Superior component compositions and antioxidant activity of Volvariella volvacea oil compared to those of Agrocybe cylindracea and two Lentinula edodes oils

Abstract The biological activity of an oil not only depends on its fatty acid composition but also the lipid composition and trace components. In this paper, to select the optimal mushroom oil, the component compositions (fatty acids, lipids, polyphenols, flavones, tocopherols, and unsaponifiable matters) and antioxidant activities in vitro of four mushroom oils (Agrocybe cylindracea, two Lentinula edodes, and Volvariella volvacea) were investigated and compared. The results showed that the four tested oils had the same fatty acid composition in different amounts, but the lipid component, minor components, and free radical scavenging activity in the tested oils varied widely depending on the type of mushroom. Overall, Volvariella volvacea oil was considered superior to the other three tested oils, as it had the largest contents of polar lipids, diglycerides, polyunsaturated fatty acids (74.38%), unsaponifiable matter (319.09 mg/kg), total phenols (124.08 mg/100 g), tocopherols (139.86 mg/100 g), as well as the highest ABTS and FRAP values (349.45 and 3801.70 μmol Trolox/100 g). This finding suggests that Volvariella volvacea oil is a promising resource that should be further researched.


| INTRODUC TI ON
With the increasing desire for greater sustainability and food security, more and more new resources have been developed to enrich food diversity (Rosmiza et al., 2016); thus, mushrooms have gained attention because of their high nutritional value (rich in protein, carbohydrates, edible fiber, vitamins, and phytosterols), medicinal activity (antitumor, hipocolesterolemic, antioxidant, antifungal, and antimicrobial), and industrial application (Kitzberger et al., 2007(Kitzberger et al., , 2009;;Sugui et al., 2003).Apart from their appearance as food ingredients at dinner tables, mushrooms are also applied in cosmetics (Wu et al., 2016), health care (Lu et al., 2020), and pharmaceutical fields (Valverde et al., 2015).At present, the mushroom industry is regarded as important for rural areas to prosper because it benefits agriculture and increases farmers' income in many countries.
Therefore, it is necessary to discuss any approach to enhance the added value of edible mushrooms.
Given the early studies on their chemical composition and nutritional qualities, mushrooms are labeled "high in proteins, vitamins, fibre and minerals" but "low in calories, fats, and essential fatty acids" (Agrahar-Murugkar & Subbulakshmi, 2005;Sanmee et al., 2003).
Compared with macronutrients such as polysaccharides, polypeptides, and dietary fiber, oil (or lipid), accounting for 1.75-15.5% of the dry weight of mushrooms (Hong et al., 1988), is often ignored by researchers, resulting in insufficient studies.However, oil (or lipid) is an important carrier of fat-soluble active constituents (i.e., tocopherols, ergosterols, terpenoids, and so on), which play an important role in the function of mushrooms, as the fat-soluble components of mushrooms have been reported to present biological activities, such as antioxidant, antitumor, and immunomodulatory activities.Therefore, to evaluate the medicinal value of mushrooms comprehensively, it is essential to study mushroom oil.
Regarding the existence of fat-soluble active constituents, the biological activity of mushroom oil is also separately affected by these trace concomitant contents, in addition to being affected by fatty acid and lipid composition (Zheng et al., 2017).Hence, when we need to explore the potential application value of mushroom oil, it is crucial to investigate the extract composition and content of the lipids and microcomponents in mushroom oil.
Early in 2013, L. edodes constituted 22% of the world's total output and was affirmed as the most widely cultivated mushroom (Royse et al., 2017).However, during the initial cultivation period, L. edodes was only cultivated in certain regions with suitable climate, its limited production could not meet the increasing demand of people, and it was the shortage of L. edodes that intensified the expansion of cultivation areas.Flower mushrooms are the product of abnormal growth of L. edodes, and unsuitable planting environmental conditions (i.e., low temperature and humidity) could promote the transformation of L. edodes to flower mushrooms.In contrast to conventional L. edodes, which have smooth pileipellis, flower mushrooms are produced only when the fleshy cells of L. edodes rupture to form chrysanthemum-like pilei (Cao, 2000).Although flower mushrooms are 5-8 times more expensive than conventional L. edodes, they are still preferred by global consumers owing to the higher nutritional value and superior characteristics (thick pilei, tender meat, and beautiful appearance) (Liu et al., 2022).With an output accounting for 5% of the world's total production, Volvariella volvacea is deemed the second most widely cultivated mushroom.Similar to L. edodes, Volvariella volvacea and Agrocybe cylindracea have also recently been accepted by more and more consumers because of their delicious taste and high nutritive value (Jia et al., 2020).Therefore, based on their enormous output and high popularity, these four mushrooms (Agrocybe cylindracea, L. edodes, flower mushroom, and Volvariella volvacea) occupy a pivotal position in the mushroom industry.
In this study, oils of Agrocybe cylindracea, conventional L. edodes, flower mushroom, and Volvariella volvacea were extracted to investigate their exact nutritional composition and free radical scavenging ability.By comparing all tested indicators, the oil with the highest development value was selected, providing theoretical data for the future application of mushroom oil.

