The Effect of Amanita rubescens Pers Developmental Stages on Aroma Profile

The dichloromethane extraction was applied to extracted volatile compounds of the six developmental stages of caps and stipes of an Amanita rubescens mushroom and the relative contents were measured with the gas chromatography-mass spectrometry. The number of identified compounds ranged between 53 and 52, respectively, with a high ratio of alkane volatiles. The significant differences between the aroma compounds were determined in caps to identify their stages of development. The fully mature stage caps were characterized by 4,6-dimethyl-dodecane (7.69 ± 1.15%), 2-hexyl-1-decanol (11.8 ± 1.61%), 1,3-di-tert-butylbenzene (11.4 ± 1.25%), heptadecyl pentadecafluorooctanoate (2.16 ± 0.31%), and 2-hexyl-1-dodecanol (13.5 ± 1.33%). Niacinamide (3.90 ± 0.07%) and glycerol (3.62 ± 1.27%) was present in the caps in the early-stage of the rotting mushroom, which represented the 10th–12th day of fructification. The caps and stipes from the 12th–15th day of fructification were characterized by 2,3-butanediol (11.7 ± 0.13% and 8.00 ± 0.10%, respectively). Moreover, the caps from this developmental stage were characterized by 2-methyl- and 3-methyl butanoic acids (0.18 ± 0.03% and 0.33 ± 0.02%, respectively) which are typical for the rotting stage. In this study, we confirmed the effect of A. rubescens developmental stages on the aroma profile.


Introduction
Aroma, formed by a combination of volatile and non-volatile compounds, is a key characteristic of food which significantly affects consumer preferences. Edible mushrooms are consumed as a delicacy not only because of their high nutritional value, but also because of their specific aroma [1]. Volatile organic compounds are synthesized as a protection of the organism or as by-products of metabolism [2]. They are released into the air and often have a characteristic odor [3]. There are many publications on fungal aroma. Altogether, 150 compounds have been identified, mainly from the categories of higher alcohols, ketones, esters, aldehydes, hydrocarbons, acids, heterocyclic and aromatic compounds [4][5][6][7][8]. In 1938, 1-octen-3-ol was first identified as a "mushroom-like flavor" and a "raw mushroom" with a characteristic earthy and sweet taste [5]. Other compounds have also been identified, specifically 1-octen-3-one, (E)-2-octen-1-ol, 1-octanol, 3-octanol, and (E)-2-octenal [9][10][11]. On the other hand, there is a lack of information around aroma profiles of different fruiting bodies and developmental stages of wild edible mushrooms, or Amanita rubescens.
European Blusher (Amanita rubescens Pers.) belongs to the Amanitaceae family. Many species of the family are inedible or toxic. On the other hand, A. rubescens, as well as some other species (A. fulva, A. baccata, A. caesarea), are edible [12][13][14][15]. The A. rubescens is a commonly collected and popular wild edible mushroom that is characterized by great sensory properties. Fruiting bodies of A. rubescens have a relatively high bioaccumulation capacity which can affect the spectrum of aromatic compounds and ultimately the increased risk of intoxication by heavy metals of the pickers [12].
According to SK Regulation 132/2014 [16] only wild edible mushrooms listed in the annex can be placed on the market. A. rubescens is absent from the annex, which contains 53 different wild edible mushroom species. In 2019, the consumption of "other vegetables and mushrooms" reached 13.3 kg per capita, according to Statistical Office of the Slovak Republic [17]. The consumption of exclusively wild edible mushrooms is not recorded in Slovakia. On the other hand, the Statistical Office of the Czech Republic recorded the consumption of 3.1 kg of "mushrooms" per capita in 2019 [18]. The aim of the work was to evaluate the effect of the various developmental stages of the Amanita rubescens Pers fruiting bodies from Slovakia on the volatile compounds, determined in a dichloromethane extract by the gas chromatography-mass spectrometry.

