1.Introduction
The two edible mushroom cultivation methods are log cultivation and sawdust-based cultivation.
Log cultivation is a method where a standing tree is felled, cross-cut into appropriate lengths and stacked, then mushroom spawn is inoculated into the logs and stimulated so mycelia spread all over the logs, and fruiting bodies grow. A feature of log cultivation is that it does not require a sterilisation process, and the nutrient source for the mushroom mycelia and the substrate are derived only from the woody parts. Edible mushroom cultivation first flourished after WWII when log-grown Shiitake bacame possible, whereby logs of konara oak (Quercus serrata) and sawtooth oak (Quercus acutissima), which were easily accessible in mountainous areas, were inoculated with spawn and fruiting bodies grown.
Sawdust-based cultivation, however, is a method where fruiting bodies are formed in medium where nutritional supplements are mixed with substrate, water is added, and the moisture content is adjusted to 60-70%. Substrate mainly plays the role of culture medium support and uses material such as wood powder (sawdust), chips, corn cobs and cottonseed hulls.  Nutrition supplementation plays the role of nutrient source for mycelia and uses cereal bran such as rice bran, bran and corn cobs. In 1960, the total amount of cultivated mushrooms in Japan was 36,000 tonnes, but increased significantly with the subsequent rapid economic growth, reaching 100,000 tonnes in 1968, 200,000 tonnes in 1977, 300,000 tonnes in 1984 and continuing to rise steadily after 2000 to 400,000 tonnes in 2004.

 In 2009 it rose to 45 tonnes, but has since remained flat at 450,000-470,000 tonnes. The total mushroom production today has increased approximately 13-fold on that of 1960 in the space of half a century(2).It is estimated that nowadays over 90% of all mushrooms produced are grown by sawdust-based cultivation(3).

The advancement of sawdust-based cultivation technology has contributed to developments in both breeding and cultivation technology. With breeding, spawn has been developed, making sawdust-based cultivation possible, and having a direct bearing on fruiting body shapes that meet consumer needs and reducing labour(4).Also with cultivation technology, the effect of the type and composition ratio of the substrate, which forms the sawdust-based medium, and nutrition supplementation on the yield and quality of the fruiting bodies is being examined, and the development of more superior medium ingredients at a lower cost is underway. This paper will show the efficacy of using industrial waste and underutilized biomass resources for the purpose of improving the sawdust-based medium for the sawdust-based cultivation of edible mushrooms, and will outline findings that have developed as practical technology.

Soy pulp is the by-product produced in the process of manufacturing tofu and other processed soy foods, with approximately 700,000 tonnes produced in Japan annually. Of that, 420,000 tonnes is used as fodder for pigs and other livestock, 160,000 tonnes in fertiliser, and 30,000 tonnes, including dried soy pulp, in food, with tens of thousands of tonnes discarded as industrial waste(5, 6). Thinking I could contribute to helping with the effective use of soy pulp and reducing the production costs of mushrooms, I examined the use of soy pulp as nutrition supplementation in sawdust-based oyster mushroom cultivation(7, 8).

I examined the applicability of fresh soy pulp as nutrition supplementation (Table 1). I used rice bran and soy pulp for the nutrition supplementation, mixed each at weight ratios of 5:0, 4:1, 3:2, 2:3, 1:4, 0:5, then used cedar sawdust for the substrate (uncomposted), mixed the substrate and nutrition supplementation at a weight ratio of 1:1, and adjusted the moisture content to 65% to create the test medium. For the test mushrooms, I used commercially available oyster mushroom spawn Meijiwase and Mori 39. With the cultivation duration, the mycelial spread duration showed a tendency to be longer when soy pulp made up at least 60% of the nutrition supplementation than the medium in which rice bran was the main constituent. The duration required for the first flush after the fungi at the top was scraped showed a tendency to decrease as the soy pulp ratio increased until the soy pulp mixing ratio was 80%. In contrast, however, the duration required for the second flush increased as the soy pulp mixing ratio increased.

