Corncob as a Substrate for the Cultivation of Lentinula edodes

Corncob contains nutrients for the growth of mushrooms. Since wood, the original substrate for mushroom production, is becoming difficult to obtain these days, the study aims to evaluate the effect of using corncob as a substrate for Lentinula edodes (shiitake) cultivation, and to provide an economic and eco-friendly approach to transform waste biomass into high quality edible mushrooms. Six formulas containing gradient proportions of corncob were assessed (100 cultivation bed-log per group) together with an oak sawdust control. Chemical profile analysis suggested corncob substitution reduced the substrate’s carbon level and increased nitrogen level. Formulas containing 18–58% corncob obtained higher yield comparing to the sawdust control, indicating a strong boosting effect of corncob. The formula composing of 50% corncob, 28% oak sawdust, 20% wheat bran, and 2% gypsum showed the best performance with the fastest mycelia growth, better log browning, the highest yield (722.08 g/log) and summit biological efficiency (80.23%). The determined carbon/nitrogen in this substrate was 67.21. The size (pileus diameter) of fruit bodies were not much affected by the change in formulas. Addition of corncob had an influence on nutritional composition of mushrooms. The highest polysaccharide content in fruit bodies (4.51 g/100 g) was found when the substrate contains 40% corncob. Corncob is a major agricultural waste in the world. These results revealed an excellent potential of corncob when used as a main substrate ingredient for L. edodes cultivation.


