Continuous Combined Intake of Polydextrose and Lactitol Stimulates Cecal Fermentation and Salivary Iga Secretion in Rats

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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; ribofavin, 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 fux 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 infuence 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 fow 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 fow rate (mL/min). In the present study, remaining in each cage at the end of each day, was the salivary IgA fow 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 fow rate. Therefore, the tized with sodium pentobarbital (65 mg/kg body weight; salivary IgA fow 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). Thereafter, pH was measured in super- were removed and weighed immediately. All samples natant fractions using a compact pH meter (HORIBA, were collected between 7:00 and 11:00 and stored at Ltd., Kyoto, Japan). −20°C until analysis. Measurement of organic acids in cecal digesta Measurement of IgA concentration Preprocessing of cecal digesta and analysis of organic The concentration of IgA in cecal digesta and saliva was acid concentrations were based on the method of Tsuka- quantified by ELISA using the Rat IgA ELISA Quantita- hara et al. (23). Samples (0.3 g) were mixed with 0.6 tion Kit (Bethyl Laboratories, Montgomery, TX, USA). mL distilled water and then with 90 μL perchloric acid Cecal digesta samples were treated with 10 volumes of (120 g/L). After centrifugation (15,000 ×g, 10 min, 4°C), distilled water containing 1 mM phenylmethylsulfonyl supernatant fractions were filtered through a 0.45-μm fuoride (PMSF) for 60 min at room temperature. The cellulose acetate membrane filter (Cosmonice Filter samples were then centrifuged (10,000 ×g, 15 min, 4°C), W, Nakalai Tesque, Kyoto, Japan) and degassed under and supernatant fractions were used for IgA measure- vacuum. Afterwards, 5 μL was injected into an SIL-10 ment. autoinjector (Shimadzu, Kyoto, Japan). Organic acids Subsequently, 96-well microtiter plates were coated were separated using two serial organic acid columns with goat anti-rat IgA diluted 1:100 with coating buffer (Shim-pack SCR-102H; Shimadzu) and a guard column (0.05 M carbonate-bicarbonate, pH 9.6) for 1 h at room (SCR-102HG; Shimadzu) at 45°C, while isocratic temperature, washed 5 times with wash solution (50 elution was performed with 5 mM p-toluenesulfonic acid mM Tris, 0.14 M NaCl, 50 g/L Tween 20, pH 8.0), and aqueous solution (0.8 mL/min) using a solvent delivery blocked with blocking solution (50 mM Tris, 0.14 M pump (LC-10ADvp; Shimadzu) with an online degasser NaCl, 10 g/L bovine serum albumin, pH 8.0) for 30 min (DGU-12A; Shimadzu). Organic acids were detected at room temperature. After washing with wash solution using an electronic conductivity detector (Waters 431; as described, samples and rat IgA standards (Bethyl Waters Corporation, MA, USA) after post-column Laboratories) were added to each well for 1 h at room dissociation (0.8 mL/min) with 5 mM p-toluenesulfonic temperature. After washing, horseradish peroxidase acid, 20 mM bis-Tris, and 100 μM EDTA using a solvent (HRP)-conjugated goat anti-rat IgA detection antibody delivery pump and quantified using a system controller (1:15,000 dilution) was added to each well for 1 h at room (CBM-20A; Shimadzu). temperature. The reaction was initiated with the addition of TMB substrate in the dark at room temperature for 15 Measurement of water content in cecal digesta min and stopped with 0.18 M H2SO4. Absorbance was Cecal digesta samples were dried in a constant tempera- then measured at 450 nm using an automated microplate ture oven (DK300; Yamato Scientific Co., Ltd., Tokyo, reader (BioRad, Hercules, CA, USA). Japan) at 135°C for 4 h until they reached stable weight. Saliva fow rate (mL/min) was determined by dividing Thereafter, water content was calculated based on the saliva weight (mg) by the sampling time (10 min), following equation: [wet weight (g) − dry weight (g)]/ assuming that the specific density of saliva was 1.00 g/ wet weight (g). 606

Fig. 1 Effects of polydextrose (PDX) and lactitol addition and Fig. 2 Effects of polydextrose (PDX) and lactitol addition and duration of feeding on weight gain (A), weight of the subman- duration of feeding for test diets on the tissue weight of the cecum dibular gland (B), and fow rate of saliva (C). Rats were fed the (A), weight of cecal digesta (B), and pH in cecal digesta (C). Rats control diet (based on AIN-76) with or without 25 g/kg PDX were fed the control diet (based on AIN-76) with or without 25 and 25 g/kg lactitol and analyzed after 0, 1, 4, and 8 weeks of g/kg PDX and 25 g/kg lactitol and analyzed after 0, 1, 4, and 8 feeding. Data are expressed as means ± SEM (n = 6 per group weeks of feeding. Data are expressed as means ± SEM (n = 6 per at each time point). No interaction effects were observed, while group at each time point). Interactions between PDX and lactitol PDX and lactitol addition had no effect on weight gain, weight addition and intake period were observed for tissue weight of of submandibular gland, and fow rate of saliva (interaction: P = the cecum, weight of cecal digesta, and pH in cecal digesta (P < 0.06, 0.09, and 0.3 and PDX and lactitol addition: P = 0.1, 0.09, 0.0001, < 0.0001, and < 0.0001, respectively, two-way analysis of and 0.8, respectively, using two-way analysis of variance). The variance). *P < 0.05 versus the control group at each week using duration of feeding affected weight gain, weight of submandibular Tukey’s multiple comparison. gland, and fow rate of saliva (P < 0.0001, < 0.0001, and < 0.0001, respectively).