The cultivation duration overall was affected by the extremely shortened duration required for the first flush after scraping due to the mixing of soy pulp, and was made 7-16 days shorter than the rice bran only group by the mixing of soy pulp. With the fruiting body yield, between the soy pulp only group and the rice bran only group, the latter had a greater fruiting body yield in the first flush. However, the fruiting body yield increased with the mixing of soy pulp with rice bran. In the test groups with a soy pulp ratio of 60-80% in particular, the Mori 39 yield was approximately double and Meijiwase was over 1.4-fold that of the rice bran only group. In the second flush, there was a greater fruiting body yield in the mixed soy pulp and rice bran groups than in the rice bran only group, with this trend remarkable in Meijiwase. In the total yield, which combined the first and second flushes, there was no remarkable difference in the rice bran only and soy pulp only groups, but the fruiting body yield increased when rice bran and soy pulp were mixed. The total yield increased significantly as the soy pulp ratio rose, particularly for a soy pulp mixing ratio of up to 80%.

Generally, the ideal C/N ratio in the medium differs depending on the mushroom variety, but because 20 is suitable for nutrient growth and 30-40 for fruiting body formation(10, 11), the medium C/N ratio improved by mixing in soy pulp. However, because the fruiting body yields of the rice bran only and the soy pulp only groups are virtually equal, it can be presumed that factors other than C/N ratio are having an effect. The ratio of contained nitrogen increased as the mixing ratio of soy pulp increased, but the water-soluble protein quantity was lower in the soy pulp only group that in the rice bran only group, and showed low values as the mixing ratio of soy pulp increased. Similarly, the reducing sugar quantity was also much lower in the soy pulp only group than in the rice bran only group, with the reducing sugar quantity values decreasing as the mixing ratio of soy pulp increased. The water-soluble protein quantity at the time of scraping was lowest in the rice bran only group, with the protein quantity increasing as the ratio of soy pulp increased. The change between the time of inoculation and the time of scraping had the greatest decrease in the rice bran only group, with the decrease declining as the mixing ratio of soy pulp increased, but was conversely greater at the time of scraping in the test groups comprising at least 80% soy pulp. The reducing sugar quantity at the time of scraping, too, was similar to the water-soluble protein quantity and increased as the proportion of soy pulp increased. The change between the time of inoculation and time of scraping was also greater at the time of scraping when the ratio of soy pulp was at least 40%.

In the incubation process of edible mushrooms, the fungi external polysaccharide-degrading enzymes beta-1.3 glucanase, end-cellulase and exo-cellulase activity rose with mycelial growth, thus the nitrogen source is thought to be absorbed by the mycelia(12, 13).Further, when the fruiting bodies grow, a considerable amount of carbohydrate and a small amount of nitrogen compound are absorbed from the medium by the mycelia and used in the formation of fruiting bodies(14, 15).From these results, when soy pulp is mixed with rice bran as nutrition supplementation, the total nitrogen quantity increases, but the content of water-soluble protein, which is easily absorbed by mycelia, and reducing sugar quantity decreases in medium prior to inoculation. However, it is presumed that, induced by soy pulp, fungi external enzymes become actively secreted, the medium compounds change to reducing sugar and are broken down, and affects the promotion of mycelial growth and the increase in fruiting body yield. It was found that fresh soy pulp is beneficial as nutrition supplementation in sawdust-based Hiratake cultivation. However, fresh soy pulp spoils quickly and needs to be used the day it is produced so it needs to be managed in a storable state for use at any time.

However, the cultivation duration was over one week longer in the dried soy pulp only group than in the rice bran only group and test groups of mixed rice bran and dried soy pulp. The duration required for fruiting body formation showed a tendency to differ depending on the test mushroom. In the test groups of mixed rice bran and dried soy pulp for Meijiwase, it was 2-6 days shorter, but for Mori 39, it actually tended to be longer. This had an effect and the cultivation duration from inoculation to fruiting body harvest showed a tendency to differ depending on the test mushroom, with the mixing of dried soy pulp with rice bran shortening this duration in Meijiwase, in particular the test groups with 60-80% dried soy pulp were approximately one week shorter than the rice bran only group. Dried soy pulp was not observed to have an effect on Mori 39. However, it was 7-10 days longer in the dried soy pulp only groups for both Meijiwase and Mori 39 than in the rice bran only groups. The fruiting body yield for Meijiwase in the 20% soy pulp mix group was similar to that of the rice bran only group, but greater in the 40-80% mix groups than the rice bran only group.In all the Mori 39 test groups mixed with soy pulp there was an increased yield compared to the rice bran only group. In both strains, the yield increased remarkably in the 60- 80% soy pulp mix groups, showing yields 1.5-1.7 times greater than that of the rice bran only groups.

