Difference in Volatile Aroma Components of Stropharia rugosoannulata under Two Cultivated Environments Investigated by SPME-GC-MS

In order to study the effect of both greenhouse and forest cultivating environments on Stropharia rugosoannulata, its volatile aroma compounds were measured by a headspace solid phase micro extractions—gas chromatograph—mass spectrometer (SPME–GC–MS). The optimal adsorption temperature was 75 °C and the optimal adsorption time was 40 min. A total of 36 volatile aroma compounds were identified by GC–MS, including 8 aldehydes, 2 ketones, 4 alcohols, 15 alkenes, and 4 alkanes. Hexanal, 3-Octanone, 2-Undecanone, (E)-Nerolidol, and (Z)-β-Farnesene made great aromatic contributions. Among them, Hexanal, 3-Octanone, 2-Undecanone were the key aroma compounds for which odor activity values (OAVs) were more than 1. (E)-Nerolidol showed odor modification in the forest samples and showed a key aroma effect in greenhouse samples. (Z)-β-Farnesene showed odor modification in greenhouse samples. 3-Octanone was the largest contributing compound for which the OAV was more than 60. The total content of volatile aroma compounds first increased and then decreased with growth time; it reached the highest level at 48 h: 2203.7 ± 115.2 μg/kg for the forest environment and 4516.6 ± 228.5 μg/kg for the greenhouse environment. The aroma was the most abundant at this time. All samples opened their umbrella at 84 h and become inedible. Principal component analysis (PCA), hierarchical cluster analysis (HCA), and orthogonal partial least squares discriminant analysis (OPLS–DA) were combined to analyze the aroma difference of S. rugosoannulata under two cultivation modes. PCA and HCA could effectively distinguish the aroma difference in different growth stages. Under different culturing methods, the aroma substances and their changes were different. The samples were divided into two groups for forest cultivation, while the samples were divided into three groups for greenhouse cultivation. At the end of growth, the aroma of S. rugosoannulata with the two cultivation modes was very similar. OPLS–DA clearly distinguished the differences between the two cultivation methods; 17 key aroma difference factors with variable importance projection (VIP) > 1 were obtained from SPLS–DA analysis.


Introduction
Stropharia rugosoannulata is a deciduous mushroom that belongs to the Strophariaceae family and is one of the top 10 mushrooms on the market [1]. It grows naturally in grassland or forest margins and is widely distributed in Asia, Europe, and America [1,2]. 30.0% wood chips. It was cultivated under the moso bamboo (Phyllostachys edulis) in the Baiyan Temple forest Farm in Hangzhou. Altitude: 50 m, slope: 15-22 • . The district has a warm, temperate, and continental monsoon climate characterized by hot and rainy summers and cold and dry winters. It has an annual mean temperature of 16.3 • C, 1467 h of sunshine, and 134 days of rain, with precipitation being 1460 mm. The average relative humidity is 78.3%. Another strain was cultivated in a greenhouse, with the temperature being 22-28 • C and the average relative humidity 75.0-90.0%.

Sample Preparation
The samples were picked and put into a clean PE food bag, placed into a 4 • C incubator, and then sent to the laboratory within 2 h. The soil and other stains on the surface were washed with pure water and the water was dried with absorbent paper. The samples were cut into 0.4-0.6 mm cubes and the 4.0 g samples were weighed and put into a 15-mL headspace bottle for SPME-GC-MS analysis.
The growth time was defined as 0 h when the S. rugosoannulata had just grown out of the surface. The samples were collected at 12,24,36,48,60,72, and 84 h to study the changes in volatility during the growth stage. The samples at the 48 h growth stage were studied to optimize the adsorption conditions of SPME.

Optimization of the Adsorption Conditions of SPME
Samples weighing 4.0 g were weighed and put into a 15-mL headspace bottle. A pretreated SPME fiber (65 µm DVB/CAR/PDMS, Supelco, Bellefonte, PA, USA) was introduced into the vial headspace to absorb the volatiles.
The adsorption temperatures were selected as 30 • C, 45 • C, 60 • C, 75 • C, and 90 • C, with treatment lasting for 30 min while the effect of the adsorption temperature on the volatile components was investigated. After the optimal adsorption temperature was obtained, the adsorption times of 10 min, 20 min, 30 min, 40 min, and 50 min were selected to further investigate the effect of the adsorption time on the volatile components.

