2014 Volume 20 Issue 2 Pages 493-497
High-temperature steam treatment of wood materials results in sterilization and improved digestibility; however, in some cases deleterious degradation products are generated. Steam-treated sawdust-based spent mushroom substrate (S-SSMS) was prepared from non-treated SSMS (N-SSMS) after the cultivation of Maitake (Grifola frondosa) mushroom by autoclaving. Wistar rats were assigned to three groups: control group, fed a basal diet; N-SSMS group, fed a 25% N-SSMS diet; and S-SSMS group, fed a 25% S-SSMS diet for 26 days. The effects of S-SSMS on the growth performance, serum biochemical parameters, fecal composition, and cecal pH of rats were investigated. There were no significant differences in body weight gain, feed efficiency, and serum biochemical parameters among the groups. However, fecal weight and protein amount of N-SSMS and S-SSMS groups were significantly higher than those of the control group, while cecal pHs of N-SSMS and S-SSMS groups were lower than that of the control group. Based on these observations, S-SSMS has potential as a feed additive for monogastric animals.
Commercial production of edible mushrooms in Japan was over 458,000 tons in 2012. Most edible mushrooms are now cultivated using mushroom beds. The basic material used in the mushroom beds is wood sawdust, agricultural straw, cotton waste, or corn-cob and is chosen depending on the mushroom species produced. Because some wood-rotting fungi (mushrooms) such as Maitake (Grifola frondosa) and Nameko (Pholiota nameko) specifically require wood material for mushroom formation, sawdust-based mushroom substrate is used for the cultivation of these mushrooms. During cultivation, sawdust-based spent mushroom substrate (SSMS), which consists of the residual substrate and mycelial remnants from mushroom cultivation, is abundantly produced and amounts to several times more than the mushroom products (Phan and Sabaratnam, 2012). Therefore, in recent years, efforts to effectively recycle SSMS have increased because of environmental and economic issues.
Among several recycling methods, utilization of SSMS as an animal feed is considered to be reasonable and promising. The digestible component (cellulose and hemicellulose) in wood sawdust is potentially an energy source for ruminants, but wood cellulose is typically not sufficiently degraded by rumen microorganisms to provide energy because of its close association with lignin. However, Suzuki et al. (1995) reported that SSMS could be utilized as an animal feed resource, since the digestibility of SSMS left after the cultivation of Nameko and Maitake mushrooms was improved through the rotting of wood materials in sawdust-based mushroom substrate during cultivation. Notably, SSMS contains the remaining mycelium. It is rich in protein, fber, vitamins, and minerals, and also includes functional components such as beta-glucans (Ruiz-Herrera, 1991; Ulziijargal and Mau, 2011). Therefore, the remaining mycelium may improve the nutritional valuable of SSMS for animal feed.
Storage of SSMS is problematic; it putrefes easily because it contains nutrient-rich organic compounds and has a high moisture content (approximately 50 – 60%) (Kwak et al., 2008). Accordingly, effective processes for uses of SSMS are needed to prolong its storage period. Among these processes, high-temperature steam treatment is applicable for sterilization of SSMS, as well as decomposition and solubilization of its residual macromolecular substrates. In fact, steam treatment of some species of hardwoods has been reported to increase their in vitro digestibility for ruminants (Bender et al., 1970). Thus, steam treatment, as well as biological treatment by mushrooms, is expected to increase the nutritional value of SSMS for animal feed. On the other hand, steam treatment has been reported to generate aldehydes that have pesticidal and bactericidal effects (Ikumo et al., 1987) and deleterious materials that may be formed from lignin (Hart et al., 1980). However, little attention has been given to biochemical tests for the safety evaluation of SSMS and wood materials used in animal feed.
Previous studies have indicated the potential of using SSMS, other material-based spent mushroom substrates, and wood materials as animal feed additives, focusing on ruminants such as sheep and calves (Phan and Sabaratnam, 2012). Few studies have dealt with monogastric animals (Britton, 1978; Song et al., 2007).
