2024 Volume 30 Issue 3 Pages 397-408
To investigate the effects of edible mushrooms on colonic luminal variables, including fecal mucins, organic acids, microbial composition, immunoglobulin A (IgA), alkaline phosphatase (ALP) and cecal organic acids, male Sprague-Dawley rats were fed a 30% lard diet containing one of 12 species of edible mushrooms for 2 weeks. We found that the dietary intake of shiitake (Lentinula edodes), enokitake (Flammulina velutipes), white button (Agaricus bisporus) and yamabushitake (Hericium erinaceus) mushrooms elevated fecal ALP activity as well as increased fecal IgA, mucins, fecal Bacteroides ratio, and cecal total organic acids. In contrast, the intake of dietary eringi (Pleurotus eryngii), maitake (Grifola frondosa), tamogitake (Pleurotus cornucopiae), wood ear (Auricularia auricula-judae) and bunashimeji (Hypsizygus marmoreus) mushrooms had minimal effects on the colonic environment factors. We speculated that edible mushrooms could be grouped according to their effects on the colonic luminal environment.
Mushrooms are widely appreciated throughout the world for their nutritional and medicinal properties. Early Greek, Egyptian, Roman, Chinese and Mexican civilizations valued mushrooms as a culinary delicacy and as medicine (Feeney et al., 2014). Around 700 mushroom species are safe for oral consumption and the most commonly grown mushrooms are white or brown button (Agaricus bisporus), shiitake (Lentinula edodes) and oyster (hiratake, Pleurotus ostreatus) mushrooms (Li et al., 2021). The global production of cultivated, edible mushrooms has increased more than 30-fold since 1978 (Royse et al., 2017). Thousands of mushroom species grow in the forests of Japan, and mushrooms have been eaten historically in Japan. The production of mushrooms in Japan reached 460 000 tons in 2020, 1.6 times higher than in 1985i). The species of mushrooms produced have also increased, such as eringi (Pleurotus eryngii) and maitake (Grifola frondosa) mushroomsi). Mushrooms belong to the kingdom Fungi, and are different from plants and animals (Feeney et al., 2014). Mushrooms contain many bioactive compounds that are peculiar to them (Feeney et al., 2014; Royse et al., 2017). So far, many studies have indicated that mushrooms and their bioactive components could have beneficial effects on human health via anti-inflammatory, anti-tumor, anti-oxidant, anti-atherosclerotic, anti-diabetic, anti-obesity and anti-neurodegenerative activities (Feeney et al., 2014; Friedman, 2016; Valverde et al., 2015; Li et al., 2021). Active research has been conducted on the cholesterol-lowering effects of mushrooms or the dietary fiber of mushrooms in Japan (Kaneda and Tokuda, 1966; Arakawa et al., 1977; Fukushima et al., 2001).
Mushrooms are generally rich in dietary fiber (Li et al., 2021). During the last two decades, several studies on the effects of dietary mushrooms on gut microbiota have shown that some mushrooms or their polysaccharides could increase the ratio of Bacteroidetes/Firmicutes and promote the growth of anti-inflammatory and short-chain fatty acid-producing bacteria (Aida et al., 2009; Jayachandran et al., 2017; Li et al., 2021). Some researchers have speculated that the beneficial activities of mushroom intake could be connected with the modulation of gut microbiota (Jayachandran et al., 2017; Li et al., 2021). Besides the effect on microbiota, there are several reports on the effects of mushrooms and their polysaccharides on the gut environment (Li et al., 2021). Some reports have shown that polysaccharides from reishi (Ganoderma lucidum), lion’s mane (yamabushitake, Hericium erinaceus) and bamboo (Dictyophora indusiata) mushrooms could ameliorate DSS-induced colitis in rats and mice by maintaining intestinal barrier integrity and increasing the levels of short-chain fatty acids (SCFAs, Li et al., 2021). Kawakami et al. (2016) reported that white button mushrooms increased cecal SCFAs and had beneficial effects on the intestinal environment in rats. It was also reported that the dietary fiber of shiitake and enokitake (Flammulina velutipes) mushrooms increased cecal SCFAs concentrations (Fukushima et al., 2001). However, studies on the functionality of mushrooms, including modulation of the gut environment, have specifically focused on certain mushrooms and their components.
Recently, it has been proposed that small intestinal alkaline phosphatase (ALP) detoxifies lipopolysaccharide endotoxins, thereby providing protection against bacterial invasion and exerting a protective effect against inflammatory disease (Lalles, 2019). We recently demonstrated that soluble dietary fiber and non-digestible oligosaccharides commonly elevate colonic and fecal ALP activity and the expression of Alpi-1, an ALP gene expressed throughout the intestine, with little effect on ALP activity in the small intestine and other organs of rats fed a high-fat (HF) diet (Lalles, 2019; Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). Our studies have also indicated that the increase in colonic ALP activity due to the dietary fiber and non-digestible oligosaccharides was significantly correlated with fecal mucins (the main macrocomponent of the mucus layer, which forms a nonspecific barrier), cecal organic acids (fermentation products) and alteration of fecal microbial composition (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). We hypothesize that the elevating effects of fermentable non-digestible carbohydrates on colonic ALP activity may be important for maintaining gut epithelial homeostasis under an HF diet condition.
In addition to characteristic bioactive compounds and dietary fibers, mushrooms contain a spectrum of nutrients, such as vitamin D, B vitamins and minerals (Feeney et al., 2014). We consider that in the field of food science and nutrition, unlike the pharmacological field, comprehensive study of the functionality of mushrooms is important and highly required. We hypothesize that edible mushrooms could be grouped by their functionality, including their modulatory effects on the gut environment. The objective of this study was to compare and evaluate the effects of 12 species of edible mushrooms on colonic luminal variables, such as fecal microbial composition, mucins, immunoglobulin A (IgA, an index of intestinal immune function), cecal organic acids and fecal and colonic ALP activity in rats.