Sampling Area and Sampling
The six developmental stages of A. rubescens fruiting bodies (morphologically characterized by the expert mycologist prof. Kunca, and in compliance with the taxonomic keys [19]) were collected in Žakýlske Pleso area (Štavnické Vrchy, Slovakia)(GPS: 48 • 31 12.8" N; 18 • 55 23.5" E) on 3 July 2020. The area of the Štiavnické Vrchy is characterized by volcanic origins. The sampling area is predominantly forested with beech and hornbeam vegetation. The altitude of the sampling point is 757 m a.s.l., with an average annual temperature of 6-7 • C, and rainfall of 700-800 mm [20]. The region is characterized as very cold and humid, with a difference between potential evaporation and rainfall <50 mm (climate indicator for the months of June to August). The soil is characterized as loamy medium-heavy soil containing organic matter >20% [21].
The samples of individual developmental stages of A. rubescens fruiting bodies were taken in the morning from one 2 × 2 m area ( Figure 1a). The advantage of such a collection is a high probability of homogeneous mycelium which creates a presumption of identical dynamics of nutrient uptake by the mycelium. Thus, identical or similar conditions for the formation of aromatic compounds were observed. After sampling, the fruiting bodies were removed from larger impurities and temporarily stored in ventilated polyethylene boxes ( Figure 1b). Upon arrival at the laboratory, the samples were rinsed thoroughly in deionized water and divided into caps and stipes. The samples were dried at a temperature of 30 • C for~22 h in a hot air dryer (Memmert UF 110m, Memmert GmbH & Co. KG, Schwabach, Germany). After thorough drying, the samples were homogenized on a rotary mill (IKA A10, IKA-Werke GmbH & Co. KG, Staufen, Germany) and stored in gas-tight 20 mL headspace vials.
commonly collected and popular wild edible mushroom that is characterized by great sensory properties. Fruiting bodies of A. rubescens have a relatively high bioaccumulation capacity which can affect the spectrum of aromatic compounds and ultimately the increased risk of intoxication by heavy metals of the pickers [12].
According to SK Regulation 132/2014 [16] only wild edible mushrooms listed in the annex can be placed on the market. A. rubescens is absent from the annex, which contains 53 different wild edible mushroom species. In 2019, the consumption of "other vegetables and mushrooms" reached 13.3 kg per capita, according to Statistical Office of the Slovak Republic [17]. The consumption of exclusively wild edible mushrooms is not recorded in Slovakia. On the other hand, the Statistical Office of the Czech Republic recorded the consumption of 3.1 kg of "mushrooms" per capita in 2019 [18]. The aim of the work was to evaluate the effect of the various developmental stages of the Amanita rubescens Pers fruiting bodies from Slovakia on the volatile compounds, determined in a dichloromethane extract by the gas chromatography-mass spectrometry.

Sampling Area and Sampling
The six developmental stages of A. rubescens fruiting bodies (morphologically characterized by the expert mycologist prof. Kunca, and in compliance with the taxonomic keys [19]) were collected in Žakýlske Pleso area (Štavnické Vrchy, Slovakia) (GPS: 48°31′12.8″ N; 18°55′23.5″ E) on 3 July 2020. The area of the Štiavnické Vrchy is characterized by volcanic origins. The sampling area is predominantly forested with beech and hornbeam vegetation. The altitude of the sampling point is 757 m a.s.l., with an average annual temperature of 6-7 °C, and rainfall of 700-800 mm [20]. The region is characterized as very cold and humid, with a difference between potential evaporation and rainfall <50 mm (climate indicator for the months of June to August). The soil is characterized as loamy medium-heavy soil containing organic matter >20% [21].
The samples of individual developmental stages of A. rubescens fruiting bodies were taken in the morning from one 2 × 2 m area ( Figure 1a). The advantage of such a collection is a high probability of homogeneous mycelium which creates a presumption of identical dynamics of nutrient uptake by the mycelium. Thus, identical or similar conditions for the formation of aromatic compounds were observed. After sampling, the fruiting bodies were removed from larger impurities and temporarily stored in ventilated polyethylene boxes ( Figure 1b). Upon arrival at the laboratory, the samples were rinsed thoroughly in deionized water and divided into caps and stipes. The samples were dried at a temperature of 30 °C for ⁓22 h in a hot air dryer (Memmert UF 110m, Memmert GmbH & Co. KG, Schwabach, Germany). After thorough drying, the samples were homogenized on a rotary mill (IKA A10, IKA-Werke GmbH & Co. KG, Staufen, Germany) and stored in gastight 20 mL headspace vials.  The specific shape and size of the six developmental stages of the mushroom Amanita rubescens Pers are shown in Figure 2. The six developmental stages were sorted, according to Falandysz et al. [22], by an expert mycologist (prof. Kunca). The first developmental stage (6 individuals) with the smallest fruiting bodies was estimated to be the 2nd-3rd day of fructification, with a size of 7 cm (Figure 2a). The 3rd to 5th day of fructification represented the second developmental stage (2 individuals), when the fruiting body reached 9 cm (Figure 2b). Figure 2c shows the third developmental stage (3 individuals), which was estimated to be the 5th-8th day of fructification, at a height of 12 cm of the mushroom body. The 8th-10th day stage (3 individuals), which is 15 cm high, is characterized as the fully mature stage of fructification and thus the fourth developmental stage (Figure 2d). Figure 2e shows the 5th developmental stage (the rotting stage) which was estimated to be the 10th-12th day of fructification, at a size of 14 cm (2 individuals). The sixth developmental stage (2 individuals) was estimated to be the 12th-15th day of fructification when the size of the fruiting body remained the same as the previous stage. The specific shape and size of the six developmental stages of the mushroom Amanita rubescens Pers are shown in Figure 2. The six developmental stages were sorted, according to Falandysz et al. [22], by an expert mycologist (prof. Kunca). The first developmental stage (6 individuals) with the smallest fruiting bodies was estimated to be the 2nd-3rd day of fructification, with a size of 7 cm (Figure 2a). The 3rd to 5th day of fructification represented the second developmental stage (2 individuals), when the fruiting body reached 9 cm (Figure 2b). Figure 2c shows the third developmental stage (3 individuals), which was estimated to be the 5th-8th day of fructification, at a height of 12 cm of the mushroom body. The 8th-10th day stage (3 individuals), which is 15 cm high, is characterized as the fully mature stage of fructification and thus the fourth developmental stage (Figure 2d). Figure 2e shows the 5th developmental stage (the rotting stage) which was estimated to be the 10th-12th day of fructification, at a size of 14 cm (2 individuals). The sixth developmental stage (2 individuals) was estimated to be the 12th-15th day of fructification when the size of the fruiting body remained the same as the previous stage.