Determination of Volatile Aroma Compounds via SPME-GC-MS
Volatile compounds were extracted via the headspace solid-phase microextraction (SPME) method and the extracts were detected via a gas chromatography/mass spectrometry (7890A/5975C, GC/MS) system (Agilent Technologies, Inc., Santa Clara, CA, USA).
GC: The chromatographic column used was the DB-5 capillary column (30 m × 0.25 mm × 0.25 µm, Agilent J&W, Santa Clara, CA, USA). The start temperature of the column was set to 50 • C and kept for 1 min; then, the temperature was increased to 260 • C and maintained for 3 min at a rate of 5 • C/min. The inlet temperature was 250 • C. The carrier gas was nitrogen (≥99.9%) and the flow rate of the carrier gas was 1.0 mL/min. The desorption time was 2 min. There was no split.
MS: The energy of the electron impact ionization was set to 70 eV; the temperature of the transfer line and ion source was set to 230 • C and 280 • C, respectively. Solvent delay was set at 0 s using the full scan mode. The mass scan ranged from 15 to 550 m/z.
Identification and quantification of volatile compounds: Identification was accomplished by matching mass spectra and odor properties to those recorded in the NIST 14.0 Library (https://www.nist.gov/nist-researchlibrary (accessed on 20 July 2022)) and the AromaChem library (https://www.alpha-mos.com/ (accessed on 13 September 2022)).
The relative content of the other volatile aroma compounds was quantified based on the ratio of its peak area relative to the peak area and concentration of the external standard. The formula of the relative content of each volatile compound was specified as: where "4" means "4.0 g samples", indicative of the quality of the sample, and "1000" is the coefficient converted from "g" to "kg".
C i -Volatile compound's relative content in the sample, µg/kg; x-The external standard content in the sample, µg; ω-The peak area of the external standard, %; ω i -The peak area of the target volatile compound, %; 2.5. Odor Activity Values for Hexanal, 3-Octanone, (E)-Nerolidol, (Z)-β-Farnesene, and 1-Hexanol in S. rugosoannulata Odor activity values (OAVs) were applied to evaluate the aroma contributions of the compounds in S. rugosoannulata. The OAV of the volatile aroma compound was specified as follows [17]: where OAV i -The odor activity values of the volatile compound (i); C i -The content of the volatile compound (i) in the sample, µg/kg; T i -The odor threshold of the volatile compound (i), µg/kg;