Animals Experimental procedures were reviewed and approved by the Ethics Committee for Animal Experimentation of the Fuji Women’s University (approved no. 2021–3). All animal experiments were conducted according to the Guidelines for Animal Experiments of the Fuji Women’s University, “Japanese Act on Welfare and Management of Animals” (law No. 105 of October 1, 1973; recent revision: law No. 38 of June 12, 2013) and “Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain” (Ministry of the Environment in Japan, Notification No. 88, April 28, 2006; recent revision: Notification No. 84, August 30, 2013).
A total of 96 male Sprague-Dawley rats (4 weeks of age, weighing 88–103 g) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). The rats were housed in individually in stainless steel cages with wire mesh bottoms in a room with controlled temperature (23–24°C), relative humidity (55–65%), and light/dark cycle (light, 08:00–20:00) in the Fuji Women’s University animal facility.
Experimental samples Shiitake, enokitake and white button mushrooms were obtained from Kitakami farm (Hokkaido, Japan), JA Nakano (Nagano, Japan) and Kamada Co. (Hokkaido, Japan), respectively. Nameko (Pholiota microspora) and maitake mushrooms were obtained from JA Kamikawa (Hokkaido, Japan). Bunashimeji (Hypsizygus marmoreus), eringi and hiratake mushrooms were obtained from Hokuto Co. (Nagano, Japan). Dried wood-ear mushroom (Auricularia auricula-judae) was obtained from Kumada Co. (Hokkaido, Japan). Mechanically dried yamabushitake, porcini (Boletus edulis) and tamogitake (Pleurotus cornucopiae) mushroom powders were obtained from Kubo Industry Co., Ltd. (Nagano, Japan), Marumiyashiitake Co. (Shizuoka, Japan) and Yaso-cha Co. (Kanagawa, Japan), respectively. Fresh shiitake, nameko, bunashimeji, enokitake, eringi, white button, hiratake and maitake were powdered after being freeze-dried. Air-dried wood-ear mushroom was powdered as is. Mechanically-dried yamabushitake, porcini and tamogitake mushroom powders were used as experimental samples as is.
Groups and treatments Following ad libitum access to a non-purified commercial rodent powder diet (CE-2, CLEA Japan; containing 9.3% moisture, 25.1 % crude protein, 4.8 % crude fat, 4.2 % crude fiber, 6.7 % crude ash, and 50.0 % nitrogen free extract; energy, 1.44 MJ per 100 g) and water for 4 d, the rats were randomized by weight and assigned to experimental groups (n = 6). Composition of the control diet in experiments 1 to 4 was as follows: lard, 30 %; casein, 20 %; L-cystine, 0.3 %; cellulose, 5.0 %; sucrose, 30 %; vitamin mixture (Reeves et al., 1993), 1.0 %; salt mixture (Reeves et al., 1993), 3.5 %, choline bitartrate, 0.25 %; and corn starch, 9.95 % (Table 1). In experiment 1, 5.0 % shiitake, nameko or bunashimeji mushroom powder was added to the control diet, and the same level of enokitake, eringi or wood ear powder was added in experiment 2. White button, hiratake or maitake mushroom powder was added to the control diet at the level of 5 % in experiment 3, and the same level of yamabushitake, porcini or tamogitake mushroom powders was added in experiment 4. The composition of shiitake, nameko, bunashimeji, enokitake, eringi, wood-ear, white button, hiratake, maitake and tamogitake mushroom powders was calculated by using the data published in the Japanese Food Composition Table 8th edition (Council for Science and Technology; Ministry of Education, Culture, Sports, Science and Technology, Japan, 2020). The composition of yamabushitake and porcini mushroom powders was estimated based on the data of Sekiya et al. (2005) and Manzi et al. (2004), respectively. The levels of dietary carbohydrate, protein, fat and fibers in the mushroom diets were adjusted by reducing the amount of dietary corn starch, casein, lard and cellulose, respectively (Table 1). The animals had free access to the experimental diets and deionized water for 2 weeks. We have confirmed that rearing for 2 weeks clearly affects the colonic luminal environment (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). Another study has also been reported in which rats were kept for about 10 days to examine the effects of dietary nondigestible carbohydrate on the intestinal environment (Morita et al., 2004). Therefore, in this study, the experimental period was set to 2 weeks. Food intake and body weight were measured daily. The welfare and general health status of the individual animals were checked every day throughout the experimental period. No adverse events were observed during this study.
| Exp.1–4 | Exp.1 | Exp.2 | Exp.3 | Exp.4 | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ingredient | Cont. | Shiitake | Nameko | Bunashimeji | Enokitake | Eringi | Wood ear | White button | Hiratake | Maitake | Yamabushitake | Porcini | Tamogitake |
| Casein | 20 | 18.675 | 18.885 | 18.535 | 18.815 | 18.585 | 19.605 | 17.625 | 18.445 | 18.665 | 18.985 | 19.118 | 17.831 |
| L-Cystine | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 | 0.3 |
| Lard | 30 | 29.83 | 29.87 | 29.675 | 29.91 | 29.8 | 29.895 | 29.755 | 29.86 | 29.665 | 29.78 | 29.647 | 29.819 |
| Mushroom powder † | - | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 |
| Cellulose | 5.0 | 2.65 | 2.83 | 2.99 | 3.29 | 3.285 | 2.13 | 3.36 | 3.775 | 2.67 | 3.425 | 3.471 | 3.012 |
| Vitamin mix (AIN-93) * | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
| Mineral mix (AIN-93G) * | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 | 3.5 |
| Sucrose | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 | 30 |
| Corn starch | 9.95 | 8.795 | 8.365 | 8.75 | 7.935 | 8.28 | 8.32 | 9.21 | 7.87 | 8.95 | 7.76 | 7.714 | 9.288 |
| Choline bitartrate | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 | 0.25 |
Procedures for collecting samples Fecal pellets were collected during the last 3 days of feeding, stored at −20 °C and then freeze-dried and milled. The powdered feces were stored at −30 °C until ALP, mucin, IgA and microbial composition analyses. At the end of the feeding period, the rats were anesthetized with 3.0 % isoflurane and euthanized. Whole blood was collected from the abdominal aorta. The serum was separated by centrifugation at 3 000 × g for 15 min and stored at −80 °C. The cecum was removed, weighed, frozen immediately with liquid nitrogen and stored at −80 °C until organic acids analysis. The colon was removed, opened longitudinally, washed with saline to remove residual luminal contents and weighed. The colon was frozen immediately with liquid nitrogen and stored at −80 °C until ALP analysis.