Volatile Compounds Analysis
The analysis was carried out using gas chromatography-mass spectrometry (GC-MS) (GC 7890B coupled by MSD 5977A; Agilent Technologies Inc.) equipped with CombiPal autosampler CTC120 (CTC Analytics AG, Zwingen, Switzerland). A column HP-5ms (30 m × 0.25 mm × 0.25 µm; Agilent Technologies Inc.) was used. One microliter of the sample extract was injected in the inlet, operated in a split mode 10:1 at 250 • C. The oven temperature program started at 40 • C. The temperature was held for 3 min, then increased to 250 • C at 3 • C/min and then held again for 10 min. Helium was used as carrier gas at the constant flow (1.2 mL/min). The mass detector parameters were as follows: ionization energy of filament: 70 eV, transfer line temperature: 250 • C, MS source temperature: 230 • C, quadrupole temperature: 150 • C. The mass spectrometer was programmed under electron impact (EI) in a full-scan mode at m/z 40-450 with a frequency of 1.8 scans/s. Each sample was measured in triplicate.

Volatile Compounds Determination
The compound identification was carried out by comparing mass spectra (over 80% match) with a commercial database NIST library 2017 (National Institute of Standards and Technology, Gaithersburg, MD, USA) and Wiley library, retention times of the reference mixture standard of n-alkanes (11 components, Restek Corporation, Bellefonte, PA, USA), and a comparison of data on the occurrence of edible fungi with the literature [23,24]. The relative percentage (%) of the determined volatile compounds was calculated by dividing the individual peak area by the total area of all peaks. Peaks under 0.1% were not counted.

Statistical Analysis
All the data obtained were analyzed by descriptive statistics arithmetic average and standard deviation. Then, all the variables were tested for normality. According to the Shapiro-Wilk test and the Kolmogorov-Smirnov test, all the tested variables did not follow the Gaussian distribution. The Kruskal-Wallis test was performed to compare significant differences between the developmental stages. The ten most numerous volatile compounds, characteristic of each stage, were used for PCA analysis. Principal Component Analysis (Spearman type) was used to find a pattern of similarity of the observations and the variables by displaying them as points on a map. Descriptive statistics, normality tests, Kruskal-Wallis test, and the PCA analysis were performed using the MS Excel and XLSTAT package program [25].
Several unique volatiles that were present in one developmental stage and not in others can be marked as markers. The cap in stage 1 had the unique volatile succinimide. The cap in the next developmental stage 2 had the unique volatiles 7-methyl-6-tridecene and 5-ethyl-5-methyl-decane. The cap in stage 3 had no specific marker. The fully mature stage had the unique volatile 2-octyl-1-dodecanol. The early-stage of the rotting mushroom had specific volatiles N-methyliminopropylbenzene and nonanoic acid. The cap in developmental stage 6 had several unique acids: 2-methyl-butanoic acid and 3-methyl-butanoic acid.

