Effects of enzymatic reaction on the generation of key aroma volatiles in shiitake mushroom at different cultivation substrates

Abstract Aroma is an important factor affecting mushroom character and quality. According to the different reaction pathway, the key aroma metabolites (sulfur and eight‐carbon volatiles) formation can be classified into enzymatic reactions and nonenzymatic reactions. Aroma volatiles are generated from precursors via the biocatalytic activities of various synthases during the growth stages of shiitake mushrooms. Understanding the specific relationships between the key aroma metabolites and their synthases is key to improving shiitake mushroom quality. At the same time, to reduce forest logging and burning of agricultural by‐products in farmland, agricultural by‐products have been applied to shiitake mushroom cultivation. Nevertheless, how to further improve the production of aroma volatiles in mushroom cultivated with agricultural waste is still a challenge. In order to understand the biosynthesis of volatiles via enzymatic reactions and screen the agricultural by‐products that can improve the production of aroma volatiles in mushroom cultivation, the mechanism of producing aroma volatiles needs to be further elucidated. In this study, the activities and gene expression levels of the key synthases involved in volatile metabolism, the contents of key aroma volatiles, and the correlations between related synthetase, volatiles, and cultivation substrate (CS) were investigated. Network models for visualizing the links between synthetase, volatiles, and CSs were built through partial least squares (PLS) regression analysis. The correlation coefficients among three related synthetase and enzymatic gene expression were high, and the combined effects of multiple synthetase promoted the production of volatiles. PLS analysis showed that the corncob and corn meal were more related to the production of volatiles and synthetase gene expression, and they can be added to the CSs as flavor promoting substances. The enrichment of key aroma volatiles in shiitake mushroom cultivated by the gradient of 20% corn meal combination CS was noticeable.


| INTRODUC TI ON
The yield of mushrooms (edible fungi) is about 39.34 million tons (2019), and shiitake mushrooms (Lentinula edodes) hold the highest market share in China (http://www.cefa.org.cn). At present, the industrial cultivation of shiitake mushroom and the industrial preparation of mushroom cultivation sticks have also risen rapidly, and the quality and scale of the development of the shiitake mushroom industry has improved significantly (Huang & Zheng, 2020;Schimpf & Schulz, 2016). The large-scale industrial production capacity of shiitake mushroom sticks is 20 million bags in 2018 (http://www.cefa.org.cn). With the continuous expansion of the production scale of shiitake mushroom, the demand for raw materials has increased significantly. Forestry is prohibited from cutting and logging, resulting in a shortage of the main raw materials (sawdust) for efficient use of mushroom cultivation. Therefore, increasing numbers of agricultural by-products are being added to cultivation substrate (CS) formulations for mushroom cultivation (Atila, 2019;Lin et al., 2015). In China, the annual output of cottonseed hull can reach more than 2 million tons (http://www.china -cotton.org/), bagasse is 12.58 million tons (Lin, 2020), and corncob is 45.9 million tons (Wang et al., 2016). Bagasse, corncob, and cottonseed hull have been gradually utilized as mushroom CSs.
These agricultural by-products have large yields and low costs, and the used CS can also be processed into biological fertilizers to form a virtuous ecological cycle.
The nutrients and unique aroma of shiitake mushroom are undoubtedly the main drivers for the increase in mushroom production/consumption. Aroma is an important factor affecting mushroom character and quality. Maintaining the flavor quality of shiitake mushrooms under the factory cultivation mode has become an important issue that needs to be studied for the shiitake mushroom industry in China. Several key aroma volatiles have been suggested to be important for the shiitake aroma. Sulfur and eight-carbon volatiles are the main flavor compounds produced during the growth of shiitake mushroom (Beluha & Ranogajec, 2011;Chen et al., 2016;Dermiki et al., 2013;Her et al., 2015;Phat et al., 2016;Schmidberger & Schieberle, 2020;Tian et al., 2016;Yang et al., 2017), and play an important role in the flavor of shiitake mushroom. Sulfur compounds have a relative content of 19.41%-21.53% in fresh mushrooms and 37.51%-62.09% in dried mushrooms. The typical mushroom and grass aroma of fresh shiitake mushroom are mainly caused by the eight-carbon compounds, and the most representative compounds are 1-octen-3-ol, 1-octen-3-one, and 3-octanone. The relative content of eight-carbon volatiles in fresh mushrooms can reach 44.13% and 4.4%-10.28% in dried mushrooms .
According to the different reaction pathway, the key aroma volatiles formation can be classified into enzymatic reactions and nonenzymatic reactions. In the enzymatic reaction, volatiles are generated from precursors via the biocatalytic activities of various synthetase during the growth stages of shiitake mushrooms. The synthetase plays a key role in the synthesis of volatiles on the enzymatic reaction biosynthetic pathway in mushrooms (Iwami et al., 1975;Li et al., 2018;Yasumoto et al., 1971). γ-glutamyl transpeptidase (γ-GGT), cysteine sulfoxide lyase (C-S lyase), and lipoxygenase (LOX) were found to be involved in the synthesis and metabolism of sulfur and eight-carbon volatiles. However, little is known about the relationship of the enzyme activity, aroma-related genes expression with the production of key aroma volatiles in the enzymatic reaction during the growth and development of shiitake mushrooms.
Therefore, the analysis of aroma-forming of shiitake mushroom based on its aroma synthesis pathways could further clarify the corresponding mechanism of volatiles to the synthetase.
In this study, we have mostly focused on shiitake mushroom key aroma volatiles formation from enzymatic reactions. Firstly, we investigated into the contents of sulfur and eight-carbon volatiles prevalent in mushrooms, which are derived from lentinic acid and unsaturated fatty acids (linoleic acid and linolenic acid).
Secondly, the activity of related synthetase and its gene expression involved in the reactions were studied. Furthermore, network models were also built to represent the links between volatiles, synthetase, and CSs. The network models will clarify the corresponding mechanism of volatiles to synthetase, volatiles and synthetase to the CSs. The results can be used to guide and regulate   the enzymatic reaction, resulting in abundant accumulation of the key aroma volatiles in shiitake mushroom during the growth and development stages.

