603 Journal of Oral Science, Vol. 59, No. 4, 603-610, 2017 Original Continuous combined intake of polydextrose and lactitol stimulates cecal fermentation and salivary IgA secretion in rats Yuko Yamamoto1), Nobuhisa Kubota2), Toru Takahashi3), Masahiro To4), Takashi Hayashi2), Tomoko Shimizu5), Yohei Kamata5), Juri Saruta2), and Keiichi Tsukinoki2) 1)Department of Dental Hygiene, Kanagawa Dental University, Junior College, Yokosuka, Japan 2)Division of Environmental Pathology, Department of Oral Science, Graduate School of Dentistry, Kanagawa Dental University, Yokosuka, Japan 3)Department of Nutrition and Health Sciences, Fukuoka Women’s University, Fukuoka, Japan 4)Division of Dental Anatomy, Department of Oral Science, Graduate School of Dentistry, Kanagawa Dental University, Yokosuka, Japan 5)Department of Highly Advanced Stomatology, Graduate School of Dentistry, Kanagawa Dental University, Yokosuka, Japan (Received November 25, 2016; Accepted January 10, 2017) Abstract: Immunoglobulin A (IgA), which plays an may upregulate salivary IgA secretion possibly by important role in infection defense, is upregulated in stimulating absorption of SCFAs produced by cecal the large intestine and oral cavity through dietary fermentation. Thus, continuous ingestion of PDX and fiber intake. However, the mechanism underlying lactitol for up to 4 weeks could increase salivary IgA salivary IgA increase through dietary fiber intake and promote immune defense against pathogen inva- remains unknown. This study investigated time- sion through the oral route. dependent effects of non-absorbable polydextrose (PDX) and lactitol intake on salivary IgA secretion Keywords: IgA; saliva; short-chain fatty acid; and cecal fermentation. Five-week-old rats were fed a polydextrose; lactitol; rat. fiber-free diet with or without 25 g/kg PDX and 25 g/ kg lactitol for 1, 4, and 8 weeks. Compared to control, those who ingested PDX and lactitol had higher Introduction salivary IgA flow rates per weight of submandibular The mucosa covers the largest surface area of the human gland tissue at 4 and 8 weeks (P < 0.05), greater cecal body, with the oral cavity being the first mucosal compart- weight and digesta at 1, 4, and 8 weeks (P < 0.05), ment of the gastrointestinal and respiratory tracts (1). and lower concentrations of short chain fatty acids Considering that the oral cavity is constantly exposed to (SCFAs) in cecal digesta (P = 0.0003). These findings the external environment, it is the primary entry route for suggest that the consumption of PDX and lactitol pathogen invasion via inhalation or ingestion (1). Saliva is continuously secreted into the oral cavity by the salivary glands, covering both the oral and pharyngeal mucosa Correspondence to Dr. Keiichi Tsukinoki, Division of (2). Moreover, it contains many important substances, Environmental Pathology, Department of Oral Science, Graduate School of Dentistry, Kanagawa Dental University, 82 Inaoka, such as electrolytes, mucus, antibacterial compounds, Yokosuka, Kanagawa 238-8580, Japan enzymes, and growth factors, which provide lubrication Fax: +81-46-822-8866 E-mail: [email protected] and initiate food digestion (3). Among various antimi- doi.org/10.2334/josnusd.16-0820 crobial agents present in the saliva, IgA antibodies play DN/JST.JSTAGE/josnusd/16-0820 an important role in the direct neutralization of infectious 604 pathogens and their toxins (4,5) and the establishment Table 1 The composition of control and polydextrose (PDX) and maintenance of mucosal homeostasis (1). A decrease + lactitol diets (g/kg) in salivary IgA concentration has been suggested as a Ingredient Control PDX + lactitol possible causal factor in the increased susceptibility to Sucrose 200.0 200.0 Casein 700.0 650.0 upper respiratory tract infection (URTI) observed among Corn oil 50.0 50.0 population groups with low immunity status, such as Mineral mixture (AIN-76)* 35.0 35.0 children, elderly people, and athletes (6). Thus, endur- Vitamin mixture (AIN-76)† 10.0 10.0 ance athletes have high incidence of URTI accounted for dl-Methionine 3.0 3.0 by a decrease in salivary IgA (7,8), and children with IgA Choline bitartrate 2.0 2.0 deficiency frequently develop bronchopulmonary infec- PDX 25.0 Lactitol 25.0 tions, bronchial asthma, gastrointestinal allergies, and Total 1,000.0 1,000.0 other pathological conditions (9). Furthermore, salivary *Mineral mixture (AIN-76) (g/kg): calcium phosphate, 500.0; sodium IgA has been suggested to play an important role in chloride, 74.0; potassium citrate, 220.0; potassium sulphate, 52.0; magnesium oxide 24.0; magnesium carbonate, 3.5; ferric citrate, 6.0; URTI prevention, given that the upregulation of IgA in zinc carbonate, 1.6; cupric carbonate, 0.3; potassium iodate, 0.01; sodium the saliva may decrease the susceptibility to infection and selenite, 0.01; chromium potassium sulphate, 0.55; and sucrose, 118.0. contribute to the maintenance of general health in people †Vitamin mixture (AIN-76) (mg/kg): thiamine, 600; riboflavin, 600; pyridoxine, 700; niacin, 300; calcium