Statistical analysis Results All statistical analyses were performed using JMP version Feeding time-dependent effects of PDX and lactitol 12 (SAS Institute Japan, Tokyo, Japan), and results were on cecal fermentation and IgA secretion expressed as mean ± SEM. Statistical analyses of time In both the control and PDX + lactitol groups, weight series data were performed using factorial two-way anal- gain, weight of the submandibular gland, and fow rate of ysis of variance, which showed two main effects and an saliva increased significantly with the duration of feeding interaction effect. Tukey’s multiple comparison test was (Fig. 1). However, there was no significant difference in used for post-hoc analysis of interaction effects, while these parameters between the groups and no interaction Spearman’s rank correlation was employed to detect a between PDX and lactitol addition and time of feeding correlation between two variables. Welch’s t-test was was detected. used to compare the PDX + lactitol and control groups In contrast, PDX and lactitol addition significantly according to SCFA concentrations and water content in increased the tissue weight of the cecum and weight cecal digesta. P values less than 0.05 were considered of cecal digesta and decreased cecal digesta pH levels statistically significant. after 1, 4, and 8 weeks of feeding (Fig. 2). In addition, 607

Fig. 4 Water content in cecal digesta. Rats were fed the control diet (based on AIN-76) with or without 25 g/kg PDX and 25 g/ kg lactitol (Lac) and analyzed after 8 weeks of feeding. Data are expressed as means ± SEM (n = 6 per group). **P < 0.0001 using Welch’s t-test. PDX + Lac, combination of 25 g/kg polydextrose and 25 g/kg lactitol.

Table 2 Concentration of organic acids in cecal contents after 8 weeks of feeding (mmol/kg wet digesta) Acids Control PDX* + lactitol P† Mean SE Mean SE Acetate 17.40 0.20 10.30 0.49 0.001 Fig. 3 Effects of polydextrose (PDX) and lactitol addition and Propionate 7.06 0.24 4.41 0.10 0.005 duration of feeding on IgA concentration in cecal digesta (A), n-Butyrate 3.58 0.25 2.35 0.11 0.100 IgA concentration in the saliva (B), and IgA fow rate of saliva SCFA§ 33.40 0.20 17.50 0.80 0.0003 per weight of submandibular gland tissue (C). Rats were fed the n = 6 animals per group. † § control diet (based on AIN-76) with or without 25 g/kg PDX and *PDX: polydextrose. Calculated using Welch’s t-test. SCFA: the sum of acetate, propionate, and n-butyrate. 25 g/kg lactitol and analyzed after 0, 1, 4, and 8 weeks of feeding. Data are expressed as means ± SE (n = 6 per group at each time point). Interactions between PDX and lactitol addition and intake period were observed for IgA concentration in cecal digesta, IgA concentration in the saliva, and IgA fow rate of saliva per weight of submandibular gland tissue (P < 0.0001, 0.0012, and < 0.0001, respectively, two-way analysis of variance). *P < 0.05 versus the control group at each week using Tukey’s multiple comparison. interaction effects between PDX and lactitol addition and Effect of PDX and lactitol addition on SCFA duration of feeding were revealed for tissue weight of the concentrations and water content in cecal digesta cecum, weight of cecal digesta, and pH in cecal digesta PDX + lactitol addition decreased the concentrations (Fig. 2). of acetate, propionate, and total SCFAs (Table 2) and PDX + lactitol addition also upregulated IgA concen- increased water content (Fig. 4) in cecal digesta at week trations in cecal digesta and the saliva and increased 8. salivary IgA fow rate per weight of submandibular gland tissue at 4 and 8 weeks (Fig. 3). In both the PDX + lactitol Discussion and control groups, the concentration and fow rate of This study investigated the effects of continuous ingestion salivary IgA increased after 4 and 8 weeks of feeding of 25 g/kg PDX with 25 g/kg lactitol for up to 8 weeks (week 0; P < 0.05, Fig. 3C). Furthermore, an interaction on salivary IgA secretion and fermentation in the rat between PDX and lactitol addition and the duration of cecum. Consistent with our previous results showing that feeding was observed for IgA concentration in cecal dietary addition with 25 g/kg PDX and 25 g/kg lactitol digesta, concentration of IgA in the saliva, and IgA fow for 3 weeks stimulated salivary IgA secretion (18), PDX rate of saliva per weight of submandibular gland tissue + lactitol increased the salivary IgA fow rate per weight (Fig. 3). of submandibular gland tissue at 4 and 8 weeks (Fig. 608