| Mushroom cultivation
We developed more types of available carbon sources (such as corncob and cottonseed hull) which can replace the sawdust, and the addition ratio and gradient of the selected nitrogen sources (wheat bran and corn meal) for clarifying the aroma-forming mechanism based on our previous study (Li et al., 2018(Li et al., , 2019. The shiitake mushroom CSs composition is listed in Table 1. Homogenized substrates were collected in mushroom bags with a moisture content of 55%, and the sterilized substrates were inoculated with colonized spawn from the selected strain L215 (strain certificate, 2,014,001). The mushrooms were cultivated in a factory cultivation mode ( Figure S1). Fresh mushrooms were harvested at the mature growth stage, and the selected fresh mushroom pileus and stipes were separated for experimental use. The mushroom harvested at CS1-CS11 were expressed as P1-P11 for pileus samples and S1-S11 for stipe samples. with at least 98% purity were obtained from Sigma-Aldrich. For unavailable standards, a tentative identification of the volatiles was achieved by comparing their mass spectrometric information of chromatographic peak with mass spectra library (similarity is greater than 75%), and manual analysis was used to identify the structure of the compound for qualitative analysis.

| Activities of synthetase analysis
The activities of LOX, γ-GGT, and C-S lyase were studied to reveal the relationship between their activities and volatiles. The activities of LOX and γ-GGT were analyzed as described by Li et al., (2019).
LOX catalyzes the reaction of linoleic acid into oxidative products, which have a characteristic absorption peak at a wavelength of 280 nm. γ-GGT catalyzes the transfer of γ-glutamyl from glutamylnitroanilide to N-glycylglycine to form p-nitroaniline, which has a characteristic absorption peak at a wavelength of 405 nm. C-S lyase catalyzes the reaction of S-methyl-L-cysteine sulfoxide to produce pyruvate, which reacts with 2,4-dinitrophenylhydrazine. It appears brownish red under alkaline conditions and has a characteristic absorption peak at 510 nm. The values of absorbance were measured to calculate enzymatic activity. The three kinds of synthetase activity assay kits (Suzhou Comin Biotechnology Co., Ltd.) were used for LOX, γ-GGT, and C-S lyase enzymatic extraction and assays according to the manufacturer's instructions.