Because lactic fermented soy pulp, where fresh soy pulp is treated with lactic acid bacteria, is something that can be used as a food ingredient even after being stored for a week at 30℃16), I examined sawdust-based oyster mushroom cultivation with lactic acid bacteria fermented soy pulp, which can be stored at room temperature (Table 4). The mixing ratio of rice bran to dried soy pulp or lactic acid bacteria fermented soy pulp as nutrition supplementation all at a dried weight ratio was rice bran:soy pulp = 3:1, 2:2 and 1:3.

The test mushroom used was Mori 39. With the cultivation duration, the cultivation duration in the previously mentioned experiment tended to get longer by mixing dried soy pulp, but by using a slightly reduced amount (about 5% less), the mycelial spread duration and the duration required for fruiting body formation in the dried soy pulp mix groups was similar to that of the rice bran only group. With lactic acid bacteria fermented soy pulp, however, when lactic fermented soy pulp comprised at least 50%, the mycelial spread duration and the duration required for fruiting body formation were five days and two days longer respectively. Lactic fermented soy pulp and dried soy pulp both similarly increased the fruiting body yield when mixed with rice bran. Up to a 75% mixing ratio for each, the dried soy pulp was 1.2-1.6 times greater and the lactic fermented soy pulp was 1.2-1.4 times greater than that of the rice bran only group.

It is suggested that lactic fermented soy pulp can also be used in sawdust-based oyster mushroom cultivation. I examined the applicability of dried soy pulp in sawdust-based shiitake cultivation (Table 5). Bran, rice bran and corn bran are used as nutrition supplementation in sawdust-based shiitake cultivation(17). Using those nutrition supplements and dried soy pulp, I mixed bran, rice bran, and corn bran each with dried soy pulp all at a dried weight ratio of 1:1, in addition to having groups comprised only of each. The test medium was oak sawdust mixed with nutrition supplementation all at a dried weight ratio of 3:1 and adjusted to a moisture content of 65%. I put 100g of the test medium into bio pots (80mm in diameter, 100mm high, manufactured by Nichii), sterilised them before inoculating them with commercially available shiitake spawn, and then incubated them at 24℃. With the cultivation duration, fruiting bodies grew in fewer days in the dried soy pulp only group than in each of the bran, rice bran and corn bran only groups. It also tended to be shorter by mixing dried soy pulp with bran and rice bran than in each of the non-mixed groups.

 Azuki bean paste waste is a by-product produced in the process that produces azuki bean paste, a processed azuki bean food. Azuki bean paste is made by boiling, mashing and then rinsing azuki beans in water, but azuki bean paste waste is the strained lees of boiled azuki beans. Foods using azuki bean paste are produced and consumed throughout Japan. At the same time, azuki bean paste waste is created in large amounts and although a small fraction is used in livestock feed, the majority is discarded. Therefore, in order to clarify its applicability in edible mushroom cultivation as part of the effective use of the underutilized resource azuki bean paste waste, I examined its applicability as a medium material in sawdust-based oyster mushroom and enoki cultivation(19, 20). In the sawdust-based oyster mushroom cultivation, I used cedar sawdust as the substrate and rice bran or bran as the nutrition supplementation. I mixed the substrate and nutrition supplementation at a total dry weight ratio of 18:17 then added tap water and adjusted the moisture content to 65% (wet basis) to form the base medium. When I substituted the rice bran, which is the nutrition supplementation of the base medium, for azuki bean paste waste and adjusted the test medium to examine the applicability of azuki bean paste waste as nutrition supplementation, the duration required for cultivation was 1-3 days shorter in the groups substituted with 25%, 50% and 75% azuki bean paste waste than in the base medium (the rice bran only group), but in the 100% substitution group (the azuki bean paste waste only group), it was actually approximately 1 day longer. The number of fruiting bodies was the greatest in the base medium with 33.9/bottle, with the number decreasing as the azuki bean paste waste substitution ratio increased, with the 100% substitution group having the least. In the 100% substitution group, the number of fruiting bodies was only 40% that of the base medium. The fruiting body yield was greatest in the base medium at 84.5g/bottle, and showed a tendency similar to that of the number of fruiting bodies to decrease as the azuki bean paste waste substitution ratio increased, with the 100% substitution group having the smallest yield at 0.7 times that of the base medium. Substituting bran with azuki bean paste waste also showed a similar trend to substituting rice bran with the duration required for cultivation increasing and the number of fruiting bodies decreasing when rice bran was substituted with azuki bean paste waste, and the fruiting body yield falling as the substitution ratio rose.