Fecal mucins and IgA Mucins were extracted by the method of Boovee-Oudenhoven et al. (1997) with some modifications (Morita et al., 2004). The fecal sample was suspended in 20 volumes of PBS. The suspension was immediately heated to 95 °C for 10 min and incubated for 90 min at 37 °C. After centrifugation at 20 000 × g for 15 min at 4°C, an equal volume of 0.4 M acetate buffer (pH 4.75) was added to the supernatant and incubated with 10 µL of amyloglucosidase for 20 min at 50 °C. After the mixture was cooled, ice-cooled absolute ethanol was added to a final concentration of 60 % by volume. The samples were allowed to precipitate overnight at −30 °C and centrifuged at 2 000 × g for 10 min at 4 °C, and finally dissolved in 2 mL of PBS. Mucins were determined using a fluorometric assay according to the method of Crowther and Wetmore (1987) with N-acetylgalactosamine (Sigma, St. Louis, MO, USA) as a standard. Feces were suspended in 40 volumes of phosphate-buffered saline (PBS) containing 50 mmol/L EDTA, 100 mg/L trypsin inhibitor and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) and incubated for 2 h at 4 °C. The suspensions were vigorously mixed and centrifuged at 9 000 × g for 10 min at 4°C and the supernatants were frozen at −80 °C until IgA quantitation by enzyme-linked immunosorbent assay (ELISA). The total IgA concentration in the feces was measured using an ELISA quantitation kit (Bethyl Laboratories, Montgomery, TX, USA).
Cecal organic acids The pH of the cecal digesta was measured directly using a compact pH meter (B-712, Horiba, Kyoto, Japan). Cecal organic acids were quantified using the internal standard method and an HPLC system (L-2130, Hitachi, Tokyo, Japan) equipped with an Aminex HPX-87H ion exclusion column (7.8 mm i.d. × 30 cm, Bio-Rad Laboratories Inc., Hercules, CA, USA) attached to a micro-guard column (Cation H Cartridge, 4.6 mm i.d. × 3 cm, Bio-Rad Laboratories Inc.) (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). Briefly, 500 mg of cecal digesta was homogenized in 5 mL of 50 mM H2SO4 containing 10 mM 2,2-dimethyl butyric acid (FUJIFILM Wako Pure Chemicals Co., Ltd., Tokyo, Japan) as an internal standard. Next, the mixture was centrifuged at 17 000 × g for 20 min at 2 °C. The supernatant was ultrafiltered using an Amicon Ultra-0.5 Centrifugal Filter Device with a 3-kDa cut-off (Merck-Millipore Ltd., Darmstadt, Germany), and the filtrate was analyzed by HPLC (column at 60 °C) (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). The mobile phase (5 mM H2SO4) was delivered at a flow rate of 0.7 mL/min using a Hitachi pump L-2130 (Hitachi). Organic acids were detected at 210 nm with a variable wavelength detector (L-2400, Hitachi) (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023).
Quantitative PCR (qPCR) analysis of fecal microbiota Bacterial genomic DNA was isolated from feces using the QIAamp DNA Fast Stool Mini Kit (QIAGEN N.V., Venlo, The Netherlands). The primer sets were 5′-ACTCCTACGGGAGGCAG -3′ (forward) and 5′-GTATTACCGCGGCTGCTG -3′ (reverse) for total bacteria (Parnell and Reimer, 2012); 5′-GTCAGTTGTGAAAGTTTGC -3′ (forward) and 5′-CAATCGGAGTTCTTCGTG -3′ (reverse) for Bacteroides (Ahmed et al., 2007); 5′- GCACAAGCAGTGGAGT -3′ (forward) and 5′- CTTCCTCCGTTTTGTCAA -3′ (reverse) for Clostridium leptum (Matsuki et al., 2004); 5′-AAATGACGGTACCTGACTAA -3′ (forward) and 5′-CTTTGAGTTTCATTCTTGCGAA -3′ (reverse) for Clostridium coccoides (Matsuki et al., 2004); 5′-GAGGCAGCAGTAGGGAATCTTC -3′ (forward) and 5′-GGCCAGTTACTACCTCTATCCTTCTTC -3′ (reverse) for Lactobacillus spp. (Delroisse et al., 2008); and 5′-CGCGTCYGGTGTGAAAG -3′ (forward) and 5′-CCCCACATCCAGCATCCA -3′ (reverse) for Bifidobacterium spp. (Delroisse et al., 2008). Bacterial species were quantified by real-time qPCR using a Light Cycler 480 System II (Roche Applied Sciences, Pleasanton, CA, USA) as previously described (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). After initial denaturation at 95 °C for 30 s, 40 PCR cycles were conducted with denaturation at 95 °C for 5 s, annealing at 55 °C (total bacteria, Lactobacillus spp., Bifidobacterium spp., C. coccoides, C. leptum and Bacteroides) for 30 s, and extension at 72 °C for 15 s (total bacteria, Lactobacillus spp. Bifidobacterium spp. and Bacteroides) or 1 min (C. coccoides and C. leptum). Melting curve analysis was performed after amplification to distinguish the targeted PCR product from the non-targeted PCR product. Data were analyzed by the second derivative maximum method using Light Cycler 480 Basic Software. The relative abundance of the microbial populations is expressed as the proportion of the total bacterial 16S rDNA gene. The amplification efficiency (e) of real time PCR was estimated for each primer set from a linear regression of the crossing point (Cp) for each fecal DNA dilution versus the log dilution using the formula: e = x−1/slope, where “x” is a fold dilution. Efficiencies of the PCR reaction of the primer sets were between 1.94 and 1.99, close to the optimum value of 2.0. The PCR efficiencies ranging from 94 % to 99 % were used in this study (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). The relative abundance of the microbial populations is expressed as the proportion of the total bacterial 16S rRNA gene, using the following equation: relative quantification = 2−(Cp target-Cp total bacteria) × (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023).