The objective of this study was to investigate the effect of steam-treated SSMS (S-SSMS) on growth performance, serum biochemical parameters of nutritional and metabolic status, fecal composition, and cecal pH in rats (as an example of a monogastric animal).
Preparation of N-SSMS and S-SSMS Fresh raw SSMS of the edible mushroom Maitake (G. frondosa) was obtained from Ichimasa Kamaboko Co. (Niigata, Japan). SSMS was oven-dried at 60°C overnight and used as N-SSMS. SSMS was treated with steam at 128°C for 120 min, then oven-dried, and used as S-SSMS. Prior to analysis, all samples were ground with a laboratory mill.
Chemical analysis of SSMS and feces Dry matter was determined by drying at 105°C for 18 h. Neutral detergent fiber (NDF), acid detergent fber (ADF) and acid detergent lignin (ADL) were determined by the method of Van Soest et al. (1991). Hemicellulose and cellulose were calculated by subtraction of ADF from NDF and ADL from ADF, respectively. Crude protein was determined by the AOAC method (AOAC, 1995), factor 6.25. Crude ash was determined by ashing at 550°C for 3 h.
Animals and diets Male Wistar rats 4 weeks of age (Charles River Japan, Yokohama, Japan) were allowed 8 days for acclimatization and were then divided into three groups (n = 8) of similar body weights: control group fed a basal diet, N-SSMS group fed a 25% N-SSMS diet, and S-SSMS group fed a 25% S-SSMS diet for 26 days. The basal diet, prepared under standards given by the American Institute of Nutrition (American Institute of Nutrition, 1977), was composed of 25% casein, 5% sucrose, 58.5% corn starch, 6% corn oil, 3.5% mineral mixture (AIN-76), 1% vitamin mixture (AIN-76), 0.2% choline bitartrate, and 0.5% cellulose powder. In the N-SSMS or S-SSMS diet, 25% of the corn starch of the basal diet was substituted for N-SSMS or S-SSMS, respectively. The rats were individually housed in an air-conditioned room at 24°C and 50 – 60% humidity with a 12 h light-dark cycle. Animals were allowed free access to drinking water and the experimental diets during the experimental period. All animal experiments were conducted in accordance with the Guidelines for Animal Experimentation of Nagaoka National College of Technology. Body weight, feed intake, and fecal wet weight of rats were measured daily from day 4 to day 26 and all feces were collected. Rats were anaesthetized with an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination from the heart. The cecum was quickly removed and stored at −20°C.
Serum biochemical parameters and cecal pH Blood samples were cooled to 4°C and centrifuged at 1,500 × g for 20 min to collect serum. Serum activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and serum concentrations of albumin, total protein, triglyceride and total cholesterol were measured with commercial kits (Wako Pure Chemical Industries, Osaka, Japan). The difference between total protein and albumin levels was regarded as the globulin level. Cecal contents (0.1 g) were homogenized in 5.0 mL of distilled water and the pH was measured using a pH meter (F-53, Horiba, Tokyo, Japan).
Statistical analysis Each data value is expressed as the mean ± SE. Statistical analysis was performed using a one-way analysis of variance (ANOVA) followed by the Tukey-Kramer HSD test for multiple comparisons. Differences were considered significant at p < 0.05.
The chemical composition of N-SSMS and S-SSMS is shown in Table 1. N-SSMS and S-SSMS were relatively high in fber and very low in protein and ash. There were no marked differences in the contents of hemicellulose, cellulose, protein, and ash between N-SSMS and S-SSMS. However, the contents of NDF, ADF, and lignin in S-SSMS were higher than those in N-SSMS. These increased contents account mainly for the increased lignin content. Likewise, it has been reported that the lignin content in corn stover and paddy straw increased with steam treatment (Oji and Mowat, 1978; Rangnekar et al., 1982). The increased lignin content in S-SSMS is presumed to be due to the production of artifact lignin, usually through non-enzymatic browning reactions during steam treatment (Van Soest, 1965). From these results, steam treatment of SSMS in this study is likely to have no apparent effects on decomposition and solubilization of digestible components (cellulose and hemicellulose).