| Gene expression of synthetase analysis
The mushroom RNA extraction and verification methods used were consistent with the literatures (Li et al., 2019;Liu et al., 2015), and the strand cDNA was obtained using a reverse transcription reac-  The OAV is defined as the ratio of the content of each volatile compound in the sample to its respective odor threshold.

| Analysis of volatiles
Five sulfur compounds and 12 types of eight-carbon compounds were selected to study the relationship between the production of key aroma volatiles and CSs. The structural formula information, odorant description, and recognition threshold of volatiles are listed in Table 2, and their respective odor activity values (OAV) were calculated to estimate their contribution to the overall aroma. The selected volatiles, dimethyl trisulfide, 1-octen-3-one, 1-octen-3-ol, 1,2,4-trithiolane, octanal, and dimethyl disulfide played an important role on the flavor profile of shiitake mushroom (OAV > 1).
Some new innovation CSs that were beneficial for the production of aroma volatiles in shiitake mushroom were found. With the change of the carbon source (sawdust, 78%-93%) and nitrogen source (corn meal, 20%-5%) gradients in the CS, the content of 1,2,4-trithiane was highest at the ratio of 20% nitrogen source (183.63 µg/kg). It also reached relatively higher contents of 125.13 µg/kg under a 5% corn meal gradient, 154.11 µg/kg under a 5% wheat bran gradient.
1,2,4,5-tetrathiane had higher and similar contents in the pileus harvested at the corn meal gradient of 20% and 10%, which were The total contents of eight-carbon volatiles in the pileus harvested at CSs with corncob and cottonseed hull were similar (103.46 µg/kg and 102.14 µg/kg, respectively). The two substrates easily promoted the production of eight-carbon compounds, and the contents were much higher than that in the pileus cultivated by adding the same proportion of bagasse in the CS (77.65 µg/kg).
To visualize the relationship between the shiitake mushroom grown in different CS formulations, a heat map and hierarchical cluster analysis (HCA) were used for the correlation, as shown in Figure 1.
It can be seen from the cluster heat map that the pileus samples P2, P3, and P10 form the cluster A group, mainly because the CSs that promoted the volatiles octanal, 1-octen-3-ol, 2-octen-1-ol, and 3-octanone synthesis were equivalent in these three pileus samples. samples were relatively high. Some eight-carbon compounds were significant in the stipe S8 cultivated with 20% corn meal in the CS.
The clustering results of stipe samples were irregular.
In summary, the use of a composite carbon source in the CS and the addition of corncobs or cottonseed hulls can promote the production of sulfur and eight-carbon volatiles in the mushroom pileus samples, which is better than adding the same proportion of bagasse to the substrate. Gradient changes of sawdust and corn meal had a greater influence on the synthesis of volatiles, and the enrichment of volatiles in the mushroom cultivated by the 78% sawdust and 20% corn meal combination CS was noticeable.

| Analysis of synthetase activities and gene expression
The shiitake mushroom fruiting bodies cultivated in different CSs had large differences in the activities of the three related synthetase and their distribution sites in the fruiting bodies. The enzymatic activities of γ-GGT and LOX were higher in shiitake mushroom fruiting bodies cultivated by adding bagasse to the CS. In the mushroom pileus cultivated with the gradient of carbon and nitrogen sources in the CSs, the activities of the two enzymes showed a downward trend as the proportion of wheat bran decreased. With the decrease of the proportion of corn meal, the activity of γ-GGT showed a slow downward trend, and LOX showed a slow upward trend. In the stipe, the activity of γ-GGT was greatly improved at the ratio of 5% wheat bran gradient. As the gradient ratio of corn meal decreased, γ-GGT was at a higher enzymatic activity level at the corn meal gradient of 20% and 15%, and then at the corn meal gradient of 10% and 5%, the activity of γ-GGT decreased significantly. The activity of LOX in the stipe fluctuated with the change of the carbon and nitrogen source gradient of the CSs, and the enzyme activity did not change significantly. The enzymatic activities of C-S lyase in the pileus were higher than those in the stipe harvested at the same CSs. The C-S lyase had a relatively higher activities in the pileus cultivated with adding cottonseed hull and corncob to the composite carbon source. With the decrease of the proportion of wheat bran in CSs, it had higher enzymatic activities in the pileus harvested at the nitrogen source gradient of 20% and 15%, then it decreased significantly at the wheat bran gradient of 10%; then, its highest enzymatic activity was at the wheat bran gradient of 5%. In the stipe, the C-S lyase enzymatic activity first decreased significantly at the nitrogen source gradients of 15% and 10%; then, it slightly increased at the ratio of 5% wheat bran gradient. With the decrease in the proportion of corn meal in CSs, C-S lyase in mushroom pileus had a slight upward trend first at the corn meal gradient of 15% and then a downward trend at lower nitrogen source proportion, and the activity level of C-S lyase in the stipe was also higher with corn meal at 15% in the CS (Figure 2).