ALP activity The colon mucosa and feces were homogenized with 10 mM Tris-buffered saline containing 1 % Triton X-100 (pH 7.3) and 1 mM PMSF. The homogenate was centrifuged at 7 000 × g at 4 °C for 15 min (Sogabe et al., 2007). The supernatant was used as an enzyme-enriched extract. Alkaline phosphatase activity was measured using a Lab Assay ALP kit (FUJIFILM Wako Pure Chemicals, Osaka, Japan).
Functional classification of the 12 species of edible mushrooms according to five colonic luminal variables The 12 species of edible mushrooms used in this study were classified into three groups according to their effects on the five colonic luminal variables (fecal mucins, fecal IgA, fecal microbial composition, cecal organic acids and fecal ALP). Mushrooms that significantly affected all five factors were classified as the high-impact (HI) group. Mushrooms that significantly affected 3 to 4 factors were categorized as the medium-impact (MI) group, and mushrooms that significantly affected only 1 to 2 factors were classified as the low-impact (LI) group. The effect on the organic acid content in the cecum was determined using the total organic acid content as an indicator. Several mushrooms and their constituent fiber have been reported to increase gut Bacteroides (Li et al., 2021). Bacteroides showed the most conspicuous change in the effects of mushrooms on fecal microbial composition in this study. Thus, the effect on fecal microbial composition was determined using the ratio of fecal Bacteroides as an indicator. Generally, a low pH in the large intestine is considered to be a favorable environmental condition (Hayakawa, 2010). To examine the validity of this classification, we compared the cecal pH among the three groups relative to the control group.
Statistical analyses Data were expressed as the mean ± SEM. Percentage data were normalized by arcsine transformation prior to data analysis. For data that followed a normal distribution, we used one-way analysis of variance (ANOVA). Data that did not follow a normal distribution were analyzed using the nonparametric Kruskal-Wallis test. Dunnett’s post-hoc test or Tukey-Kramer post-hoc test was performed when a significant effect was detected using one-way ANOVA. The Steel’s post-hoc test was performed when a significant effect was detected using the Kruskal–Wallis test. Some data were subjected to a Spearman rank correlation analysis (Rs; Spearman rank correlation coefficient). Data analysis was performed using BellCurve for Excel software (version 3.10, Social Survey Research Information Co., Ltd., Tokyo, Japan). P values < 0.05 were considered statistically significant.
Growth, food intake, relative organ weights and fecal weight Initial and final body weight (wt.), food intake, relative organ wts. and fecal dry wt. are shown in Table 2. Final body wt. was slightly decreased by dietary nameko and enokitake mushrooms. Shiitake, nameko, enokitake, eringi and wood ear mushrooms significantly decreased food intake. Colon wt. was increased by eringi, wood ear, white button and hiratake mushrooms intake. Cecum wt. was increased by dietary shiitake, nameko, enokitake, white button and hiratake mushrooms. Fecal dry wt. was decreased by dietary bunashimeji mushrooms.
| Initial body wt. (g) | Final body wt. (g) | Food intake (g/2 weeks) | Relative weight of colon (g/100 g body wt.) | Relative weight of cecum (g/100 g body wt.) | Fecal dry wt. (g/3 days) | |
|---|---|---|---|---|---|---|
| Experiment 1 | ||||||
| Cont. | 141 ± 2 | 269 ± 6 | 237 ± 7 | 0.392 ± 0.019 | 0.99 ± 0.03 | 4.24 ± 0.26 |
| Shiitake | 141 ± 2 | 258 ± 2 | 218 ± 4 * | 0.422 ± 0.028 | 1.27 ± 0.05 * | 4.30 ± 0.16 |
| Nameko | 141 ± 1 | 251 ± 4 * | 207 ± 4 ** | 0.453 ± 0.019 | 1.46 ± 0.13 * | 4.12 ± 0.16 |
| Bunashimeji | 141 ± 2 | 260 ± 5 | 222 ± 5 | 0.402 ± 0.019 | 1.24 ± 0.10 | 3.33 ± 0.18** |
| Experiment 2 | ||||||
| Cont. | 138 ± 3 | 257 ± 4 | 229 ± 4 | 0.367 ± 0.019 | 1.03 ±0.03 | 3.90 ± 0.17 |
| Enokitake | 138 ± 2 | 233 ± 7 * | 188 ± 6 * | 0.408 ± 0.036 | 1.48 ± 0.10 * | 3.80 ± 0.21 |
| Eringi | 138 ± 2 | 241 ± 4 | 204 ± 6 * | 0.462 ± 0.017 * | 1.22 ± 0.09 | 3.67 ± 0.16 |
| Wood ear | 138 ± 2 | 238 ± 6 | 208 ± 7 * | 0.445 ± 0.005 * | 1.24 ± 0.11 | 4.11 ± 0.26 |
| Experiment 3 | ||||||
| Cont. | 144 ± 3 | 265 ± 7 | 226 ± 9 | 0.276 ± 0.010 | 1.10 ± 0.06 | 3.55 ± 0.30 |
| White button | 144 ± 3 | 250 ± 7 | 208 ± 6 | 0.347 ± 0.019 * | 1.70 ± 0.08 ** | 3.10 ± 0.18 |
| Hiratake | 144 ± 2 | 246 ± 6 | 201 ± 5 | 0.389 ± 0.025 ** | 1.38 ± 0.07 * | 4.24 ± 0.19 |
| Maitake | 144 ± 2 | 258 ± 6 | 225 ± 8 | 0.316 ± 0.009 | 1.33 ± 0.08 | 4.24 ± 0.19 |
| Experiment 4 | ||||||
| Cont. | 137 ± 3 | 255 ± 11 | 209 ± 11 | 0.347 ± 0.018 | 1.08 ± 0.05 | 3.61 ± 0.28 |
| Yamabushitake | 137 ± 2 | 243 ± 7 | 195 ± 6 | 0.339 ± 0.020 | 1.20 ± 0.11 | 3.49 ± 0.24 |
| Porcini | 137 ± 2 | 251 ± 6 | 198 ± 3 | 0.330 ± 0.008 | 1.16 ± 0.10 | 3.52 ± 0.11 |
| Tamogitake | 137 ± 2 | 243 ± 7 | 215 ± 10 | 0.328 ± 0.014 | 1.00 ± 0.05 | 4.32 ± 0.31 |
Mean values with their standard errors; n = 6. * p < 0.05, ** p < 0.01, significant difference from control group (Dunnetts’ post hoc test or Steel post hoc test).