Introduction
Lentinula edodes (Berk.) Singer (Shiitake in Japanese or Xiang-gu in Chinese) is a nutritious edible and medical mushroom. They provide protein, essential amino acids, dietary fibers and vitamins (B1, B2, B12, C, D, and E), and are also reported to present medicinal molecules such as polysaccharides, terpenoids, sterols and lipids, which possess anti-hypertensive, anti-viral, antioxidant, anti-tumor, and immuno modulatory activities [1]. As a world's leading cultivated mushroom, L. edodes has an annual output over 7 billion kg, contributing 22% of the world's mushroom supply [2]. China has a history of artificial cultivation of Xiang-gu for over 800 years (County Record of Qingyuan, 1209 AD), and is now the major producer of this species. Many communities in China benefited from economic profits of L. edodes production and lifted themselves from poverty.
In early days, L. edodes was usually grown on natural logs of the shii tree (Castanopsis cuspidate). Nevertheless, the sawdust-based substrate invented in late 1960 s in China-a breakthrough technique in L. edodes cultivation-largely increased the production efficiency by shortening crop cycle and improving nutrient utilization, and it is now upgraded and widely adopted by growers in all producing countries. Currently in the United States, most L. edodes are cultivated on nutrient-supplemented sawdust substrate, using a 16-20 day spawn run and then browning outside or inside the bag [2]. Approximately 90% of the L. edodes production in Japan is using blocks made from solidified woodchips [3]. In Brazil, L. edodes is cultivated on logs of Eucalyptus species as well as using synthetic bags cultivation with sawdust as the basic ingredient in the formula [4]. In China, a typical substrate formula for L. edodes cultivation composes of 78% oak sawdust, 20% wheat bran and 2% gypsum, which highly relies on wood supply [5]. However, the increasing logging and export bans in many countries (Albania, Laos, Ukraine and other supplier countries) and the forest resource control regulation according to the China Forest Law implemented in 2018, as preventive measures against deforestation and related environmental impacts, have resulted in dwindling wood supply and soaring wood prices. Meanwhile in China, most of corncobs are burned in rural areas or stacked for later fuel use, which leads to air pollution, space occupation and other environmental burdens. It is therefore necessary to explore alternative substrates using agricultural wastes such as corncobs for L. edodes cultivation with ecological and economic advantages.
L. edodes is reported to be grown on several agricultural byproducts and forest residues, such as ground wheat straw [6,7], hazelnut husk [8], peanut shell [7], corn cob and vine pruning waste [7], enabling biotransformation of wastes. It is reported that ground wheat straw has potential for L. edodes cultivation: when applying 8 and 16% wheat straw (44 and 32% oak sawdust in the formula respectively), mushroom yields were 11 and 19% higher as compared with reference formula (52% oak sawdust), while mushroom sizes were not affected by formula change [6]. Mycelial growth measurements revealed adding 5-20% rice bran, wheat bran and soya bran to eucalyptus residues supported faster growth of 3 L. edodes strains, suggesting nitrogen input and bioavailability were related to mycelium running [9]. Hazelnut husk was reported as a competent new basal ingredient in substrate for L. edodes cultivation, the highest yield (202.96 g/kg substrate) was achieved when using 75% hazelnut husk:15 beech wood-chip:10 wheat bran formula, which was not significant different from the control group (60 beech woodchip:20 wheat straw:20 wheat bran), though the biological efficiency (BE) was lower [8].
Corn is a major crop in the world. As the most widely produced feed grain in the U.S., it accounts for more than 95% of total production and use (USDA, https:// www. ers. usda. gov/ topics/ crops/ corn-and-other-feedg rains/). Corncob is a major byproduct from corn industry, which is easy to collect from the field. China is a world's major corn producer that produces 45,900,000 tons of corncobs after corn harvesting, among which 8,000,000 are utilized as forage or burned as fuel, there is still much room to develop new applications with eco-benefits. Corncobs (dried biomass) contain 5-36% cellulose, 32-40% hemicellulose, 15-20% lignin and 1.0-1.7% ash [10,11], with water holding capacity and solidity showing a potential for fungi cultivation. A previous research suggested addition of corncob (39%) was related to improve the production of volatiles of L. edodes and synthetase gene expression, and they can be added as flavor promoting substances [12]. Pleurotus eryngii (king oyster mushroom) was reported to grown on a substrate containing 70% corncob and other ingredients including cotton hull, bran, corn flour, lime, gypsum, and potassium dihydrogen phosphate, the optimal biological efficiency reached 60.5%, and mushroom products contained 1.56% polysaccharides [13]. A comparative study investigated Agaricus brunnescens cultivated using corncob substrate or rice straw substrate, and found that mushrooms harvested from corncob substrate showed higher content of protein, while those harvested from rice straw substrate showed higher content of fiber and free amino acids; further nutritional analysis showed amino acid composition of A. brunnescens cultivated on corncob substrate were more close to the WHO/ FAO recommended reference pattern and got higher amino acid scores, indicating better nutritional value [14]. EIRA et al. [4] reported both ground and whole corncobs can be used in L. edodes production, however, whole corncob substrates resulted in small mushrooms, the biological efficiency reached 43.87% when combining 90% corncob and 10% rice bran. In this study, corncobs were assessed as a substrate ingredient for L. edodes cultivation at various proportions substituting sawdust. The effects of different formulas on production efficiency and agronomic traits were investigated, and nutritional quality of mushrooms were determined, hence to provide a useful alternative approach to grow L. edodes and enhance biotransformation of agriculture wastes.

Isolate and Spawn
Isolate of L. edodes (Shenxiang215, strain number CCMJ2806) was provided by the National Engineering Research Center of Edible Fungi, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences. The isolate was maintained on potato-dextrose agar (PDA) at 23 ℃ in the dark before use. Then spawn was prepared by cultivating isolates on substrates composing of 78% oak sawdust, 20% wheat bran and 2% gypsum at 23 ℃ in the dark, running for 25 days to allow substrates get fully colonized by the selected strain.

Substrates and Preparation
Substrates containing different ratios of corncobs were prepared using formulas in Table 1. The formulas were designed referring to the traditional formula (A) and substituting corncob for oak sawdust at gradient ratios (B-G). Corncobs (dried and chopped to particle size of 0.5-0.8 cm) were supplied by corn growers from Xiayi county, Henan province, China. Oak sawdust, wheat bran and gypsum were prepared as described in our previous publication [5]. Ingredients were mixed thoroughly, after which tap water was added until moister content reaching 55%. Homogeneous substrate mixtures were prepared after fully stirred.