Statistical Analysis
All experiments were conducted in triplicate and the results were expressed as mean ± standard deviation. The data statistics were obtained using WPS 2019 (Beijing Kingsoft Office Software, Inc. Beijing, China). Significance analysis, Pearson analysis, and principal component analysis (PCA) were performed using SPSS 20.0 (SPSS Inc., Chicago, IL, USA). Graphics were produced using Origin 9.5 (Originlab, Northampton, MA, USA). Hierarchical cluster analysis (HCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted at the following web link (https://cloud.metware.cn/#/tools/tool-list (accessed on 20 May 2023)).   Figure 1, the adsorption capacity of Hexanal, (E)-Nerolidol, (Z)β-Farnesene, and the total volatile compounds was positively correlated with temperature, while the adsorption capacity of 3-octanone was negatively correlated with temperature. When the temperature was 30 • C and 45 • C, only 3-octanone was detected. 3-octanone is a natural fragrance with characteristic mushroom and cheese aromas [18] which are common aromas of edible mushrooms [19]. Hexanal, (E)-Nerolidol and (Z)-β-Farnesene are also common in food, and they are the four aromas with the higher content of volatile components in the mushroom. At the adsorption temperature of 90 • C, the number of volatile compounds reached the maximum and the total adsorption capacity decreased. Compared with 75 • C, the alkanes and fatty acid methyl esters increased at 90 • C and the high temperature may lead to aroma decomposition and esterification. Considering the number of adsorption compounds and the adsorption capacity of the main compounds, 75 • C was selected as the optimal adsorption temperature. a natural fragrance with characteristic mushroom and cheese aromas [18] which are common aromas of edible mushrooms [19]. Hexanal, (E)-Nerolidol and (Z)-β-Farnesene are also common in food, and they are the four aromas with the higher content of volatile components in the mushroom. At the adsorption temperature of 90 °C, the number of volatile compounds reached the maximum and the total adsorption capacity decreased. Compared with 75 °C, the alkanes and fatty acid methyl esters increased at 90 °C and the high temperature may lead to aroma decomposition and esterification. Considering the number of adsorption compounds and the adsorption capacity of the main compounds, 75 °C was selected as the optimal adsorption temperature.  perature. With the increase in the adsorption temperature and adsorption time, the volatiles adsorbed are more abundant, meaning the total adsorption increases. When the adsorption time exceeds 30 min, the adsorption rate of Hexanal and 3-Octanone tends to be balanced and (E)-Nerolidol and (Z)-β-Farnesene continue to rise. When the adsorption time exceeds 40 min, the adsorption of (E)-Nerolidol and (Z)-β-Farnesene tends to be balanced and the increases in the total adsorption capacity of the volatiles have the same trend. The adsorption time was selected as 40 min.
The changes in content for each category of volatile aroma compound in S. rugosoannulata are shown in Figure 3. Combined with Tables 1 and 2, it confirmed that ketones, alcohols, and aldehydes were the main volatile aromas in S. rugosoannulata and that they were mainly C6-C15 compounds. The alcohols had pleasant woody and fragrant aromas, aldehydes had a low threshold value and fatty aromas, and the ketones had floral and fruity aromas, which contributed significantly to the overall flavor of the S. rugosoannulata [17,20]. No matter if it was cultivated under a forest or under a greenhouse, the content of the ketones in S. rugosoannulata was always the highest ( Figure 3). The total content of the volatiles' aroma compounds first increased and then decreased with the growth time for S. rugosoannulata under greenhouse or forest cultivation. Its content reached its highest at 48 h, when the harvest aroma was the most abundant. At 84 h, the volatile components decreased to their lowest content; at this point, the mushrooms had all opened in such a way as to become umbrella-like, with the edible flavor of the compounds having turned bad. The quantity and content of the volatile compounds of the samples in the greenhouse were higher than those in the forest. The difference was significant (p < 0.01) at the early growth stage (12-48 h). Their content difference reached the largest at 48 h and the content of the former was 2.14 times that of the latter. At the later growth stage (60-84 h), the volatile components decreased sharply with time for two cultivation modes and their content had no significant difference (p > 0.1). The difference in the content of the volatile components under two cultivation modes may be related to the growing environment. Studies have shown that rainfall, air humidity, soil moisture, and atmospheric pressure are significantly related to the quality and the yield of S. rugosoannulata [21]. The stable environment with sufficient moisture in the greenhouse conditions is more conducive to the growth of S.rugosoannulata as it is good for promoting flavor formation for S. rugosoannulata. Moreover, the slow air circulation in greenhouses results in less loss of volatile components, while the situation is the opposite in a wild environment. This may be one of the reasons for the difference in the quantity and content of the volatile components of S. rugosoannulata under the two cultivation modes.  