Next, to examine the applicability of azuki bean paste waste as a medium base, I substituted the cedar sawdust, which was the substrate of the base medium, with azuki bean paste waste and conducted cultivation experiments. Using azuki bean paste waste as the substrate, normal fruiting bodies formed even when the substitution ratio of azuki bean paste waste to cedar sawdust variably changed.

The duration required for cultivation was 47.0-48.5 days in the groups substituted with azuki bean paste waste compared to 48.0 days in the base medium, so the duration was virtually equal in the base medium and the groups substituted with azuki bean paste waste. The number of fruiting bodies in the base medium was 35.3/bottle with no significant difference observed in the 25% substitution group of the azuki bean paste waste substitution groups, but there were more fruiting bodies in the 50%, 75% and 100% substitution groups than in the base medium.

In sawdust-based enoki cultivation, I conducted test cultivation substituting Ezo spruce sawdust with azuki bean paste waste. The duration required for cultivation with Ezo spruce medium was 60 days, and in the azuki bean paste waste substitution medium it was two days longer than the Ezo spruce medium. In the azuki bean paste waste substitution medium, the mycelial spread duration in the incubation process was two days longer and this period affected the duration required for cultivation. The fruiting body yield increased with the substitution of Ezo spruce sawdust with azuki bean paste waste. The fruiting body yield increased with the increase in the substitution ratio of the azuki bean paste waste, with the greatest yield at a 60% substitution ratio. In 20% and 40% azuki bean paste waste substitution medium it was approximately 1.1 times, and in 50-80% azuki bean paste waste substitution medium it was approximately 1.2 times that of the 133.0g/bottle in Ezo spruce medium.

With the enoki fruiting bodies cultivated in medium in which Ezo spruce sawdust was replaced with azuki bean paste waste, the crude protein and crude fat content showed a tendency to decrease slightly by substituting with azuki bean paste waste, and no impact due to the substitution of azuki bean paste waste on ash content was observed. The content of chitin and total dietary fibre was about the same in the Ezo spruce medium and the azuki bean paste waste substitution medium. With the free sugar and sugar alcohol content, arabinitol and glycerol showed a tendency to increase slightly in the azuki bean paste waste substitution medium compared to the Ezo spruce medium, and the total amount showed a tendency to be higher in azuki bean paste waste substitution medium. The total organic acid amount was 35-50% higher in the azuki bean paste waste substitution medium compared to the Ezo spruce medium, and the content of malate, pyroglutamate, citrate and succinate increased remarkably. The citrate content in particular was 3.7-4.7 times higher than that of the Ezo spruce medium. With the free amino acid content, the total amount was 10-20% higher in the azuki bean paste waste substitution medium compared to the Ezo spruce medium. Alanine and glycine are particularly sweet, and glutamate has umami(23), but alanine, glycine and glutamate increased 10-30%, 5-20% and 5-30% respectively in the azuki bean paste waste substitution medium compared to the Ezo spruce medium. When actually tasted, the enoki fruiting bodies cultivated in azuki bean paste waste substitution medium had traces of sweetness, so it is presumed that these compounds contributed to the expression of sweetness. In dried shiitake, organic acids have been reported to play a modulatory role in which they add an accent to flavour(24), so because the content of malate and citrate, which contribute to taste, increased in the enoki fruiting bodies cultivated in azuki bean paste waste substitution medium, it is possible that the increase in these organic acids are also affecting the expression of sweetness. In sawdust-based lion's mane mushroom cultivation, it has been reported that differences in strain and nutrition supplementation affect the content of fruiting body compounds(25), and it has been noted that differences in fruiting body compounds arise in log cultivation and sawdust-based cultivation in maitake and shiitake cultivation(26, 27).In this study, too, it is clear that differences in substrate in sawdust-based enoki cultivation affects fruiting body compounds.

It is suggested that it is possible to cultivate "sweet enoki" by using azuki bean paste waste as substrate. Therefore, when I examined cooking methods for enoki cultivated in azuki bean paste waste substitution medium, the sweetness was enhanced in cooking where it was steamed, stir-fried and deep-fried, but the sweetness disappeared when cooked in soup. This suggested that compounds that contribute to the expression of the sweetness of enoki fruiting bodies are water-soluble compounds, and this was consistent with the findings of this research. Therefore, when harnessing those traits, attention needs to be paid to cooking and processing methods of enoki cultivated using azuki bean paste waste as "sweet enoki".

