NH4 and H2 (Oku, 2005). This action is similar to that of prebiotics such as non-digestible oligosaccharides and sugar alcohols ingested orally. These short-chain fatty acids are energy sources for the host and improve intestinal microflora. In this way, sucrose (the digestion of which in the small intestine is inhibited by

of sucrose is reduced, the prevalence of dental caries is also reduced. Phytochemicals are commonly added to the diet to evaluate anticariogenic effects. After breeding, the molar is removed and the degree of dental caries is scored. The details of the experimental protocol have been described (Ooshima et al, 1981; Tsunehiro et al, 1997). The typical procedure of

According to the established method, we have carried out animal experiments using phytochemicals that revealed anticariogenic effects *in vitro*. Young rats were fed with diet #2000 containing phytochemicals with anticariogenic effects for 60 days. However, our results were inconclusive. Therefore, we would like to describe some key points for the

If oral infection by mutans streptococci is not sufficient, dental caries is not induced in the experimental animal despite feeding with a caries-inducing diet. The amount of mutans streptococci adhered on teeth also influences the development of caries. Accordingly, the amount of mutans streptococci in the oral cavity should be measured periodically until the end of the experimental schedule. According to several studies, dental caries is definitely induced if the amount of mutans streptococci is >105 colony-forming units (CFU)/mL

Dental caries is positively correlated with the amount of sucrose intake (Sreebny, 1982). Therefore, the amount of diet that the animals ingest needs to be equal among feeding groups. Furthermore, the Vipeholm Dental Caries Study clarified that the texture of food containing sucrose influences the occurrence of dental caries (Gustafsson et al, 1954). In that study, subjects ate several foods (e.g., bread, chocolate, caramel) containing sucrose. The incidence of caries was higher in the group consuming "gooey" foods between meals than in the control group. Namely, the ingestion of sucrose that causes the adhesion to the teeth surface becomes a high risk of dental caries induction. Therefore, the texture and configuration of test materials containing phytochemicals added to the animal experimental diet are important to get significance. If the texture and taste of test substances are unique and likely to influence intake and adhesion, the method to reduce these factors should be

**2.2.4 The indirect effect of anticariogenic substances on body except for tooth** 

The test substance might have multiple functions apart from anticariogenic effects. Test substances such as ELM, ES and Alg53, which have been used in our experiments, have inhibitory effect on α-glucosidase. Therefore, if experimental animals are given an αglucosidase inhibitor and sucrose, the latter is not digested by intestinal disaccharidases and reaches the large intestine, where it is fermented by microbiota. These intestinal microbiota produce short-chain fatty acids, CO2, NH4 and H2 (Oku, 2005). This action is similar to that of prebiotics such as non-digestible oligosaccharides and sugar alcohols ingested orally. These short-chain fatty acids are energy sources for the host and improve intestinal microflora. In this way, sucrose (the digestion of which in the small intestine is inhibited by

caries scoring is the Keyes Caries Score (Keyes, 1958).

planning of animal experiments based on our experience.

**2.2.2 Amount of mutans streptococci** 

(Ooshima et al, 1993; Tsunehiro et al, 1997).

implemented.

**2.2.3 Amount of sucrose intake and texture of diets** 

α-glucosidase) provides many beneficial effects. However, if a large amount of sucrose and α-glucosidase are ingested simultaneously, transient diarrhea is caused because of an increase in osmotic pressure in the large intestine. This mechanism is thought to identical to that of lactose intolerance.

When a diet containing ELM or ES is added to Diet #2000 and given to rats, most of the rats suffer osmotic diarrhea during the experimental period and growth is slightly suppressed. ELM and ES strongly inhibit sucrase activity. Hence, a lot of sucrose of Diet #2000 is transferred to the lower intestine and may cause osmotic diarrhea. Osmotic diarrhea may reduce the immune response, and disturb anticariogenic effect of phytochemicals in experimental animals. If experimental animals catch illness except for dental caries during the experiment, the risk of dental caries infection may increase. Therefore, the properties and functional effects of test substance apart from anticariogenic effects need to be examined, and the concentration and form of test substance added to diets should be investigated carefully before carrying out animal experiments.