Discussion
In this study, the fruit bodies of A. rubescens were divided into six developmental stages. According to Kalač [30], the lifetime of the fruiting body is estimated to be only 10-14 days. In this study, the sixth developmental stage was estimated to be the 12th-15th day of fructification, characterized by a dark brown color. The results showed that the caps contained higher quantities of volatile compounds than the stipes. On the other hand, the rotting mushroom cap stages 5 and 6 were statistically (PCA and Kruskal-Wallis test) characterized by unique aroma markers (2,3-butanediol (p < 0.0001), glycerol (p < 0.0001),

Discussion
In this study, the fruit bodies of A. rubescens were divided into six developmental stages. According to Kalač [30], the lifetime of the fruiting body is estimated to be only 10-14 days. In this study, the sixth developmental stage was estimated to be the 12th-15th day of fructification, characterized by a dark brown color. The results showed that the caps contained higher quantities of volatile compounds than the stipes. On the other hand, the rotting mushroom cap stages 5 and 6 were statistically (PCA and Kruskal-Wallis test) characterized by unique aroma markers (2,3-butanediol (p < 0.0001), glycerol (p < 0.0001),

Discussion
In this study, the fruit bodies of A. rubescens were divided into six developmental stages. According to Kalač [30], the lifetime of the fruiting body is estimated to be only 10-14 days. In this study, the sixth developmental stage was estimated to be the 12th-15th day of fructification, characterized by a dark brown color. The results showed that the caps contained higher quantities of volatile compounds than the stipes. On the other hand, the rotting mushroom cap stages 5 and 6 were statistically (PCA and Kruskal-Wallis test) characterized by unique aroma markers (2,3-butanediol (p < 0.0001), glycerol (p < 0.0001), and niacinamide (p = 0.0068)). The 2-methyl and 3-methyl butanoic acids (p = 0.0074) were characterized only for the 6th developmental stage and according to the literature, it is known to be sweaty [28]. In general, the acid compounds (i.e., nonanoic acid, and 2-methyl-and 3-methyl-butanoic acids) are responsible for the sweaty attribute, which is unpleasant and had a negative effect on the aroma profile of the mushrooms [31][32][33]. Most publications have focused on the sensory aroma quality of mushrooms from a technological aspect [11,[34][35][36][37], while very few publications examined the sensory properties of mushroom developmental stages [38]. Lu et al. [38] characterized the aroma profile of two truffle species (T. indicum and T. pseudohimalayense) and the influence of the maturation stage on volatile organic compounds. The unripe stage was characterized by volatile compounds from groups: alcohols and phenols (7), esters (9), aldehydes and ketones (6), hydrocarbons (3), and 1 N-containing compound. In the mature stage, volatile compounds such as esters (2), alcohol (1), aldehydes and ketones (4), benzenes and methoxy compounds (5), hydrocarbons (2), and N-containing compounds (2) were identified. The same compound groups were also identified in the dichloromethane extracts of Amanita rubescens in our study.

Conclusions
This study provided a thorough comparison of the volatile compound profiles of Amanita rubescens at various developmental stages of fruiting bodies. Our results for the volatile compounds in A. rubescens show a minor change for caps and stipes during the fruit body maturation (from the smallest fruit body to full maturity). On the other hand, the eight compounds (4-methyl-dodecan-1-ol, 2-hydroxy-2,N-dimethyl-butanamide, 1,3,5-trimethy-cyclohexane, 4-methylene-decane, heptadecane, 5-methyl-tridecane, 4,6dimethyl-undecane, and 5-methyl-undecane) were identified in the earlier stages of the fruit body while they were absent at the full-maturity stage. Statistical analysis showed that several compounds displayed different relationship patterns in the cap the rotting mushroom stage (2,3-butanediol, glycerol, and niacinamide) from that of the full-maturity stage (4,6-dimethyl-dodecane, 2-hexyl-1-decanol, 1,3-di-tert-butylbenzene, heptadecyl pentadecafluorooctanoate, and 2-hexyl-1-dodecanol). We confirmed that, based on aromatic profiles, it is possible to distinguish the initial developmental stage of a fruiting body from the fully mature and the decomposition stage. At the same time, it is better to determine the aromatic profiles of mushrooms in caps than stipes. For the first time, this study specifies the individual developmental stages of the wild edible mushroom Amanita rubescens. In conclusion, there is still a need for further confirmation of volatile organic compounds on more individual fruiting bodies. We recommend studying developmental stages of A. rubescens from different localities in Slovakia (possible effect of chemical-physical, and geochemical parameters of soil) or comparing the results with different developmental stages of other wild edible mushrooms.