Synergistic effect of dietary glycerol galactoside and porphyran from nori on cecal immunoglobulin A levels in mice

Abstract

We investigated the effects of dietary porphyran, an indigestible sulfated polysaccharide and the main component of nori, Porphyra yezoensis, and glycerol galactoside (GG; floridoside: 2-O-glycerol-α-D-galactopyranoside, and isofloridoside: 1-O-glycerol-α-D-galactopyranoside), which is abundant in low quality discolored nori and has prebiotic activity, on the concentration of immunoglobulin A (IgA) and microbiota in the cecum of mice. Porphyran increased the IgA concentration and contents in the cecum more than 2-fold. GG alone had little effect on these indices, but synergistically enhanced the IgA-elevating effect of porphyran greatly. Porphyran and GG both altered the cecal microbiota, and an interactive effect of porphyran and GG on the cecal microbiota was observed. Therefore, GG and porphyran are considered to have a synergistic effect on the intestinal immune system through the intestinal microbiota and their metabolites.

Introduction

The red algae Porphyra spp., especially Susabinori Porphyra yezoensis and Asakusanori Porphyra tenera, are processed into a sheet-like dried food called nori. Nori is largely consumed in East and Southeast Asia, and due to the recent popularity of sushi, it is now available in many countries. Nori contains various physiologically functional components, such as porphyran, taurine, mycosporine-like amino acids and vitamin B₁₂ (Bito et al., 2017). Anti-tumor, immunostimulatory, anti-allergic, anti-mutagenic, and anti-diabetic effects of nori have been reported (Noda et al., 1989, Yoshizawa et al. 1993; Okai et al., 1996, Ishihara et al., 2005, Kitano et al., 2012).

Discolored nori is caused by a decrease in nutrient concentration in the habitat area (Sakaguchi et al. 2003). Discolored nori has a low content of proteins, photosynthetic pigments, and free amino acids, resulting in poor blackness and low umami components (Saito et al., 1975; Ishihara et al., 2008). Therefore, discolored nori is low priced and underutilized (Tsuchiya et al., 2007). Discolored dried nori contains approximately 30% porphyran and more than 10% glycerol galactoside (GG; floridoside: 2-O-glycerol-α-D-galactopyranoside, and isofloridoside: 1-O-glycerol-α-D-galactopyranoside) on a dry basis (Ishihara et al. 2008, Sugano et al. 2009). We previously showed that GG has prebiotic activity (Muraoka et al., 2008; Ishihara et al., 2010). In vitro, GG was not hydrolyzed by digestive enzymes and was not adsorbed in the everted sac of rat small intestine. Dietary GG (5% of the diet) significantly increased Lactobacillus spp. and Bifidobacterium spp. in the cecum of rats. GG may regulate intestinal function through modulation of the microbiota. In our analysis, low quality discolored nori contained more than 20% GG (data not shown). Porphyran is one of the sulfated polygalactans composed of galactose and 3, 6-anhydrogalactose units, which is sometimes substituted with galactose-6-sulfate, and 6-O-methyl-galactose (Bito et al., 2017). Porphyran is a major component of nori and is also a soluble dietary fiber. It has been reported that porphyran has an activating effect on the intestinal immune system, such as increasing the ability of mesenteric lymphocytes to produce immunoglobulin (Ig) A in rats (Tsuge et al., 2005). However, the functionality of GG and porphyran when ingested simultaneously, as in the case of ingestion of discolored nori as a food, has not been studied. In this study, we investigated the effects of dietary GG and porphyran individually and in combination on cecal and fecal IgA concentrations as indicators of intestinal immune activity in Balb/c mice.

Materials and Methods

Reagents    ELISA kits for antibodies were purchased from Bethyl Laboratories (Montgomery, TX, USA). Other reagents were obtained from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan).

Preparation of GG and porphyrin    GG was extracted from discolored nori by the method described previously (Muraoka et al., 2008). The purity of GG was assessed by HPLC as > 95% (Ishihara et al., 2008). Porphyran was extracted from the residue of GG extraction. The residue (approximately 300 g) was added to 0.5 L of water and autoclaved at 120 °C for 30 min. Water was added to the resultant solution up to 5 L and stirred overnight at 5 °C. The extract was collected by a centrifugal dehydrator (H-122, Kokusan Co., Saitama, Japan) and concentrated to approximately 1 L using a rotary evaporator. Porphyran was precipitated from the concentrated extract by successively adding a 1/10 volume of 3M sodium acetate and three volumes of ethanol. After centrifuging at 10 000 × g for 15 min, the supernatant was discarded and the precipitant was washed twice with 70% aqueous ethanol. The precipitant was dried under vacuum and milled by a grinder 800DG (Iwatani Co., Tokyo, Japan) to give a white powder. Only galactose was detected by sugar composition analysis of the powder with an ABEE Labeling Kit Plus S (J-Oil Mills Co., Tokyo, Japan) after hydrolysis with 4M trifluoroacetic acid. Sulfate content was 8.1% by the rhodizonate method (Silvestri et al., 1982). Thus, the powder was judged as pure porphyran.