I mixed beech sawdust or oak sawdust with nutrition supplementation at an absolute dry weight ratio of 1:1, added tap water and adjusted the moisture content to 63% (wet basis) to make the base medium. I substituted the beech sawdust or the oak sawdust of the base medium with spent maitake substrate at an absolute dry weight of 25%, 50%, 75% and 100% to make the test medium. In all the test groups in which I substituted beech sawdust of the base medium (beech sawdust and bran medium) with spent maitake substrate, normal fruiting bodies with long pins formed similar to those in the base medium.

Even when nutrition supplementation other than bran such as corn bran and super bran were used, normal lion's mane fruiting bodies formed in the test groups substituted with spent maitake substrate at all the substitution ratios. The fruiting body yield showed a tendency to increase as the substitution rate of spent maitake substrate rose in all the nutrition supplementation of bran, corn bran and super bran. It increased significantly in the bran with a substitution rate of over 50% compared to the beech sawdust, reaching equilibrium at a substitution rate of over 75% and increasing approximately 30%.  It increased significantly in the corn bran with a substitution rate of over 25% compared to the beech sawdust, reaching equilibrium at a substitution rate of over 50% and increasing approximately 30%.

The fruiting body growth increased significantly in the super bran with a substitution rate of over 25% compared to the beech sawdust, with the greatest yield at 100% substitution rate and increasing approximately 40%. Using oak sawdust for the substrate in the base medium, when I substituted the oak sawdust with spent maitake substrate and cultivated lion's mane mushrooms, the fruiting body yield in all the nutrition supplementation showed a tendency to increase as the substitution rate of the spent maitake substrate increased. It increased significantly in the bran with a substitution rate of over 25% compared to the oak sawdust, reaching equilibrium at a substitution rate of over 50% and increasing approximately 30%. It increased significantly in both the corn bran and super bran with a substitution rate of over 25% compared to the oak sawdust, reaching equilibrium at a substitution rate of over 75% and increasing approximately 20-30%. It has been reported that it is possible to use spent shiitake growing logs as sawdust for sawdust-based cultivation of oyster mushrooms, enoki and shiitake, and that the nitrogen source content increases in the sawdust of spent shiitake growing logs, and that the C/N ratio improves by using the logs(30, 31). Because the proteins accumulate in the medium as the incubation process progresses in sawdust-based enoki cultivation(32), it is presumed that the proteins and nitrogen sources derived from mycelium compounds similarly accumulated in the medium in the incubation process in sawdust-based maitake cultivation, that the nitrogen source content in the medium increased with the use of spent maitake substrate in the substrate, and that this contributed to an increase in fruiting body yield. After examining the mixing ratio of each nutrition supplementation when I used spent maitake substrate only for the substrate and used bran, corn bran, super bran and rice bran for nutrition supplementation, the fruiting body yield showed a tendency to increase as the mixing ratio of the nutrition supplementation rose in all the nutrition supplementation. The optimal mixing ratio was 45-50% in the bran and rice bran, 45% in the corn bran, and 40-50% in the super bran. By composting spent maitake substrate for 28-42 days at 15 degrees Celsius and 25 degrees and for 14-28 days at 35 degrees, the fruiting body yield increased compared to when fresh spent substrate was used. Composting will be required when using spent enoki substrate in sawdust-based maitake cultivation or using spent oyster mushroom or nameko substrate in sawdust-based hatake-shimeji (Lyophyllum decastes) cultivation because the medium will have excessive nutrition if any of the spent substrates is used fresh as-is(33,34). Sawdust-based lion's mane mushroom cultivation differs to sawdust-based shiitake and hatake-shimeji cultivation in that fresh spent substrate can be used as-is (33, 34), so this is thought to be a feature of lion's mane mushrooms. Because the composting effect appears in a shorter period when treating spent maitake substrate at 35 degrees Celsius rather than at 15 or 25 degrees, it is presumed that the composting effect is derived from the activity of the fungi in the sawdust-based medium.