Cultivation and Fruiting
The well-mixed substrates were put into low pressure polyethylene mushroom bags (17 cm × 55 cm × 0.05 cm, purchased from Zhengdashiye, Yingkou, China), 2300-2400 g per bag, to make artificial bed-logs. Then bed-logs were sterilized in an autoclave for 16 h at 100 ℃. After cooled down to below 28 ℃, the sterilized substrates were inoculated with 40 ± 1 g prepared spawn. The inoculated bed-logs were maintained in a ventilated and darkened spawn running room at 23 ℃, 60-70% relative humidity (Rh) until mycelium running accomplished (approximately 35-40 days). In order to reduce impact of environmental differences, bedlogs of each group were randomly distributed and set on the shelves in different areas of the cultivation room to promote browning, the inside conditions were controlled as 21 ± 2 ℃, 60-70% Rh, CO 2 concentration ≤ 3000 ppm and baglogs were exposed to a 12-h light/dark regime (white light, 300 lx). Bed-logs were pricked to allow inflation of oxygen as we reported before [15]. Mycelium then gradually matured, forming tumor-shape nodules. After browning of bed-logs, they were moved to a growing room, the inside conditions were controlled as 18 ± 2 ℃, 85-90% Rh, CO 2 concentration ≤ 2500 ppm and bag-logs were exposed to a 12-h light/dark regime (white light, 300 lx) to encourage fruiting. "Bud thinning" was conducted according to the standardized production process, the number of primordia on each bed-log was controlled, and about 15-20 fruit bodies were left on each bed-log. Fresh L. edodes fruit bodies with pilei not fully unfolded were picked. It took 75 ± 5 days from the formation of primordium to the end of harvest for each substrate group, a total of 3 flushes of mushrooms were  A  78  0  20  2  B  58  20  20  2  C  48  30  20  2  D  38  40  20  2  E  28  50  20  2  F  18  60  20  2  G  0  78  20  2 acquired. One hundred bed-logs were applied for each substrate group (a total of 700 bed-logs). Yields were calculated as the sum of the weight of 3 flushes of fresh mushrooms, weighing using an electronic balance with an accuracy of 0.01 g. BE (%) was calculated as the ratio of grams of fresh mushroom that harvested per gram of dry substrate [6]. All 100 bed-logs of each substrate group were investigated for yield and BE.

Chemical Analysis of Substrates
Before and after sterilization, pH of substrates was determined by a handheld pH meter (Horiba, Fukuoka, Japan). Homogenized substrates (100 g for each formula, original water amount of 50-52%) were dried in an oven at 50 ℃ until reaching a constant weight. Total carbon content was determined using a commercial test kit (Comin biotechnology, Suzhou, China) based on ferrous sulfate reaction and spectrophotometry, all procedures were conducted following manufacturer's instruction. Total nitrogen content was determined using the Kjeldahl method (Kjeltec™ 8000, Foss, Hilleroed, Denmark). C/N was calculated by dividing total carbon content by total nitrogen content in triplicate. Determination of lignin, cellulose, hemicellulose was performed according to the manuals of commercial test kits (Comin biotechnology, Suzhou, China). Experiments were performed in triplicate.

Assessment of Mycelial Growth
Mycelia growth rate was determined at 23 ℃ in glass tubes using the method modified from Zou et al. [16], substrates were equally distributed to each tube by weight and pressed to the same height, mycelia growth rate (mm/day) was calculated as the average growth rate between the day once mycelia began to grow (Day 1) and 6 days after day 1 (Day 7). Data were measured in pentaplicates.