Animal experiment    The animal experiment was approved by the Animal Care and Use Committee of the National Research Institute of Fisheries Science (H27-1). Twenty-four Balb/c female mice (4 wk old) were obtained from Charles River Japan (Yokohama, Japan). After a one-week acclimation period, mice were divided into 4 groups (Control, GG, Por, GG+Por). Each group of mice was fed the test diet (Table 1) for 21 days. In our previous study, the amount of GG added to the diet to assess prebiotic activity was set at 5%. In the present study, due to limitations in the amount of test samples, the amounts of GG and porphyran added to the diet were set at 4%. To exclude the effect of other dietary fibers, the amount of cellulose added was set at 1%. Our preliminary study showed that at least 1% cellulose was required to maintain stool consistency. On the 19th day after starting the test diet, feces excreted during 24 h were collected. On the 21st day, mice were anesthetized by isoflurane inhalation. Total blood was withdrawn from the inferior vena cava and serum was collected after centrifuging at 2 500 × g for 15 min. Liver, spleen, large intestine, and cecum were excised and weighed. Cecal contents were collected and subjected to pH determination as described previously (Ishihara et al., 2010), and then the samples were stored at −80 °C until the microbiota analysis.

Table 1. Composition of Test Diets.

	Control	GG	Por	GG+Por
Ingredient	g/kg
β-Cornstarcha	427	395	397	370
α-Cornstarcha	142.5	134.5	132.5	119.5
Caseina	200	200	200	200
Sucrosea	100	100	100	100
Soy oilb	70	70	70	70
Cellulosea	10	10	10	10
Mineral mixtureac	35	35	35	35
Vitamine mixtureac	10	10	10	10
L-Cystineb	3	3	3	3
Choline bitartrateb	2.5	2.5	2.5	2.5
t-BHQb	0.014	0.014	0.014	0.014
Glycerol galactoside	0	40	0	40
Porphyran	0	0	40	40

a  Oriental Yeast Co. Ltd.(Tokyo Japan).

b  FUJIFILM Wako Pure Chemical Co. Ltd. (Osaka, Japan).

c  AIN-93G formulation.

Antibody measurement    After pH measurement, the cecal contents were diluted with 0.05% Tween 20 / PBS (-) (PBS-T) supplemented with 0.01% trypsin inhibitor and 0.1% proteinase inhibitor cocktail (Wako). Feces were freeze-dried and ground with a motor and pestle, then aliquots of the fecal powder were extracted with the same buffer used for the cecal content extraction. The extracts and serum were appropriately diluted with 1% bovine serum albumin / PBS-T, and then cecal and fecal IgA, and serum IgG, IgM, IgA and IgE concentrations were quantitated by ELISA.

Analyses of cecal microbiota    DNA of the microbiota in the cecal contents was extracted by the method previously reported (Ishihara et al., 2010). Briefly, a 10-mg portion of the cecal contents was washed with PBS 3 times. The washed content was extracted with extraction buffer after shaking with glass beads (1-mm diameter) for 1 min. The extracted DNA was recovered by isopropanol precipitation and stored at −80 °C until use. The cecal microbiota was analyzed by quantification of 16S rDNA gene sequences using genus- and cluster-specific primers and probes with a real-time polymerase chain reaction (PCR) system, as described previously (Ten Bruggencate et al. 2005, Matsuki et al. 2004, Ezaki et al., 1999; Lyons et al., 2000; Huijsdens et al., 2002). Lactobacillus spp., Bifidobacterium spp., Bacteroides fragilis group, Clostridium coccoides group, Escherichia coli, and Enterococcus spp. were enumerated and expressed as log CFU/g cecal content.

Statistical procedure    Data were statistically analyzed by two-way ANOVA (GG, Por, GG × Por) using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) (Kanda 2013), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). When the interaction between Por and GG was significant by ANOVA, multiple comparisons between dietary groups were performed by the Tukey-Kramer test (p < 0.05) with EZR.