the growth of S.rugosoannulata as it is good for promoting flavor formation annulata. Moreover, the slow air circulation in greenhouses results in less lo components, while the situation is the opposite in a wild environment. Thi of the reasons for the difference in the quantity and content of the volatile co S. rugosoannulata under the two cultivation modes. Two ketones were detected, including 3-Octanone and 2-Undecanone has earthy and mushroom notes and 2-Undecanone has milky and fruity n samples in the forest, the content of 3-Octanone was 1855.1 ± 208.7 μg/kg, a 91.2% of the total volatiles at 12 h, and the content of 3-Octanone was 1732.1 accounting for 78.6% of the total volatiles at 48 h. For samples under the gr content of 3-Octanone was 2861.0 ± 234.7 μg/kg, accounting for 87.9% of the Two ketones were detected, including 3-Octanone and 2-Undecanone. 3-Octanone has earthy and mushroom notes and 2-Undecanone has milky and fruity notes [17]. For samples in the forest, the content of 3-Octanone was 1855.1 ± 208.7 µg/kg, accounting for 91.2% of the total volatiles at 12 h, and the content of 3-Octanone was 1732.1 ± 192.7 µg/kg, accounting for 78.6% of the total volatiles at 48 h. For samples under the greenhouse, the content of 3-Octanone was 2861.0 ± 234.7 µg/kg, accounting for 87.9% of the total volatiles at 12 h, and the content of 3-Octanone was 1688.4 ± 108.8 µg/kg, accounting for 37.4% of the total volatiles at 48 h. From Table 3, the OAV values of 3-Octanone under the under-forest cultivation and greenhouse cultivations methods are both more than 60.0, showing that 3-Octanone has the greatest contribution to the flavor of S. rugosoannulata. 2-Undecanone first increased and then decreased with the growth time, with its content reaching its highest values at 48 h, with 30.4 ± 2.3 µg/kg for the forest conditions and 31.7 ± 4.7 µg/kg for the greenhouse conditions. The OAV values of 2-Undecanone are more than 4.3 (Table 3), showing that it also has a large role in aroma contribution. (E)-Nerolidol has a fragrant, floral, and fruity aroma [22]. It ranked second place in terms of the content of the volatile compounds of S. rugosoannulata (Tables 1 and 2). Its content increased and then reached its highest value at 48 h, which was 12.7 ± 32.4 µg/kg for the forest conditions and accounting for 9.7%, and 1362.6 ± 188.7 µg/kg for the greenhouse conditions and accounting for 30.2%; after that, its content decreased with time. The content of (E)-Nerolidol in the forest samples is six times lower than that in the greenhouse samples. The aroma effect of this component is relatively lower in the forest products but more obvious in greenhouse products. Wei et.al [10] had reported that (E)-Nerolidol was present in essential oils from fresh and dried S. rugosoannulata. The content of 3-Octanone and (E)-Nerolidol in the forest samples was lower than that in the greenhouse samples, with the difference being more than 1000 µg/kg in samples detected at the 12 h point. This situation may be caused by the stable environment found in greenhouses. 1-Hexanol has a floral and fatty aroma [17] but may be a sign of corruption for S. rugosoannulata. At the growth stage of 12-60 h, its content was very low: less than or equal to 1.0 ± 0.1 µg/kg in the forest samples and 3.4 ± 0.3 µg/kg in the greenhouse samples. At the growth stage of 72-84 h, its content rose rapidly, with the largest content being 49.8 ± 7.3 µg/kg in the forest samples and 63.9 ± 6.5 µg/kg in the greenhouse samples, with this latter value having increased 30-48 times. Hexanal, Benzaldehyde, and Nonanal had a relatively higher content in the two cultivation modes compared with other aldehydes. Hexanal had the highest concentration, showcasing a grassy and apple fragrance aroma [17,22]. The OAV values of Hexanal were more than 8.6 and it ranked second place for aroma contribution in key volatile compounds of S. rugosoannulata (Table 3). (Z)-β-Farnesene is a major component of several plant essential oils with a slight balm aroma [23], mainly existing in rose oil, orange oil, and other essential oils. It is also one of the raw materials for the synthesis of vitamin E [24]. Li et Al. had detected (Z)-β-Farnesene in the pleurotus eryngii [25]. The (Z)-β-Farnesene OAV values of samples under the forest and greenhouse differed greatly: 0.0 for the forest samples and 0.3 for the greenhouse samples. Studies had shown that compounds with an OAV range of 0.1-1.0 can modify the aroma and that a value greater than 1.0 indicates that the compound is a key aroma substance; indeed, the greater the value, the greater the contribution to the overall odor characteristics of the sample [26]. Therefore, (Z)-β-Farnesene had a certain aroma modification effect upon the greenhouse samples. In conclusion, for forest samples, the key aroma substances of S. rugosoannulata are Hexanal, 3-Octanone, and 2-Undecanone and the modification aroma substance is (E)-Nerolidol; for the greenhouse samples, the key aroma substances of S. rugosoannulata are Hexanal, 3-Octanone, 2-Undecanone, and (E)-Nerolidol and the modification aroma substance is (Z)-β-Farnesene.