Evaluation of Fruit Body Quality
Fruit bodies harvested in the second flush were applied for morphological and chemical tests. 60 Fruit bodies from 20 bed-logs were randomly selected (excluding deformities), and their pileus thickness, pileus diameter, stipe length and stipe diameter were measured using a Vernier caliper. 500 g of fruit bodies (randomly selected) were dried in an oven at 50 ℃ until reaching a constant weight. Samples were grinded and kept at 4 ℃ for later use. Ash, protein, and fat were determined referring to national standard methods issued by National Health Commission of P. R. China: Standard GB 5009.4-2016 for ash analysis, Standard GB5009.5-2016 for protein analysis, and Standard GB 5009.6-2016 for fat analysis. The N-to-P factor used in protein determination is 4.5 as reported by Mattila et al. [17]. Polysaccharides were determined as outlined by Haltiwanger [18]. Contents of Ca, K and Na were determined using commercial detection kits (Comin biotechnology, Suzhou, China) and flame spectrophotometry, all procedures were performed following the manufacturer's manual. Phosphorus content was determined adopting a commercial detection kit (Comin biotechnology, Suzhou, China) and spectrophotometry, according to a protocol provided by manufacturer. Tests were performed in triplicate.

Statistical Analysis
Data were expressed as mean ± SD. The original data were processed using Microsoft Excel (Microsoft Inc., Redmond, WA, USA). Differences among the means of groups were analyzed by analysis of variance (ANOVA) invoking SPSS 17.0 (IBM Inc., Armonk, NY, USA). Duncan's multiple range tests was performed at 95% confidence level (p < 0.05).

Chemical Profile of Substrates
Before sterilization, substituting corncob for sawdust significantly increased pH of the substrate, reaching a peak at 8.23 when sawdust was totally replaced by corncob, however no significant difference was observed in pH after sterilization (6.27-6.67), suggesting an excellent buffer effect of the substrates ( Table 2). Total carbon contents of substrates were 46.12-49.58%, total nitrogen contents were 0.61-0.77%, corncob substitution reduced substrate's carbon level while elevating nitrogen level, leading to a notable decline in C/N ( Table 2). In this study, corncob provided more nitrogen and less carbon as compared with oak sawdust, which is consistent with that reported by Hoa et al. using sawdust made from acacia wood [19]. A suitable C/N ratio and a physical structure that allows gas exchange are important characteristics of substrates for growth of wood-degrading mushrooms, such as P.ostreatus [20] and A. bisporus [21]. L. edodes has wide adaptability to C/N of substrate, the reported suitable C/N for its vegetative growth (mycelial growth) was reported to be 25-40, and C/N for its reproductive growth (fruit body differentiation and growth) was reported to be 73-600 [22,23]. High concentration of nitrogen inhibits the transformation of L. edodes from vegetative growth to reproductive growth, reducing primordial differentiation [22]. The current cultivation mode for L. edodes cultivation harvests multiple flushes of fruit bodies on one substrate, our study suggested the initial substrate C/N of 60.39-81.28 supported L. edodes harvesting for at least 3 flushes. Biopolymers in woody biomass such as lignin and cellulose are essential 1 3 aliment for fungi growth, L. edodes is known for efficient capacity of degradation of lignocellulosic materials in nature [24]. In this study, we determined lignin, cellulose and hemicellulose in different substrates before inoculation and in spent mushroom substrates after harvesting (Fig. 1). Before inoculation, lignin content did not change significantly with varying formula, yet decrease in cellulose and increment in hemicellulose were found when changing sawdust to corncob (Fig. 1). After L. edodes production, cellulose and hemicellulose contents decreased notably in each spent mushroom substrate as compared with those in the uninoculated substrates. The degradation rate of cellulose ranged from 15 to 34%, and that of hemicellulose ranged from 10 to 26%, suggesting these carbon sources were consumed and converted to small molecule sugars for mycelium growth and fruit body development. Lignin changed only slightly after mushroom cultivation. The observations were similar to those reported in P. tuoliensis [25]. The act of bioconversion of lignocellulosic biomass to monosaccharide by white rot fungi majorly attributes to their hydrolytic enzyme productivity, including lignin-degrading enzymes (such as laccases, lignin peroxidases and manganese peroxidases) and hemicellulose and cellulose-degrading enzymes (such as xylanase, cellulases and cellobiose dehydrogenase) [26,27], and the efficiency is also related with hysicochemical properties of the substrate [28]. The data suggested even if a small amount of lignin was degraded, cellulose and hemicellulose were accessible for L. edodes. The further enzymatic hydrolysis would accelerate the depolymerization of the lignocellulosic biomass [25].