| Ingredient | N-SSMS (%) | S-SSMS (%) |
|---|---|---|
| NDF | 67.5 | 73.5 |
| ADF | 53.0 | 60.0 |
| Hemicellulose (NDF − ADF) | 14.5 | 13.5 |
| Cellulose (ADF − ADL) | 33.6 | 35.3 |
| Lignin (ADL) | 19.4 | 24.7 |
| Crude protein (N × 6.25) | 6.4 | 6.6 |
| Crude ash | 2.5 | 2.4 |
Abbreviations: N-SSMS, non-treated sawdust-based spent mushroom substrate; S-SSMS, steam-treated sawdust-based spent mushroom substrate; NDF, neutral detergent fber; ADF, acid detergent fber; ADL, acid detergent lignin
To investigate whether ingestion of S-SSMS affects the growth performance of rats, we determined the body weight, feed intake, and feed efficiency of rats fed the experimental diets. Significant differences in body weight among the three test groups were not observed during the experimental feeding period and all the rats in each test group grew normally (data not shown). There were no significant differences in body weight gain and feed efficiency among the three test groups during the experimental feeding period, although the feed intake in the S-SSMS group was significantly higher than that in the control group (Table 2). In some feeding studies with ruminants, the daily body weight gain and voluntary feed intake were reported to be decreased by inclusion of wheat straw-based spent mushroom substrate into the diets (Adamović et al., 1998; Fazaeli and Talebian Masoodi, 2006). In contrast, the results of this study suggested that the ingestion of N-SSMS and S-SSMS has no infuence on the growth performance of rats. Also, the potential of steamed hardwood materials as a feed for ruminants has been evaluated by in vivo animal feeding studies. Slight increases or decreases in feed intake and body weight of ruminants were observed when steamed birch (30 or 60%) or aspen (30%) was added to the diet (Heaney et al., 1973; Kajikawa et al., 1987); however, based on overall results, it was concluded that steamed aspen or birch could be used as a roughage substitute in maintenance rations for ruminants. Accordingly, although there are differences in nutritional value depending on the tree species, evaluation of the feeding value of S-SSMS for livestock is worthy of further study.
| Item | Group | ||
|---|---|---|---|
| Control | N-SSMS | S-SSMS | |
| Initial body weight (g) | 181.3 ± 3.6 | 181.5 ± 3.0 | 181.9 ± 3.9 |
| Final body weight (g) | 364.4 ± 6.3 | 378.5 ± 8.2 | 379.4 ± 8.5 |
| Body weight gain (g/25 d) | 183.1 ± 5.6 | 197.0 ± 7.4 | 197.5 ± 5.1 |
| Slaughter weight (g) | 282.0 ± 5.5 | 290.5 ± 4.8 | 293.3 ± 6.3 |
| Feed intake (g/d) | 20.9 ± 0.6a | 22.7 ± 0.6ab | 23.8 ± 0.7b |
| Feed efficiency1 | 0.35 ± 0.01 | 0.35 ± 0.01 | 0.33 ± 0.01 |
Values are expressed as the mean ± SE (n = 8).
Values with different superscript letters indicate significantly different ( p < 0.05).
1Body weight gain (g/25 d)/feed intake (g/25 d).
Abbreviations: N-SSMS, non-treated sawdust-based spent mushroom substrate; S-SSMS, steam-treated sawdust-based spent mushroom substrate
To investigate whether ingestion of S-SSMS affects the liver-function, nutritional status, and lipid metabolism of rats, we determined the serum enzyme activities and the concentrations of protein and lipid in rats fed the experimental diets. There were no significant differences in the activities of AST and ALT, liver-function marker enzymes, in rat serum among the test groups (Table 3). These results suggest that supplementation with N-SSMS and S-SSMS did not induce liver injury in rats, although there are reports of the possibility that steam treatment generates toxic materials from lignin or carbohydrates (Hart et al., 1980; Ikumo et al., 1987). There were no significant differences in the concentrations of serum albumin and total protein among the test groups, indicating that the ingestion of N-SSMS and S-SSMS had no effect on the nutritional status of rats. There were no significant differences in the concentrations of serum triglyceride and total cholesterol among the test groups, indicating that the ingestion of N-SSMS and S-SSMS did not affect the lipid metabolism of rats.