Bitterness was weakest in spent maitake substrate and strongest in konara oak sawdust. The bitterness was significantly weaker in the spent maitake substrate than in the konara oak sawdust and beech sawdust. Of all the fruiting body compounds, the nutrients that most affect taste are free amino acids. Free amino acids affect umami, sweetness and bitterness. After calculating the total content of free amino acids related to bitterness, konara oak sawdust had the highest content followed by beech sawdust. The content in spent maitake substrate was half that in the konara oak sawdust and beech sawdust. From these results, it was presumed that by cultivating lion's mane mushrooms in spent maitake substrate that had been composted, the lion's mane fruiting bodies had less free amino acids which present bitterness thus, as a result, the bitterness was reduced. Please see the reviews(36-38) for the relationship of fruiting body compounds and functionality with the growth stage of lion's mane fruiting bodies cultivated with spent maitake substrate, and for the applicability of spent nameko, enoki, bunashimeji and shiitake substrate on sawdust-based lion's mane mushroom cultivation.

Because the content of free glucose and low-molecular a-glucan and b-glucan in the medium increased with the process of preparing spent maitake substrate as the medium, it was presumed that the polysaccharide-degrading enzymes derived from the spent substrate reacted with the nutrition supplementation in the medium and increased the low-molecular glucans so, as a result, contributed to the increase in fruiting body yield(39). Based on this, I thought that by adding polysaccharide-degrading enzymes when preparing the medium and deliberately increasing the low-molecular glucans in the medium processing process, I could expect an increase in fruiting body yield. Therefore, I examined the impact the addition of polysaccharide degrading enzymes would have on fruiting body formation and behaviour of glucans in the medium in sawdust-based lion's mane mushroom cultivation(40). Further, because sawdust-based nameko cultivation should not use anything other than hardwood sawdust such as beech as a substrate and thus has limited substrate options, needs a lengthy incubation period, and differs greatly to sawdust-based lion's mane mushroom cultivation, I also examined the effect of adding polysaccharide-degrading enzymes on sawdust-based nameko cultivation.

I tested amylase and b-glucanase as polysaccharide-degrading enzymes. For the amylase, I used the commercially available enzymes of mesophilic amylase (BAN made by Novozymes, herein abbreviated as MPA) and thermostable amylase (Termamyl made by Novozymes, herein abbreviated as HRA). For the b-glucanase, I used endo-1, 3-b-D-glucanase (made by Megazyme, herein abbreviated as GLU).

By adding suitable amounts and types of polysaccharide-degrading enzymes when preparing the medium for sawdust-based lion's mane mushroom and nameko cultivation, fruiting bodies formed normally in both strains and fruiting body yield increased. The effective enzymes and the degree of their effect appeared differently in both strains. In sawdust-based lion's mane mushroom cultivation, adding 100-500ppm of the amylase MPA and HRA, the fruiting body yield increased 1.3-1.4 times that of the non-addition group. Further, with GLU, the yields were slightly higher than amylase, increasing 1.5 times that of the non-addition group with the addition of more than 500ppm, and HRA GLU showed a similar tendency to GLU. In sawdust-based nameko cultivation, although the degree of yield increase was slightly lower than for lion's mane mushroom in the case of MPA and HRA, it showed a clear yield increase effect. However, with GLU the yield was of the same degree as that of the non-addition group and showed no effect on yield increase effect. Meanwhile, unlike lion's mane mushrooms, with HRA GLU the fruiting body yield was greater than the addition of each individually, and a complementary effect was observed due to amylase and glucosidase. Further, in the amylase addition groups, the individual weight of the fruiting bodies that grew showed a tendency to be greater, and a tendency was observed for the fruiting bodies that grew to be bigger. There needs to be detailed examination of the types enzymes and the amount in which they are added, but if we focus on the fact that the fruiting bodies that grow increased in size, then it is possible that enzymes are suitable for cultivating king oyster mushrooms (eryngii), shiitake, oyster mushrooms and other mushrooms in which large fruiting bodies are preferred. From glucan analysis of the lion's mane mushroom cultivation test medium, the content of free glucose, low-molecular a-glucan and low molecular b-glucan in the test medium increased with the addition of MPA, HRA and GLU, and the content of high-molecular a-glucan and amorphous high-molecular b-glucan showed a tendency to decrease. This suggests that high-molecular glucans decomposed due to the enzymatic reaction between the nutritional supplement bran and the polysaccharide-degrading enzymes, and free glucose and low-molecular glucans were produced. It is presumed that the increased free glucose and low-molecular glucans had a beneficial effect on the fruiting body formation of lion's mane mushrooms, and showed a similar trend to the behaviour of glucans with fruiting body yield when spent lion's mane mushroom substrate was recycled and used(39). From these results, an increase has been demonstrated in fruiting body yield by the degrading of high-molecular glucans into lower molecules due to the enzymatic reaction between the polysaccharide-degrading enzymes and the nutritional supplement bran in the spent lion's mane mushroom substrate. The fruiting body yield and behaviour of glucans in HRA GLU showed a similar trend to GLU, and no complementary effect was observed. In nameko, the amylase MPA and HRA showed a similar trend to lion's mane mushrooms in the fruiting body yield and behaviour of glucans, but GLU was similar to the non-addition group and had no effect on the fruiting body yield and behaviour of glucans. However, with HRA GLU the fruiting body yield was higher than with HRA and the content of free glucose and low-molecular glucans in the medium was also higher, so it can be presumed that GLU has a complementary effect on HRA.