Results

Growth and relative organ weight    Initial and final body weight (wt.), and relative organ wt. are shown in Table 2. Final body wt. was significantly affected by dietary porphyran. Liver and spleen wt. were not changed by diets. Cecal wt. was increased by both GG and porphyran, but no interactive effect of GG+porphyran was observed. Large intestine wt. was increased by GG+porphyran.

Table 2. Body weight (wt) and Relative Organ wt.

	Control	GG	Por	GG+Por	Significance Level by ANOVA
					GG	Por	GG × Por
Initial body wt*1	17.5 ± 0.3	17.5 ± 0.2	17.5 ± 0.3	17.4 ± 0.2	0.950	1.005	0.950
Final body wt*1	21.8 ± 0.3	21.0 ± 0.4	21.9 ± 0.4	22.7 ± 0.5	0.980	0.044	0.052
Liver wt.*2	4.97 ± 0.11	4.73 ± 0.11	4.88 ± 0.11	4.78 ± 0.10	0.139	0.813	0.528
Spleen wt.*2	0.53 ± 0.04	0.50 ± 0.02	0.54 ± 0.05	0.49 ± 0.04	0.393	0.948	0.780
Cecum wt.*2	0.74 ± 0.04	1.16 ± 0.07	1.13 ± 0.06	1.54 ± 0.07	< 0.0001	< 0.0001	0.927
Large intestine wt.*2	1.10 ± 0.05a	1.26 ± 0.06a	1.43 ± 0.14a	2.08 ± 0.15b	0.001	< 0.0001	0.034

*1  grams (g),

*2  % of body wt.

Values are expressed as mean ± SEM (n = 6). Values with different superscripts within each row indicate a significant difference (p < 0.05).

Fecal weight and cecal contents    Cecal content wt. and pH value, and fecal wt. are shown in Table 3. Cecal content wt. was increased by dietary GG and porphyran, but no interactive effect was observed. Cecal content pH was reduced by dietary GG, but this reducing effect was attenuated by dietary porphyran. Porphyran increased fecal wt. and fecal dry wt. Dietary GG alone did not influence fecal wt. and fecal dry wt., but synergistically augmented the effect of porphyran.

Table 3. Properties of Cecal Content and Feces in Mice fed GG and Por.

	Control	GG	Por	GG+Por	Significance Level by ANOVA
					GG	Por	GG × Por
Cecal content weight (g)	0.069 ± 0.008	0.150 ± 0.013	0.154 ± 0.014	0.258 ± 0.019	< 0.0001	< 0.0001	0.434
Cecal content pH	7.52 ± 0.34	6.44 ± 0.24	7.73 ± 0.14	7.30 ± 0.32	0.000	0.004	0.070
Fecal weight (g/day)	0.20 ± 0.02a	0.20 ± 0.01a	0.27 ± 0.01b	0.35 ± 0.03c	0.012	< 0.0001	0.029
Fecal dry weight (g/day)	0.19 ± 0.02a	0.18 ± 0.01a	0.25 ± 0.01b	0.33 ± 0.02c	0.042	< 0.0001	0.027

Values are expressed as mean ± SEM (n = 6). Values with different superscripts within each row indicate a significant difference (P < 0.05).

Serum immunoglobulin concentration    Serum immunoglobulin concentrations are summarized in Table 4. Dietary GG and porphyran had no effect on serum IgG, IgM and IgE concentrations, whereas serum IgA concentration was suppressed by dietary porphyran.

Table 4. Serum Antibody Concentrations in Mice fed GG and Por.

	Control	GG	Por	GG+Por	Significance Level by ANOVA
					GG	Por	GG × Por
IgA (mg/ml)	506 ± 32	455 ± 51	341 ± 25	390 ± 29	0.971	0.004	0.170
IgG (mg/ml)	317 ± 31	321 ± 23	287 ± 8	314 ± 20	0.507	0.424	0.594
IgM (mg/ml)	785 ± 51	711 ± 35	737 ± 58	834 ± 44	0.820	0.449	0.089
IgE (ng/ml)	57 ± 10	36 ± 5	37 ± 2	35 ± 2	0.063	0.111	0.129

Values are expressed as mean ± SEM (n = 6). Values with different superscripts within each row indicate a significant difference (p < 0.05).