pantothenate, 160; folic acid, 200; with decreased immunity (10). biotin, 200; cyanocobalamin, 10; retinol, 24,000; cholecalciferol, 2.5; Ingestion of dietary fiber promotes fermentation in tocotrienols, 5,000; menadione, 5; and sucrose, 979,000. the large intestine (11). Short-chain fatty acids (SCFAs), such as acetic, propionic, and butyric acids, are the major metabolites of anaerobic microbial fermentation of poly- of PDX and lactitol intake on salivary IgA secretion and saccharides in the forestomach and hindgut (12). SCFAs cecal fermentation in rats. are implicated in lipid and protein metabolism, colonic health, and immunity, suggesting that gut microflora Materials and Methods may modulate host immune status through fermentation Animals of dietary fiber to SCFAs (13). Polydextrose (PDX) and The experimental protocol used in this study was lactitol are fermentable peptic carbohydrates known to reviewed and approved by the Ethics Committee for increase SCFA production (14,15,16). PDX, a synthetic Animal Experiments of Kanagawa Dental University oligosaccharide containing in average 12 randomly (#20140724). All experiments were performed in accor- polymerized glucose units, is a commercially avail- dance with the Guidelines for Animal Experimentation able dietary fiber widely used as a multi-purpose food of Kanagawa Dental University and the ARRIVE guide- supplement (14), while lactitol is a non-absorbable sugar lines for reporting animal research. All efforts were made alcohol produced industrially by lactose hydrogenation to minimize animal suffering and reduce the number (16,17). A previous study showed that the combination of of animals used. Four-week-old male Wistar rats were PDX and lactitol caused a 10-fold increase in IgA secre- purchased from CLEA Japan (Tokyo, Japan) and housed tion in the cecum (17), suggesting that these compounds in wire mesh cages without bedding material at 22 ± 3°C modulate IgA production in the gastrointestinal tract. with a 12-h light/dark cycle. The rats had free access to Our previous study indicated that the ingestion of PDX a commercial diet (CE-2; CLEA Japan) and water for 7 and lactitol for 3 weeks increased IgA levels both in the days before starting the experiment. large intestine and saliva of rats, demonstrating, for the first time, the interrelationship between the consumption Diets and sampling of indigestible carbohydrates and salivary IgA levels (18). The compositions of the fiber-free control diet and Using Bayesian network analysis, we also showed that experimental diet are shown in Table 1. The control the increase in the secretion of salivary IgA per weight of diet was based on solidified AIN-76 formulation, where submandibular gland tissue induced by fructooligosac- corn starch and cellulose were converted to sucrose to charides correlated with SCFA concentration in cecal eliminate the flux of carbohydrates into the large intes- digesta (19). However, whether continuous ingestion tine, given that corn starch and cellulose produce SCFAs of PDX and lactitol is associated with fermentation in (20,21), while sucrose is absorbed in the small intestine the large intestine, SCFA production, and salivary IgA and does not influence fermentation in the large intestine levels still remains unclear. Therefore, the purpose of the (22). The PDX + lactitol diet (CLEA Japan) contained present study was to examine the time-dependent effects 25 g/kg PDX (Litesse Ultra; Danisco Japan Ltd., Tokyo, 605 Japan) and 25 g/kg lactitol (lactitol MC; Danisco Japan mL. Salivary IgA flow rate (μg/min) was calculated by Ltd.), with rats having free access to diet and water. On multiplying the absolute IgA concentration (μg/mL) average, 30 g of food, calculated based on the amount by the saliva flow rate (mL/min). In the present study, remaining in each cage at the end of each day, was the salivary IgA flow rate positively correlated with the provided to each rat daily. weight of the submandibular gland tissue (rs = 0.78, P The animals were randomly divided into eight groups < 0.0001, n = 48, Spearman’s rank correlation coeffi- (n = 6 per group) receiving either the control or PDX + cients), suggesting that the weight of the submandibular lactitol diets for 0, 1, 4, or 8 weeks. Rats were anesthe- gland affected the salivary IgA flow rate. Therefore, the tized with sodium pentobarbital (65 mg/kg body weight; salivary IgA flow rate was normalized to the weight of Kyoritsu Seiyaku Corp., Tokyo, Japan), and saliva the submandibular gland tissue calculated as the median secretion was induced after 5 min by intraperitoneal weight of the right and left glands. injection of pilocarpine (8 mg/kg body weight; Nacalai Tesque, Inc., Kyoto, Japan). Approximately 5 min after Measurement of pH in cecal digesta pilocarpine injection, whole saliva was collected for 10 Cecal digesta samples were thoroughly mixed with equal min using a pipette. The rats were then sacrificed, and volumes of distilled water before centrifugation (10,000 cecal digesta, cecal tissues, and submandibular glands ×g, 15 min, 4°C).