Effect of Substrate Formula on Mycelia Growth, Log Browning and Mushroom Yield
Different chemical profile of formula resulted in significant variance of mycelial growth rate (Table 3). Among all tested groups, the fastest growth rate was found when adopting formula E, reaching 2.96 ± 0.13 mm/day, which was 30.97% higher than the control (A), indicating supplementation of corncob at an appropriate proportion effectively boosted mycelia growth. Mycelial running is an extension and colonization of fungal mycelium throughout the substrates, which is affected by fungal strain, growth environment and carbon source and nitrogen source of the substrate [28]. Mycelial growth requires abundant nutrients and oxygen, as well as appropriate pH, temperature and moisture conditions. L. edodes growth could be accelerated by addition of CaSO 4 and MgSO 4 at concentrations less than 0.5%, while Fe and Cu addition impedes L. edodes mycelial growth [22]. Browning, the light-induced brown film formation process in morphogenesis peculiar to L. edodes [29], largely affects yield, number of fruiting flushes, and resistance potential against diseases and insects [30]. The mechanism of browning has not been fully revealed yet. The blue-light photoreceptor genes Le.phrA and Le.phrB, the white collar complex, and phytochrome have been reported to play key roles in light-induced the brown film formation of L. edodes [31,32]. A previous study [33] suggested cations of calcium or manganese are involved in the formation of brown mycelial film in liquid culture medium in the presence of necessary amino acids. Brown mycelial film extracts have higher lectin activity, which may be affected by nitrogen source [31]. Our study suggested that partly substituting corncob for sawdust promoted log browning. However, the optimal browning effect was not obtained in substrate with only corncob. This might indicate that some sawdust is needed for a minimum requirement of physical properties of the substrate, such as air permeability and water holding capacity.
Three flushes of mushrooms were collected. The highest total yield and biological efficiency both obtained when applying formula E (Table 3). In group E, 722.08 g/log of mushroom were harvested, which increased by 40.39% as compared with the control, showing an excellent boosting effect. Besides, other substrates supplemented with corncob (B, C, D, F) also exposed various increase in yield, suggesting that partial substitution with corncob probably improved the C/N of substrate and made it more suitable for L. edodes growth. Most of the corncob substrates had maximum mushroom harvesting in the second flush, while sawdust control substrate showed a growing yield per flush (Fig. 2). Mushroom yield is highly related with the utilization of nitrogen, carbohydrates as well as other macronutrients and micronutrients, the moisture of the substrate and uniformity of the substrate [21]. The current business model for L. edodes production include household cultivation by farmers and factorized cultivation by enterprises. For enterprises, the pursuit is to harvest as many mushrooms as possible in fewer flushes, so as to reduce time cost and labor cost caused by log recuperation and water supplementation as well as improve the utilization rate of mushroom houses. Hence, 2-3 flushes of L. edodes are usually harvested in factories to meet the current needs for intensive production. However, for farmers, they may prefer to harvest 4-6 flushes of mushrooms, since the cost of mushroom bags is a one-time input, more flushes indicate more output. In the current study, we took samples of the first three flushes of mushrooms, the yield performance of the new substrate was promising, suggesting corncob was well decomposed and utilized. However, we are not sure how the yield performance will be when more flushes are picked. Therefore, for further use, how many flushes to be harvested should be considered comprehensively according to the actual production and management mode.
The biological efficiency in this study ranged from 37.56% (group G) to 80.23% (group E), suggesting formula E (50% corncob, 28% oak sawdust, 20% wheat bran, 2% gypsum) provides the optimized nutritional and physiochemical properties for L. edodes growth. The results are higher than those reported using formula consisting of corncob and rice bran (biological efficiency of 43.87% for 3 flushes), or consisting of corncob and eucalyptus sawdust (biological efficiency of 18.88%~27.88% for 3 flushes) or whole corncob only (39.30% for 3 flushes) [4]. Combining our study with the reference [4], it is suggested that both corncob and sawdust provide a good source of carbon, however, to achieve a good yield performance and biological efficiency, nitrogen source is required in L. edodes substrate, and wheat bran is an excellent choice.