Cecal and fecal IgA levels    Cecal IgA concentration and total IgA content are shown in Fig. 1. Fecal IgA concentration and total IgA excretion are shown in Fig. 2. Two-way ANOVA showed that both factors (GG and porphyran) and their interaction were all significant in the cecal IgA concentration. Especially, dietary GG greatly strengthened the effect of porphyran on the cecal IgA concentration when they were combined, although GG alone did not change the cecal IgA concentration compared to the control. In the feces, the effects of GG and porphyran were not apparent as in the cecum (Fig. 2). However, the potentiating effect of GG on the effect of porphyran on fecal IgA excretion was observed (Fig. 2B).

Fig. 1.

Immunoglobulin (Ig) A concentration (A) and content (B) in cecal content of mice fed glycerol galactoside and porphyran. Values are expressed as mean ± SEM, n = 6. Values with different superscripts indicate a significant difference (p < 0.05).

†Significant levels of the dietary effect (GG, Por, GG × Por) by two-way ANOVA.

Fig. 2.

Fecal immunoglobulin (Ig) A concentration (A) and excretion (B) in mice fed glycerol galactoside and porphyran. Values are expressed as mean ± SEM, n = 6. Values with different superscripts indicate a significant difference (p < 0.05).

†Significant levels of the dietary effect (GG, Por, GG × Por) by two-way ANOVA.

Cecal microbiota    Cecal bacterial counts are shown in Table 5. GG significantly increased the total bacterial count and the C. coccoides group. Although not significant, GG tended to increase Bifidobacterium spp. (p = 0.055). Porphyran increased Lactobacillus spp. and E. coli. Porphyran also increased the B. fragilis group; however, this increment was cancelled by GG.

Table 5. Cecal microbiota in mice fed GG and Por.

	Control	GG	Por	GG+Por	Significance Level by ANOVA
	log CFU/g				GG	Por	GG × Por
Total Bacteria	10.22 ± 0.07	10.68 ± 0.08	10.19 ± 0.11	10.59 ± 0.03	< 0.001	0.501	0.732
Lactobacillus spp.	6.97 ± 0.20	7.08 ± 0.17	7.49 ± 0.08	7.83 ± 0.04	0.112	< 0.001	0.406
Bifidobacterium spp.	8.05 ± 0.20	8.33 ± 0.07	7.95 ± 0.10	8.17 ± 0.08	0.055	0.325	0.825
Bacteroides flagilis Group	8.31 ± 0.15a	8.33 ± 0.26a	9.34 ± 0.10b	7.84 ± 0.38a	0.007	0.290	0.006
Clostridium cocoides Group	8.55 ± 0.07b	8.91 ± 0.10bc	8.13 ± 0.12a	9.03 ± 0.09c	< 0.001	0.130	0.010
Enterococcus spp.	6.75 ± 0.23	7.09 ± 0.13	6.80 ± 0.09	6.68 ± 0.11	0.443	0.228	0.134
E. coli	6.49 ± 0.10	7.20 ± 0.60	9.17 ± 0.08	8.62 ± 0.18	0.807	< 0.001	0.062

Bacterial counts were expressed as log CFU / g cecal content. Values are expressed as mean ± SEM (n = 6). Values with different superscripts within each row indicate a significant difference (p < 0.05).

Discussion

GG is a component particularly abundant in discolored nori compared to nori of normal quality, and it has been reported to have prebiotic activity by increasing Bifidobacterium spp. in the cecum of rats (Ishihara et al., 2010). Porphyran is another main component of nori and is an indigestible sulfated polysaccharide. Many research studies on the functionality of porphyran have also been performed (Cho and Rhee, 2020). Tsuge et al. discovered the intestinal immunostimulatory effect of porphyran in rats (Tsuge et al. 2008). Prebiotics such as fructooligosaccharides (FOS) have been reported to have intestinal immunostimulatory activity (Nakamura et al., 2004; Nawaz et al., 2018). Therefore, we considered the possibility that GG, which has prebiotic activity, and porphyran may have some interactive actions on intestinal immunity. Then, we evaluated cecal IgA levels and fecal IgA excretion as indicators of the intestinal immune activity in mice following simultaneous administration of dietary GG and porphyran. Dietary porphyran elevated the cecal content and concentration, and fecal excretion of IgA, whereas GG alone had little effect on those measurements. However, GG potentiated the effect of porphyran to increase cecal IgA antibody levels. The effects of porphyran and GG on fecal IgA excretion were less clear than those on cecal IgA content. In rodents, fecal IgA levels might not accurately correlate to the intestinal IgA secretion (Nakamura et al., 2004). This phenomenon was confirmed in the present mouse study.