Fecal microbial composition Table 3 shows fecal microbial composition. Relative abundance of Bacteroides was significantly increased by dietary shiitake, nameko, enokitake, white button, hiratake, maitake, yamabushitake and porcini mushrooms. Relative abundance of C. leptum was decreased by dietary enokitake, eringi, white button, hiratake and maitake mushrooms. Relative abundance of C. coccoides was decreased by dietary enokitake, eringi and wood ear mushrooms. Dietary nameko, hiratake and maitake mushrooms significantly decreased the relative abundance of Lactobacillus spp. Relative abundance of Bifidobacterium spp. was not affected by any of the dietary mushrooms.
| Bacteroides | Clostridium leptum group | Clostridium coccoides group (% of total bacteria) | Lactobacillus spp. | Bifidobacterium spp. | |
|---|---|---|---|---|---|
| Experiment 1 | |||||
| Cont. | 5.54 ± 1.29 | 0.676 ± 0.127 | 6.95 ± 1.02 | 14.78 ± 0.72 | 1.536 ± 0.301 |
| Shiitake | 31.19 ± 4.47 ** | 0.617 ± 0.263 | 14.03 ± 4.60 | 8.23 ± 4.58 | 0.654 ± 0.304 |
| Nameko | 31.06 ± 6.02 ** | 0.301 ± 0.072 | 7.88 ± 0.72 | 6.45 ± 1.66 * | 0.853 ± 0.280 |
| Bunashimeji | 13.66 ± 2.75 | 0.771 ± 0.276 | 8.50 ± 1.56 | 14.44 ± 4.45 | 1.947 ± 0.916 |
| Experiment 2 | |||||
| Cont. | 13.46 ± 3.08 | 1.490 ± 0.402 | 24.05 ± 4.06 | 7.85 ± 2.87 | 0.954 ± 0.566 |
| Enokitake | 28.02 ± 2.73 ** | 0.150 ± 0.031 * | 13.53 ± 1.33 * | 10.39 ± 1.75 | 1.152 ± 0.194 |
| Eringi | 21.73 ± 3.16 | 0.193 ± 0.041 * | 8.03 ± 1.27 ** | 5.17 ± 1.20 | 0.588 ± 0.179 |
| Wood ear | 7.59 ± 1.82 | 1.865 ± 0.226 | 9.40 ± 1.67 ** | 12.15 ± 2.21 | 0.695 ± 0.146 |
| Experiment 3 | |||||
| Cont. | 7.76 ± 1.65 | 1.121 ± 0.262 | 13.79 ± 2.22 | 5.30 ± 1.16 | 0.707 ± 0.242 |
| White button | 21.16 ± 2.81 ** | 0.322 ± 0.050 ** | 12.40 ± 1.54 | 7.46 ± 1.99 | 1.241 ± 0.172 |
| Hiratake | 25.88 ± 3.30 ** | 0.211 ± 0.046 ** | 9.71 ± 1.30 | 1.56 ± 0.54 * | 0.202 ± 0.067 |
| Maitake | 27.52 ± 3.03 ** | 0.327 ± 0.080 ** | 16.72 ± 2.39 | 2.50 ± 0.66 * | 0.165 ± 0.047 |
| Experiment 4 | |||||
| Cont. | 7.99 ± 3.90 | 0.787 ± 0.069 | 7.08 ± 2.24 | 5.83 ± 2.55 | 0.231 ± 0.101 |
| Yamabushitake | 45.19 ± 3.83 ** | 0.786 ± 0.136 | 10.81 ± 2.12 | 2.01 ± 0.76 | 0.267 ± 0.080 |
| Porcini | 29.18 ± 3.97 ** | 0.777 ± 0.239 | 9.00 ± 1.97 | 3.35 ± 0.93 | 0.131 ± 0.051 |
| Tamogitake | 17.06 ± 3.22 | 1.003 ± 0.208 | 9.48 ± 2.06 | 2.24 ± 0.98 | 0.092 ± 0.052 |
Mean values with their standard errors; n = 6. * p < 0.05, ** p < 0.01, significant difference from control group (Dunnetts’ post hoc test or Steel post hoc test).