| D ISCUSS I ON
Japanese scholars discovered that lentinic acid formed the characteristic volatiles of lenthionine under the action of γ-GGT and C-S lyase, accompanied by by-products (Iwami et al., 1975;Yasumoto et al., 1971). Combet et al., (2006) had also reviewed the formation pathway of eight-carbon volatiles in fungi. Therefore, based on the synthetic pathway of characteristic volatiles of shiitake mushroom, our research results can provide a guide that can be used to regulate the synthesis of characteristic volatiles during the factory cultivation process by analyzing the response mechanism of volatiles synthesis and synthetase changes to the CS. Masakazu et al., (2006), Masakazu et al., (2010) had studied the smell and odorous components of dried shiitake mushroom and found rice bran was the main source of sulfur, or added amino acids to sawdust media, which both had the most effect on 1,2,4-trithiolane content. In our study, the correlation coefficient of 1,2,4-trithiane and corn meal was the highest, followed by corncob. These results provided practical basis for the development of mushroom CSs.
Scholars in some Asian countries have studied the correlation between shiitake cultivation and mushroom flavor. Asia is the main area for shiitake mushroom cultivation and consumption, and research scholars from various countries had also done a lot of work on the yield production of mushrooms (Azman et al., 2019;Barshteyn & Krupodorova, 2016;Ozcelik & Pesken, 2007;Philippoussis, 2009;Rigoberto et al., 2020;Rossi et al., 2003), or investigated the flavor of shiitake mushrooms during processing (Tian et al., 2016;Wen et al., 2020;Xu et al., 2019). Along with flavor enhancement or agricultural recycling and resource development, there is still a lot of work to be done in the fields of mushroom cultivation and flavor quality research in the future.

| CON CLUS IONS
The results showed that shiitake mushroom aromas, and their biosynthesis, are various and complex. The combined effects of multiple synthetase promoted the production of volatiles, and shitake mushroom had its own complex networks of volatile biosynthesis. γ-GGT and its encoding genes were involved in the biosynthesis of eight-carbon ketones, C-S lyase and genes closely related to eight-carbon aldehydes, alcohols, sulfur volatiles formation. Enzymatic assay also confirmed that LOX had positive effects on aroma volatiles production. The large variety of synthetase contributing to mushroom aroma in enzymatic reaction suggest that more remains to be learned. The carbon and nitrogen source material base and the addition ratio in the CS also had an important influence on the synthesis of characteristic volatiles in shiitake mushroom. On the CS containing corncob, it was easier to promote the production of the sulfur and eight-carbon volatiles. Also, adding corn meal to the CS promoted better production of these volatiles and synthetase gene expression. The recommended order for the addition of carbon source to the CS is corncob, cottonseed hull, and bagasse; nitrogen source order is corn meal, wheat bran; the nitrogen source gradient order is 20% corn meal, 5% wheat bran, and 5% corn meal. In summary, as optimized and more environmentally friendly cultivation substrate constituents, corncob and corn meal can be added to the CSs as flavor promoting substances for the industrial cultivation of shiitake mushroom to obtain mushroom fruiting bodies rich in flavor compounds.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.