Aroma Difference Analysis of S. rugosoannulata
Data analysis mainly includes unsupervised analysis and supervised analysis. Unsupervised analysis can cluster samples in the absence of unknown sample information, such as via PCA and HCA. Unsupervised analysis methods cannot ignore intra-group errors, while supervised analysis methods can reduce intra-group random errors and highlight systematic errors between each group via partial least squares-discriminant analysis (PLS-DA) and OPLS-DA. PCA, HCA, and OPLS-DA will be combined to discuss the aroma difference of S. rugosoannulata here.
A PCA based on 36 aromas detected via GC-MS is shown in Figure 4. It expresses the difference in the aroma characteristics of S. rugosoannulata at different growth stages more directly. A total of five principal components with eigenvalues greater than 1 were extracted and the cumulative variance contribution rate was 91.4%. Figure 4 shows the scores of the first two principal components; the contribution rate of PC1 was 41.5% while the contribution rate of PC2 was 26.6%. The cumulative variance contribution rate was 68.1%. PC1 and PC2 contained a large amount of information on the samples and could reflect the characteristic aromas for S. rugosoannulata. The distance between samples in Figure 4 represents the aroma difference. From Figure 4, it shows that the aromas of the forest samples at each growth stage were relatively similar, while the aromas of greenhouse samples were greatly different. Furthermore, the greenhouse samples at 24-48 h have a similar aroma, and the greenhouse samples at 60-72 h also have a similar aroma.     Figure 5 shows the HCA heat map of the volatile aroma compounds in S. rugosoannulata during growth. It shows that the forest samples at 12-60 h can be regarded as one group and the forest samples at 72-84 h can be regarded as the other group. The greenhouse samples had relatively large aroma differences. Samples at 12 h can be taken as a single group, samples at 24-48 h can be taken as one group, and samples at 60-84 h can be taken as another group. There is a large difference between the two cultivation methods at the early stage of growth, while at the end of growth (especially at the 72 h and 84 h stages) the samples under the two forms of cultivation are very similar. Figure 6 shows the score chart of the OPLS-DA mode, with R 2 X = 0.59, R 2 Y = 0.93, and Q 2 = 0.83 indicating that the model is stable and reliable. There was a significant difference between the two cultivation methods and the 95.0% confidence interval was obviously divided into two blocks. It indicates that there were significant differences in volatile aroma substances between those under greenhouse cultivation and those under the forest cultivation environment. With variable importance projection (VIP) > 1 as the selecting criterion, the main potential component causing aroma differences between the two cultivation methods could be obtained. The larger the VIP value, the greater the influence of this component on aroma differences. The results show that there are 17 key factors that influence aroma differences between the two cultivation modes, which are (E,E)-2,4-Nonadienal (VIP = 1.

Conclusions
In order to analyze the aromas of S. rugosoannulata, the effect of adsorption t ture and time on the volatile components of S. rugosoannulata via GC-MS has been The optimal adsorption temperature was selected as 75 °C and the optimal time lected as 40 min.
A total of 36 aroma compounds were identified in the studied S. rugosoannu forest cultivation, 22 aroma compounds were identified, including 8 aldehydes, 2

Conclusions
In order to analyze the aromas of S. rugosoannulata, the effect of adsorption temperature and time on the volatile components of S. rugosoannulata via GC-MS has been studied. The optimal adsorption temperature was selected as 75 • C and the optimal time was selected as 40 min.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.