Agronomic Traits of Mushrooms
Agronomic traits of mushrooms hugely affect their commercial value. The results showed using corncob did not affect diameters of mushroom pileus or stipe (Fig. 3). Average stipe length was not significantly varied either, except that when using corncob only (substrate G), remarkably long stipes were observed. The thickness of pileus was most affected by formula change among agronomic traits, thickest pileus and thinnest pileus were observed in group A and G, respectively. Previously it is reported that change in substate formula sometimes affected the morphology and size of mushrooms, such as the studies conducted in P. eryngii [16], Pholiota microspora [34] and A. bisporus [21]. However, in our study, application of corncob did not notably affect the shape and size of L. edodes fruit body, which is consistent with the previous study using a formula consisting of corncob and rice bran [4]. It is speculated to be related to the "bud thinning" process in our fruiting management. We manually controlled the number of primordia on each bedlog to 15-20, referring to standardized protocols for scale production. The agronomic traits of fruit bodies, especially their sizes, are highly linked to the number of primordia [5,28]. Hence, in the present study, the controlled primordia number probably resulted in mushrooms with a more uniform size.

Nutritional Profile of Fruit Bodies
Edible fungi are excellent source of polysaccharide and protein, and they do not contain much fat. It was found that different formulas in this study significantly affected contents of ash, polysaccharide, protein, and fat in dried mushrooms (Fig. 4). The ash content ranged from 6.19-6.94 g/100 g, increasing with the concentration of corncob in the formula, and group G had 12.12% more ash than control. Among minerals, K is an abundant element in L. edodes, similar to that reported in other mushrooms such as P. ostreatus and P. cystidiosus and P. sajor-caju [19,35]. In this study, P and K levels in L. edodes, were not significantly affected by formula change, while Na and Ca contents were increased as sawdust gradually replaced by corncob (Fig. 5). The ash content reflects the mineral supply of the fruit bodies, which is affected by substrate formula, such as its element concentration and electrolyte conductivity, materials enriched with minerals are sometimes employed to both boost mushroom growth and improve nutritional quality [27]. Our results suggested corncob provided more minerals than sawdust, which is consistent with the previous study [19] reporting P. ostreatus and P. cystidiosus cultivated using formulas with corncob and sugar bagasse showed higher values of ash content than those using sawdust. The ash content of corncob was also reported to be higher than substrates made from water straw or water hyacinth [35]. Corncob addition increased Ca, K and Mg levels without affecting Na level in fruit bodies of pleurotus mushrooms [19]. It is confirmed that these elements are naturally occurred in the substrate materials, and corncob contained higher levels of several minerals comparing to saw dust, such as Ca, Cu, Fe, K, Mg, Mn, P, Zn [19,35]. However, similar to our results, even the substrates sometimes contain a higher level of some elements such as phosphorus, the fruit bodies may not achieve higher levels of the element, which might be explained by that high electrolyte conductivity affected fungal mineral uptake  [19]. Protein content in mushrooms (13.4-15.9 g/100 g) decreased with the increase of corncob, and protein content in mushrooms produced by substrate G was 18.72% lower than control. The determined protein content in foods is largely affected by N-to-P conversion factor. However, the commonly used conversion factor of 6.25 (standard) does not accurately reflect the protein level of mushrooms [17,20]. Because in fungi, chitin of the cell walls largely contributes to the non-protein nitrogen pool, hence we adopted a determined factor for L. edodes as reported by Mattila et al. [17] in our calculation. Only crude protein is reported in the present study, amino acid profile of