Cecal digesta, cecal pH and cecal organic acid concentrations Cecal digesta wt., pH value and organic acids are shown in Table 4. Cecal digesta wt. was increased by dietary white button mushrooms. Cecal digesta pH was reduced by dietary shiitake, nameko, enokitake, eringi, white button, yamabushitake and porcini mushrooms. Cecal succinate was increased by dietary shiitake, nameko and maitake mushrooms. Cecal lactate and n-butyrate were increased by dietary enokitake and white button mushrooms. Cecal propionate was increased by dietary bunashimeji and porcini mushrooms. Cecal acetate was not significantly affected by dietary mushrooms. Dietary shiitake, nameko, bunashimeji, enokitake, white button, yamabushitake and porcini mushrooms significantly increased cecal total organic acids.
| Cecal digesta (g) | Cecal pH | Succinate | Lactate | n-Butyrate | Propionate | Acetate | Total organic acids | |
|---|---|---|---|---|---|---|---|---|
| (µmol / g cecal digesta) | ||||||||
| Experiment 1 | ||||||||
| Cont. | 1.90 ± 0.09 | 7.57 ± 0.16 | 7.72 ± 3.43 | 1.20 ± 0.41 | 11.0 ± 1.09 | 9.5 ± 0.82 | 27.4 ± 3.7 | 56.8 ± 6.1 |
| Shiitake | 2.36 ± 0.16 | 6.23 ± 0.0.8 ** | 47.59 ± 8.01 * | 3.82 ± 1.50 | 17.1 ± 2.4 | 11.4 ± 2.09 | 36.9 ± 5.6 | 116.8 ± 7.5 * |
| Nameko | 2.75 ± 0.30 | 6.87 ± 0.17 * | 39.50 ± 8.47 * | 4.35 ± 1.18 | 17.9 ± 2.1 | 13.7 ± 1.02 | 35.5 ± 5.9 | 110.9 ± 11.8 * |
| Bunashimeji | 2.36 ± 0.22 | 7.03 ± 0.2 | 19.84 ± 7.11 | 2.74 ± 0.92 | 13.9 ± 1.8 | 15.3 ± 1.98 * | 38.8 ± 3.2 | 90.5 ± 5.6 * |
| Experiment 2 | ||||||||
| Cont. | 2.07 ± 0.10 | 7.18 ± 0.09 | 18.71 ± 5.79 | 1.22 ± 0.70 | 11.7 ± 2.5 | 12.9 ± 1.12 | 40.5 ± 2.0 | 85.0 ± 5.1 |
| Enokitake | 2.69 ± 0.22 | 6.23 ± 0.21 ** | 27.94 ± 4.05 | 5.63 ± 0.67 ** | 20.1 ± 1.6* | 15.8 ± 1.02 | 36.9 ± 4.4 | 106.4 ± 7.1* |
| Eringi | 2.31 ± 0.22 | 6.68 ± 0.10 * | 17.01 ± 4.31 | 2.35 ± 0.65 | 15.3 ± 1.5 | 12.5 ± 1.24 | 31.8 ± 3.9 | 78.9 ± 5.7 |
| Wood ear | 2.40 ± 0.25 | 7.20 ± 0.13 | 6.08 ± 3.06 | 1.59 ± 0.35 | 16.6 ± 1.5 | 10.7 ± 0.97 | 40.2 ± 4.7 | 75.2 ± 4.8 |
| Experiment 3 | ||||||||
| Cont. | 1.92 ± 0.17 | 7.28 ± 0.09 | 7.24 ± 3.30 | 0.79 ± 0.62 | 9.6 ± 1.4 | 12.4 ± 1.5 | 29.9 ± 1.9 | 59.9 ± 5.9 |
| White button | 3.01 ± 0.21 * | 6.18 ± 0.07 * | 26.32 ± 4.10 | 6.80 ± 0.90 ** | 18.9 ± 1.8 ** | 13.0 ± 1.4 | 31.8 ± 2.6 | 96.9 ± 6.8 * |
| Hiratake | 2.22 ± 0.11 | 6.93 ± 0.20 | 22.56 ± 8.13 | 2.94 ± 1.11 | 12.3 ± 1.5 | 12.9 ± 1.0 | 29.5 ± 4.3 | 80.2 ± 10.9 |
| Maitake | 2.36 ± 0.17 | 6.97 ± 0.17 | 31.23 ± 8.97 * | 2.82 ± 0.79 | 12.2 ± 1.9 | 12.6 ± 1.2 | 34.1 ± 3.7 | 92.8 ± 12.2 |
| Experiment 4 | ||||||||
| Cont. | 1.87 ± 0.14 | 7.02 ± 0.22 | 13.86 ± 7.17 | 0.53 ± 0.34 | 12.6 ± 3.2 | 13.2 ± 1.7 | 31.4 ± 3.2 | 71.6 ± 10.2 |
| Yamabushitake | 2.07 ± 0.25 | 6.12 ± 0.17 ** | 41.46 ± 10.26 | 7.82 ± 3.71 | 12.6 ± 2.1 | 26.5 ± 3.3 | 52.0 ± 8.8 | 140.5 ± 21.6 * |
| Porcini | 2.10 ± 0.27 | 6.27 ± 0.14 ** | 22.57 ± 11.81 | 4.38 ± 2.11 | 14.2 ± 2.5 | 34.3 ± 4.6 ** | 65.1 ± 9.5 | 128.9 ± 19.4 * |
| Tamogitake | 1.78 ± 0.16 | 7.15 ± 0.08 | 2.34 ± 0.71 | 0.69 ± 0.4 | 18.1 ± 6.7 | 21.3 ± 6.6 | 66.7 ± 19.6 | 97.2 ± 24.1 |
Mean values with their standard errors; n = 6. * p < 0.05, ** p < 0.01, significant difference from control group (Dunnetts’ post hoc test or Steel post hoc test).
Fecal mucins and IgA level, and ALP activity Fecal mucins and IgA level, and ALP activity are shown in Fig. 1. All 12 species of mushrooms significantly increased the concentration of fecal mucins. Fecal IgA level was enhanced by dietary shiitake, nameko, enokitake, white button, hiratake and yamabushitake mushrooms. Fecal ALP activity was increased by dietary shiitake, enokitake, white button, hiratake and yamabushitake mushrooms. In the present study, colonic ALP was not significantly affected by dietary mushroom intake (data not shown).