3C). However, short-term (1 week) ingestion of PDX and turn increases diffusion and absorption therein (38,39). lactitol did not affect the salivary IgA fow rate (Fig. 3C), Hence, decreased SCFA levels in cecal digesta of PDX + indicating that IgA secretion is upregulated only after lactitol-fed rats could be due to the increased absorption continuous and extended PDX + lactitol intake. in the cecum because of elevated water content in cecal PDX + lactitol addition also upregulated IgA secretion digesta produced through fermentation. in the cecum (Fig. 3A) while increasing the weight of In our previous study on the injection of fructooli- cecal tissues and digesta and decreasing pH (Fig. 2). gosaccharide, an indigestible carbohydrate, a Bayesian These findings are consistent with a previous report in network, which is a probabilistic graphical model (40), which the consumption of indigestible carbohydrates revealed that the salivary IgA ﬂow rate per weight of induced fermentation in the cecum (24), which has been submandibular gland tissue was directly affected by shown to positively affect microbial composition and SCFA concentration in cecal digesta (19). The present immune responses in the large intestine of rats (17). The study showed that PDX and lactitol ingestion induced upregulation of IgA secretion in the cecum is caused by fermentation in the cecum, which should promote SCFA various antigens, such as bacterial cell wall constituents production and absorption, thus increasing salivary IgA or decomposed and fermented products of indigestible secretion. The decrease in SCFA concentration in cecal carbohydrates, generated as a result of increased fermen- digesta may be attributed to higher SCFA absorption tation by intestinal microfora (25). In addition, colonic rates in the cecum, which could then upregulate salivary bacterial fermentation of carbohydrates, such as PDX and IgA secretion. Thus, continuous PDX and lactitol inges- lactitol, produces organic acids, mainly SCFAs, and gas tion for up to 4 weeks could increase salivary IgA and

(CO2 and hydrogen) (15,26). Higher SCFA concentra- promote immune defense against pathogen invasion tions in the large intestine have been known to increase through the oral route. the weight of cecal tissue (27,28) through the stimulation The present study showed that the ingestion of PDX of epithelial cell proliferation and augmentation of the and lactitol decreased acetate and propionate levels in absorptive surface area (29,30). Hence, dietary supple- cecal digesta but did not affect n-butyrate concentra- mentation with PDX + lactitol stimulates fermentation, tions (Table 2), although reports have shown that lactitol which increases SCFA production in the cecum. strongly shifted bacterial fermentation toward n-butyrate In the present study, the ingestion of PDX and lactitol (15). In the present study, n-butyric acid-producing increased the weight of cecal tissue (Fig. 2A). However, bacteria might have been increased in the cecum. the concentration of SCFAs in cecal digesta was reduced Furthermore, the increase in n-butyrate production may (Table 2), which is at variance with previous findings not have been detected because of increased absorption wherein the intake of PDX, lactitol, or both increased in the cecum. SCFA concentrations in cecal digesta and feces (15,31- Given that the control diet used in the present study 33). On the other hand, reports have also shown that PDX had no fiber, the cecal digesta pH therein was signifi- decreased SCFA levels in cecal digesta of rats and pigs cantly higher than that in fiber-supplemented diets (41) (14,34). The discrepancy may be explained by consid- (Fig. 2C). PDX addition in a high-cholesterol diet ering that SCFA concentrations in cecal digesta and reduced fecal pH and decreased putrefactive biomarkers feces may not accurately refect SCFA production in the in humans (17). Hence, the reduction of cecal digesta pH cecum because of the immediate absorption of SCFAs by by PDX + lactitol addition observed in the present study epithelial cells in the large intestine. Thus, reports have would result in the reduction of digesta putrefaction. shown that SCFAs could hardly be detected in the cecum Carpenter et al. (42) have reported that the rate of IgA even after direct infusion of large amounts (35). This secretion into the saliva is regulated by autonomic nerves suggests that regardless of the level of SCFA production supplying the glands, both in vitro and in vivo. Given that in the cecum, SCFA concentrations in cecal digesta could the increase of SCFA levels in the intestinal tract affects vary depending on the absorption rate. Therefore, the the activity of autonomic nerves (30,43,44), which in lower SCFA concentrations in cecal digesta of the PDX turn should promote the function of salivary glands (45), + lactitol group in our experiments could be attributed to the stimulation of fermentation and SCFA production by higher SCFA absorption rates. Furthermore, the PDX + PDX and lactitol in the cecum should affect the activity lactitol group had higher water content in cecal digesta of autonomic nerves and induce the secretion of salivary than the control group (Fig. 4). Dietary fiber ingestion IgA. Therefore, further experiments are needed to clarify has been reported to increase the water content in the the relationship between SCFA production in the large digesta residue of the large intestine (36,37), which in intestine and the increase in salivary IgA through the 609

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