| Item | Group | ||
|---|---|---|---|
| Control | N-SSMS | S-SSMS | |
| Serum | |||
| AST (Karmen unit) | 135.9 ± 9.8 | 128.6 ± 11.8 | 127.8 ± 15.9 |
| ALT (Karmen unit) | 30.5 ± 1.1 | 33.7 ± 1.0 | 33.3 ± 2.1 |
| Albumin (g/100 mL) | 3.6 ± 0.0 | 3.4 ± 0.1 | 3.6 ± 0.1 |
| Total protein (g/100 mL) | 6.3 ± 0.1 | 6.2 ± 0.0 | 6.0 ± 0.1 |
| Albumin/globulin ratio | 1.34 ± 0.02ab | 1.28 ± 0.05a | 1.54 ± 0.07b |
| Triglyceride (mg/100 mL) | 132.1 ± 30.2 | 125.7 ± 13.0 | 116.4 ± 18.7 |
| Total cholesterol (mg/100 mL) | 101.8 ± 7.1 | 109.5 ± 3.1 | 102.0 ± 5.5 |
| Fecal | |||
| Wet weight (g/d) | 0.87 ± 0.04a | 5.13 ± 0.19b | 4.96 ± 0.12b |
| Moisture (%) | 28.8 ± 2.1 | 34.0 ± 2.8 | 28.4 ± 2.0 |
| Crude protein (g/d) | 0.19 ± 0.01a | 0.58 ± 0.03b | 0.64 ± 0.02b |
| Crude ash (g/d) | 0.22 ± 0.01a | 0.36 ± 0.02b | 0.37 ± 0.01b |
| NDF (g/d) | 0.21 ± 0.01a | 3.38 ± 0.12b | 3.28 ± 0.08b |
| Cecal | |||
| pH | 7.63 ± 0.06a | 7.18 ± 0.04b | 7.17 ± 0.03b |
Values are expressed as the mean ± SE (n = 8).
Values with different superscript letters indicate significantly different ( p < 0.05).
Abbreviations: N-SSMS, non-treated sawdust-based spent mushroom substrate; S-SSMS, steam-treated sawdust-based spent mushroom substrate; AST, aspartate aminotransferase; ALT, alanine aminotransferase; NDF, neutral detergent fber
To investigate whether ingestion of S-SSMS affects the fecal excretion and induction of cecal fermentation of rats, we determined the fecal weight and composition and cecal pH of rats fed the experimental diets. Daily fecal wet weight and the concentrations of protein, ash, and NDF in the N-SSMS and S-SSMS groups were significantly higher than those in the control group (Table 3). The abundant feces excreted from groups N-SSMS and S-SSMS were largely attributable to abundant fber concentrations in dietary N-SSMS and S-SSMS. The pH of the cecal contents was significantly lower in the N-SSMS and S-SSMS groups than in the control group. The increased fecal protein concentration and decreased cecal pH, respectively, are probably due to increases in total enteric bacterial mass and in the production of organic acids, through the promotion of cecal fermentation in the rats by SSMS ingestion.
In conclusion, there were no significant differences in rat growth performance, serum biochemical parameters, and fecal composition when the N-SSMS and S-SSMS groups were compared. These results indicate that the long-term steam treatment at a comparably low temperature in this study probably has no negative dietary infuence on S-SSMS. Therefore, it seems possible for S-SSMS to be used as feed additive for monogastric animals.
Acknowledgements We are grateful to Nobuhisa Kawaguchi for his valuable advice and support. Also, we thank Shungo Toyama and Masatoshi Yasui for their excellent technical support. This study was supported by the Nakajo Biomass Consortium (Niigata, Japan).