Effects of edible mushrooms on fecal mucins, IgA and ALP activity in rats fed a high-fat diet in Experiment 1–4.
Data in box-plots are presented as median, minimum and maximum values, n = 6. * p < 0.05, ** p < 0.01, vs Control (Dunnett test or Steel test). C, Control; SH, Shiitake; NA, Nameko, BU; Bunashimeji, EN; Enokitake, ER; Eringi, WE; Wood ear, WB; White button, HI; Hiratake, MA; Maitake, YA; Yamabushitake, PO; Porcini, TA; Tamogitake.
Functional classification of the 12 species of edible mushrooms according to the five colonic luminal variables Table 5 shows the presence or absence of effects of the 12 species of edible mushrooms on the five colonic luminal variables (fecal mucins, fecal IgA, fecal microbial composition, cecal organic acids and fecal ALP). Dietary shiitake, enokitake, white button and yamabushitake mushrooms significantly affected all five variables (fecal mucins, fecal IgA, fecal microbial composition, cecal organic acids and fecal ALP) (HI group). Dietary tamogitake affected only fecal mucins, and maitake, eringi, wood ear and bunashimeji mushrooms affected only one or two variables (fecal mucins and fecal microbial composition or cecal organic acids) (LI group). Dietary nameko, porcini and hiratake mushrooms affected three to four variables (MI group). In the present study, significant relationships were found between fecal mucin level and fecal relative ratio of Bacteroidetes (Rs = 0.560, n = 96, p < 0.001), cecal level of total organic acids (Rs = 0.487, n = 96, p < 0.001), fecal IgA level (Rs = 0.230, n = 96, p = 0.0245) and fecal ALP activity (Rs = 0.509, n = 96, p < 0.001). Figure 2 shows the comparison of cecal pH among the three groups compared to the control group. There was no significant change in cecal pH in the LI group compared to the control group (Fig. 2). Cecal pH in the MI group was significantly decreased compared to the control group, and even more so in the HI group.
| HI group | MI group | LI group | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Shiitake | Enokitake | White button | Yamabushitake | Hiratake | Nameko | Porcini | Eringi | Maitake | Tamogitake | Wood ear | Bunashimeji | |
| Mucins (mg/g feces) | p < 0.05 | p < 0.05 | p < 0.001 | p < 0.05 | p < 0.001 | p < 0.05 | p < 0.05 | p < 0.05 | p < 0.001 | p < 0.05 | p < 0.05 | p < 0.05 |
| Bacteroides (% of total bacteria) | p < 0.001 | p < 0.001 | p < 0.01 | p < 0.001 | p < 0.001 | p < 0.001 | p < 0.001 | p < 0.001 | ||||
| IgA (mg/g feces) | p < 0.01 | p < 0.05 | p < 0.01 | p < 0.05 | p < 0.001 | p < 0.01 | ||||||
| Total organic acids (µmol/g cecal digesta) | p < 0.05 | p < 0.05 | p < 0.05 | p < 0.05 | p < 0.05 | p < 0.05 | p <0.05 | |||||
| Fecal ALP (kunits/mg protein) | p < 0.05 | p < 0.05 | p < 0.01 | p < 0.05 | p < 0.01 | |||||||
Statistically significant p-values vs control group are shown in the table (p < 0.05, p < 0.01 or p < 0.001; Dunnett test or Steel test, n = 6). HI, high-impact; MI, medium-impact: LI, low-impact.
Comparison of cecal pH in each edible mushroom group.
Data in box-plots are presented as median, minimum and maximum values.
a, b, c Values without a common letter differ, p < 0.05 (Tukey-Kramer post hoc test).
LI group: Eringi, Maitake, Tamogitake, Wood ear, Bunashimeji (n = 30); MI group: Hiratake, Nameko and Porcini (n = 18); HI group: Shiitake, Enokitake, White button and Yamabushitake (n = 24). Cont., n = 24.
LI, low-impact; MI, medium-impact; HI, high-impact.
In Japan, several biologically similar foods are grouped into “food groups”, such as vegetables and meat, as shown in the Japanese Food Composition Table (Council for Science and Technology; Ministry of Education, Culture, Sports, Science and Technology, Japan, 2020). Foods from the same food group generally contain similar nutritional components (Council for Science and Technology; Ministry of Education, Culture, Sports, Science and Technology, Japan, 2020). Over the last few decades, much research has been done on the health effects of functional ingredients in foods. The value of a food is generally determined by the types and amounts of nutrients and functional ingredients it contains. We have recently shown that consumption of fermentable non-digestible soluble fiber or oligosaccharides commonly elevated ALP activity and expression of Alpi-1, an ALP gene expressed throughout the intestine (specifically in the colonic mucosa), and the elevation was significantly correlated with fecal mucins, changes in microbial composition and cecal organic acids in rats fed an HF diet (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). In this study, for the purpose of classifying mushrooms by their functionality, we compared and evaluated the effects of 12 species of edible mushrooms on colonic luminal variables, such as fecal and colonic ALP activity, fecal microbial composition, mucins, IgA and cecal organic acids in rats fed an HF diet.
It has been reported that many dietary fibers, whether soluble or insoluble, increase gut mucin content (Morita, 2022; Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). Mushrooms are rich in dietary fiber, but the data are limited regarding their effect on gut mucin content. Yang et al. (2017) reported that feeding of a water extract from reishi mushrooms to rats increased the fecal mucin content. The present study indicated that all 12 species of mushrooms significantly increased the fecal mucin content. We speculated that mushrooms generally could increase the gut mucin content. In addition, it was suggested that the mucin content may be the most susceptible factor among the colonic environmental factors examined in this study.