The mechanisms by which porphyran increases the concentration of IgA in the cecum and by which GG enhances the action of porphyran are not clear. However, soluble dietary fibers are known to be fermented by intestinal bacteria to produce short chain fatty acids (SCFA), such as acetate, propionate, and butyrate (Alexander et al. 2019). SCFA are reported to activate the NLRP3 inflammasome via GPR43 and GPR109A receptors, and to promote the production of cytokines in intestinal epithelial cells. It has also been reported that SCFA promote IgA production from B cells of the lamina propria (Wang et al., 2019). Soluble dietary fiber also increases B. fragilis group bacteria in the gut, thereby increasing IgA production (Nakajima et al., 2020). Prebiotics, such as FOS, have been suggested to promote IgA production by increasing the concentration of SCFA and increasing the number of lactic acid bacteria in the gut (Ito et al., 2011).

GG was shown in our previous study to increase Bifidobacterium spp. and Lactobacillus spp. as well as butyrate concentrations in the rat cecum (Ishihara et al., 2010). The reason why GG had little effect on cecal IgA concentrations in the present study is unclear, but it may be due to the fact that the amount of GG added to the diet in our previous study was 5%, compared to 4% in the current study, and that the prebiotic activity of GG is weaker than that of FOS (Ishihara et al., 2010). In fact, the effect of GG on bifidobacterium counts in the cecum was not significant in the present study. The concentration of SCFA in the cecum was not measured due to sample volume limitations, but the cecal pH was lower than that of the control, suggesting that the concentration of organic acid was elevated. Further research, including dose-testing of GG, is needed in the future.

Porphyran significantly increased IgA concentrations in the cecum compared to the control. As mentioned above, soluble dietary fiber promotes IgA production via intestinal B. fragilis group bacteria and SCFA. Porphyran may have promoted IgA production through a similar mechanism. In fact, porphyran increased the number of B. fragilis group in the cecum. Furthermore, porphyran also increased the number of Lactobacillus spp. in the cecum. It is possible that SCFA may also be involved. However, this is unlikely because the pH in the cecum was not lowered compared to the control by porphyran in this study, and Kawadu et al. reported that SCFA concentration in the cecum of 1% porphyran-treated rats was unchanged (Kawadu et al., 1995).

In the present study, GG potentiated the increasing effect of porphyran on IgA concentration in the cecum, while the effects of GG and porphyran on the gut microbiota, blood antibody concentrations, and cecal content pH were complex. The increase in B. fragilis group by porphyran was reduced by the coexistence of GG. In addition, the GG-induced decrease in cecal content pH was cancelled by porphyran. Porphyran also reduced serum IgA concentrations relative to the control; however, the reduction was mitigated by GG. Further studies involving comprehensive analyses of metabolites of intestinal contents and microbiota are necessary to clarify the interaction of dietary porphyran and GG.

In this study, GG and porphyran were administered at a concentration of 4% each. Nori contains about 30–40% porphyran (Sugano et al., 2009). Further, in our analysis, discolored nori contains GG around 20% on a dry basis (data not shown). Therefore, the dietary composition of GG and porphyran used in this study is different from that of discolored nori. In addition, the dose-effect of GG and porphyran on the concentration of cecal IgA has not yet been examined. These points will need to be examined in future studies. A number of papers have shown positive health effects of dietary porphyran (Bito et al., 2017; Cho and Rhee, 2020; Ishihara et al., 2005; Jiang et al., 2012; Kawadu et al., 1995; Noda et al., 1989; Okai et al., 1996; Tsuge et al., 2005; Yoshizawa et al., 1993). In the present study, dietary porphyran increased IgA concentrations in the cecum but decreased serum IgA concentrations. Although the physiological significance of the reduction in serum IgA concentrations is unclear, GG may reduce these non-positive effects of porphyran. This study suggests that discolored nori, which is rich in GG and porphyran, could be used as a new functional food material to regulate the intestinal immune system.

Acknowledgements    We thank Mr. T. Yamada, the Federation of Japan Fisheries Cooperatives, and Dr. M. Fujiwara, Kagawa Prefectural Fisheries Experimental Station, for providing us with the discolored nori samples. We also thank Prof. T. Kuda, Tokyo University of Marine Science and Technology, for useful discussions. The authors would like to thank Ms. Y. Sato for her skillful technical support.

Abbreviations

GG

glycerol galactoside

Por

porphyran

Ig

immunoglobulin

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