Thus, the rise of differences in addition effects on lion's mane mushrooms and nameko due to differences in the type of polysaccharide-degrading enzymes suggests that differences in the type, amount and composition ratio of nutrition supplementation in the medium has an impact depending on the sawdust-based cultivation of both types of mushroom.

With unprocessed bamboo sawdust, there were striking differences in the cultivation duration among the test groups due to the complementary relationship where the duration required for fruiting body formation in the test groups with a short mycelial spread duration showed a tendency to be longer, and the duration required for fruiting body formation in the test groups with a long mycelial spread duration showed a tendency to be shorter. For fruiting body yields, there were no significant differences in the groups substituted with 25%, 75% and 100% bamboo sawdust compared to the control group, with the greatest yield in the 50% substitution group. Because the cultivation duration and fruiting body yield with bamboo only as the medium base is comparable to that of the base medium, from the perspective of proactively using bamboo sawdust, using bamboo sawdust only is recommended, and using it at a 50% substitution rate is recommended if we focus on productivity. With unprocessed bamboo sawdust, because the amounts of alcohol and benzene extract and soluble sugar are high compared to hardwood sawdust, it is presumed that it has antimicrobial compounds and nutrients for mycelial growth, but the fact that the fruiting body yield for oyster mushrooms was high in the 50% substitution group in contrast to the fruiting body yield for nameko decreasing as the substitution rate of bamboo sawdust increased(44) is thought to be the effect of the difference in sensitivity of nameko and oyster mushrooms to antimicrobial compounds and soluble sugars, as well as the fact that oyster mushrooms have a wide range of substrate options.

For composted bamboo sawdust, in the group substituted with 100% bamboo that has been composted for at least two months and in the group substituted with 75% bamboo that had been composted for at least three months, there was no significant difference in cultivation duration compared to the base medium.  The fruiting body yield increased in both the 75% and 100% substitution groups composted for at least one month compared to the base medium. The yield was greatest in the 100% substitution group that had been composted for four months, at approximately 1.3 times that of the base medium. The substitution rate of bamboo sawdust, where the fruiting body yield is greatest per composted period, showed a tendency for the substitution rate to increase with an increase in time composted. From these results, it is believed that composting is effective in the use of bamboo sawdust as a substrate in sawdust-based oyster mushroom cultivation.

With composted bamboo sawdust, because the types and amount of bacteria increase after eight weeks of composition, it is presumed that bamboo sawdust that has been composted for eight weeks or more is affected by bacteria and that extracellular enzymes derived from bacteria in bamboo sawdust being composted accumulate. In sawdust-based lion's mane mushroom cultivation until now, fruiting body yield increases when spent substrate from sawdust-based maitake, nameko, bunashimeji and shiitake cultivation is composted and are used as substrate, so it is presumed that the increase in low-molecular glucans from spent substrate-derived extracellular enzymes and nutrition supplementation in spent substrate in the medium preparation process are a factor in fruiting body yield(37, 39). From these results, it is possible that low-molecular glucans increased due to bacteria-derived extracellular enzymes in composted medium.

Conclusion

In sawdust-based edible mushroom cultivation, I have demonstrated that the type of substrate and nutrition supplementation that form sawdust-based medium and their composition ratios affect the yield and quality of fruiting bodies, and in particular I have shown that industrial waste and underutilized biomass resources can be used in sawdust-based medium and are beneficial. In sawdust-based cultivation, it is thought that suitable medium material changes depending on social conditions, the economic situation and local circumstances. Currently, there is a need to provide "safe" and "delicious" mushrooms at a low price, but I believe various issues can be resolved by gradually building knowledge on medium material relating to sawdust-based cultivation knowhow.