mushrooms might be further studied in future to have a better understanding of the nutritional quality of the product. Previously, it is reported that adding nitrogen enriching ingredients to the substrate, such as legumes with high capability for nitrogen fixation, would increase protein level in mushrooms [20]. However, the mushroom performance applying high protein supplements is not consistently higher than when using low-protein supplements [21]. The nitrogen in L. edodes cultivation substrate is mainly supplied by wheat bran [12]. Our study found that, corncob, as a new substrate material, provided a higher level of nitrogen than oak sawdust, yet it may affect the form and availability of nitrogen source, as reflected by the protein content in the fruit bodies. The mechanism for nitrogen utilization and protein synthesis is a direction for future research. The fat content in mushrooms ranged from 1.57 g/100 g (group G) to 1.89 g/100 g (group E and group F). The results were similar to those reported in other edible fungi species, suggesting mushrooms are healthy snack material with low amount of fat [17,36]. The mushrooms also contained 3.56-4.51 g/100 g polysaccharides, and substrate D produced mushrooms with highest polysaccharide level, which was 26.69% more than control. L. edodes is an important edible fungus for both culinary and clinical use, its polysaccharides (such as lentinan and β-glucan) have been reported with health-promoting properties [37]. Polysaccharide content in mushrooms is linked to the cultivation substrate and growing conditions [38,39]. Oak log-grown L. edodes were reported to yield greater amount of high molecular polysaccharides than oak sawdust-grown L. edodes [38]. Polysaccharide yield was higher when obtained from spent L. edodes substrate originated from wood chips as compared with that from synthetic substate containing wood chips (59%), cottonseed hulls (25%) and wheat bran (15%), which is attribute to the higher contents of cellulose and hemicellulose in wood chips [39]. Similarly, we also found substrate D with higher amount of cellulose and hemicellulose produced mushrooms with more polysaccharides. As consumers generally have more pursuit on health and wellness today, the products with abundant bioactive components attract customer interest and stimulate purchase. Our results revealed substrate formula had great impact on nutritional profiles of mushroom products, it is probably an effective and feasible approach to obtain high-yield and high-quality products through formula improvement.

Conclusions
Corn is the most widely produced crop in the world, leaving corncob as a major agriculture waste. In this study, we assessed the possibility of using corncob to cultivate L. edodes in this study, monitored the chemical profile and pH of substrates, and investigated the influence of formula on mycelia growth, log browning, mushroom yield, BE, and agronomic traits and nutritional profile of mushrooms. The results revealed the feasibility to substitute corncob for sawdust in L. edodes production in terms of ecological and economic benefits, and a high-yield formula (E: 50% corncob, 28% oak sawdust, 20% wheat bran, 2% gypsum) was obtained. Using formula E to produce L. edodes, the single log yield was as high as 722.08 g/log, increased by 40.39% comparing to the control, showing an excellent boosting effect. The proportion of corncob in this formula reaches 50%, which can effectively reduce the use of sawdust and make good use of agricultural waste, thus lowering material cost and reducing environment burden.
In recent years, consumer's demand for L. edodes has gradually shifted from an adorable appearance to health benefits. The mushrooms produced by corncob-sawdust mixture substrates were with competent agronomic and nutritional quality. Among our tests, using formula D (40% corncob, 38% oak sawdust, 20% wheat bran, 2% gypsum) produced mushrooms with polysaccharide level higher than other tested formulas. Hence this formula may serve as an alternative approach to meet the market demand.

Conflict of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.