When soluble dietary fiber is fermented by gut bacteria, organic acids or SCFAs are produced and cecal pH is decreased in rats (Hayakawa, 2010; Morita, 2022). In the present study, dietary shiitake, nameko, enokitake, white button, yamabushitake and porcini mushrooms increased cecal total organic acids and decreased cecal pH. On the other hand, bunashimeji mushrooms significantly increased cecal total organic acids, but did not significantly affect cecal pH. The reason for this may be that the cecal organic acid-increasing effect of bunashimeji mushrooms was not sufficient to significantly lower the cecal pH. As an exception, eringi mushrooms did not affect cecal organic acids, but significantly decreased cecal pH. The reason for this is currently unknown.
It has been reported that several species of mushrooms, such as shiitake, yamabushitake, maitake and reishi, or their polysaccharides increase the proportion of intestinal Bacteroides in animal models (Li et al., 2021). This study also showed that the relative abundance of Bacteroides in feces was significantly increased by dietary shiitake, nameko, enokitake, white button, hiratake, maitake, yamabushitake and porcini mushrooms. On the other hand, in this study, dietary bunashimeji, eringi, wood ear and tamogitake mushrooms did not significantly affect the ratio of Bacteroides in the feces. Li et al. (2021) considered that the beneficial activities of mushrooms are connected with the modulation of gut microbiota. In our previous research, dietary glucomannan, inulin and fructo-oligosaccharides significantly increased cecal organic acids, fecal mucins and fecal and cecal ALP activity, but did not increase fecal Bacteroides in rats (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). In addition, dietary chitosan remarkably increased the ratio of Bacteroides in feces, but its effects on fecal mucins and ALP activity were nominal compared with dietary inulin (Okazaki and Katayama, 2022). Therefore, we speculate that the effects of edible mushrooms on the colonic luminal variables measured in this study may be difficult to explain solely by modulation of the gut microbiota.
Secretory IgA (sIgA) released from the gut mucosal surface has a central role in the mucosal immune system by inhibiting the adherence of pathogenic bacteria and neutralizing biologically active antigens (Mestecky and Russell, 2003). Some researchers indicated that soluble dietary fibers and indigestible oligosaccharides increased gut IgA production, and cecal and fecal IgA content (Tanabe et al., 2004; Ito et al., 2011). Regarding the effects of mushrooms on IgA secretion, it has been reported that reishi mushrooms can induce IgA secretion in the rat small intestine (Li et al. 2021). In a pilot clinical study, Nishimoto et al. (2023) indicated that dietary supplementation with a mixture of three mushroom species (eringi, bunashimeji and maitake) tended to increase the amount of intestinal IgA. This study found that fecal IgA level was significantly enhanced by dietary shiitake, nameko, enokitake, white button, hiratake and yamabushitake. However, the other mushrooms assessed in this study did not significantly affect fecal IgA levels. These results suggest that the effects of mushrooms on IgA production from the intestinal tract differ considerably depending on the species of mushroom.
We have demonstrated that soluble dietary fiber and non-digestible oligosaccharides commonly elevate colonic and fecal ALP activity and the expression of Alpi-1, an ALP gene expressed throughout the intestine (Okazaki and Katayama, 2017; 2019; 2021; 2022; 2023). In the present study, despite the high fiber content, intake of the 12 species of edible mushrooms did not have a significant effect on colon ALP activity. A possible reason for this is that the dietary fiber contained in the mushrooms is mainly composed of insoluble dietary fiber (Council for Science and Technology; Ministry of Education, Culture, Sports, Science and Technology, Japan, 2020). This study showed that dietary shiitake, enokitake, white button, yamabushitake and hiratake mushrooms significantly elevated fecal ALP activity, but the other mushrooms examined did not have a significant effect. The authors have repeatedly observed a high positive correlation between fecal ALP activity and fecal mucin excretion. Regarding the mechanism of increased mucin secretion by dietary fiber, it is thought that the promotion of turnover of small intestinal epithelial cells is involved (Morita, 2010). It is possible that the enhancement of fecal ALP activity by mushrooms is related to the promotion of intestinal cell turnover. Fecal ALP activity was the least affected of the large intestine environmental factors examined in this study.
This study demonstrated that the effect on the colonic environment in rats fed an HF diet could vary greatly depending on the species of mushroom. The 12 species of mushrooms were classified into HI, MI and LI groups based on their effects on the five colonic luminal variables in rats fed an HF diet. A significant correlation was observed between fecal mucin content and other environmental factors in the colon, indicating the possibility of a relationship among the environmental factors. Comparing the cecal pH of each group, the LI group did not show a significant difference from the control group, but the MI and HI groups showed a significant stepwise decrease. Therefore, we suggest that the classification of mushrooms in this study has a certain significance. This classification is based on the degree of diversity in the effects of mushrooms on the colonic environment, and does not indicate the superiority or inferiority of mushrooms as foods, but is rather an indicator of their characteristics. We speculate that these results may serve as a reference when selecting mushrooms to ingest based on physiological conditions and palatability. Given the progress in research and development of functional foods, it is considered important to evaluate, characterize and classify food groups from the aspect of functionality. We suggest that the classification used in this study requires further careful validation in terms of accuracy and content.
The classification of mushrooms in this study could not be explained by dietary fiber content. Mushrooms contain various bioactive compounds such as resistant protein, ergosterols, lectins, terpenoids, phenolics and polyphenolics. It is possible that the effects of mushrooms on the colonic environment are due to the combined contribution of dietary fiber and these compounds. The causes of differences in the effects of mushrooms on the colonic environment are currently under investigation.
Acknowledgements This research was supported in part by a research grant from the Hokuto Foundation for Bioscience. The authors thank Reika Honma, Rubi Awazu, Mai Ozaki, Rina Takahashi, Rika Kimura and Yuka Honma for their technical assistance.
Conflict of interest There are no conflicts of interest to declare.
alkaline phosphatase
FOSfructo-oligosaccharides
HFhigh-fat
HIhigh-impact
IgAimmunoglobulin A
LIlow-impact
MImedium-impact