| Mushroom oil preparation
The solvent extraction method was used to prepare the mushroom oil.Specifically, the mushroom sample was washed, dried, and smashed through an 80-mesh sieve to extract oil using petroleum ether (boiling range, 60-90°C) at a ratio of 4:1 (V/W), after 4 h of oil extraction at room temperature, and the mushroom oil was mainly distributed into the supernatant, which needed to be isolated from the mushroom sediment by centrifugation.After removing the solvent from the supernatant, mushroom oil was obtained and stored at −20°C for analysis.

| Oil yield
Oil yield (%) was calculated by the mass ratio of oil to the mushroom sample after oil extraction.

| Lipid separation
According to Heinzelmann et al. (2014), a silica gel column was applied to separate the polar lipids (phospholipids and glycolipids) from the neutral lipids (glycerides) of mushroom oil, namely the polar lipids and neutral lipids of mushroom oil were separated by a silica gel column (30 × 400 mm) containing approximately 35 g active silica gel.After adding 25 mg/mL oil chloroform solution into the column, chloroform, acetone, and methanol were used to wash the column in turn to obtain fractions of glycerides, glycolipids, and phospholipids, respectively.

| Fatty acid composition
GC-MS was applied to determine the fatty acid composition of the mushroom oil and the procedures were performed according to Kang et al. (2019) with some modifications.In brief, a 20 mg oil sample and 3 mL sodium methoxide solution (0.5 M) were mixed together to enable saponification for 30 min at 70°C, and then, 1 mL boron trifluoride diethyl ether solution was added into the mixture for water bath heating at 80°C for 30 min, which enhanced the production of the fatty acid methyl ester.After the alkaline reaction liquid cooled to room temperature, 3 mL of heptane was added to extract the fatty acid methyl ester from mixture.After the evaporation of solvent, the dried oil extract was re-dissolved, filtered (13 mm, 0.22 μm), and injected into the GC-MS for fatty acid analysis.GC-MS for determining the fatty acid composition was equipped with a DB-5 column (30 m × 0.25 mm × 0.25 μm), and the temperatures of the injector and detector were both set at 290°C.At a sample loading of 1.0 μL, the sample was analyzed at a split ratio of 100/1 and an oven temperature program containing 180°C (2 min), 10°C/min to 230°C (1 min), 3°C/min to 236°C (1 min), 6°C/min to 260°C (2 min), and 5°C/min to 270°C (2 min).Temperatures of ion source and transmission line were 250°C and 280°C, respectively, for EI Ionization mode at a scan range of 100-350 amu.
The mass spectrometer was operated in both positive and negative ion modes.At maximum injection times of 100 ms and 80 ms, precursor spectra (70-1050 m/z) and fragment spectra were collected at resolutions of 70,000 and 17,500, respectively, to hit AGC targets of 3e 6 (precursor) and 1e 5 (fragment).DDA mode was brought to acquire data in the top three configurations, and the stability of the UPLC-MS was evaluated by a quality control sample after every 10 tested samples.

| Total phenol, flavone, and tocopherol content
0.5 mL of 90% aqueous methanol was used to extract total phenol and flavone from 50 mg of mushroom oil successively three times, and the methanol supernatant was collected to evaporate to dryness.After redissolving the dryness into 0.5 mL of methanol, the mixed methanol solution (i.e., the oil methanol extract) was filtered TA B L E 2 Fatty acid compositions of four test mushroom oils.Note: Datas with different superscript letter mean a significant difference (p < .05)betwwen two samples, while that with same letter signifys no significant difference.

TA B L E 1
Oil yield and lipid category distribution of four test mushroom oils.
A 0.1 g/mL oil n-hexane solution was filtered for tocopherol analysis, which requires an HPLC device equipped with a silica gel column ZORBAX RX-SIL (4.6 mm × 250 mm, 5 μm) and ultraviolet detector (Cunha et al., 2006).At a flow rate of 1 mL/min, the temperature of the column was set at 35°C, with n-hexane/isopropanol/ glacial acetic acid at 99/0.2/0.8 (V/V/V) as the only mobile phase, and 10 μL of sample solution was analyzed by a 20 min isocratic elution procedure.The tocopherol content in mushroom oil was determined by the external standard method.

| Unsaponifiable matter
After saponifying the mushroom oil by alkaline hydrolysis, the mixture containing alcoholic potassium hydroxide was extracted by nhexane to obtain unsaponifiable matter.To ensure full saponification and precise quantification, the oil sample (0.2 g) was mixed with 3 mL of potassium hydroxide solution (2 M) and 0.5 mL of internal standard (5α-cholestane, 0.1 mg/mL) and heated for 30 min at 80°C.The hexane solvent was evaporated to dryness and 200 μL BSFA-TMCS was added for silanization.Based on the internal standard method of quantitative analysis, GC-MS was applied to analyze the exact composition of the unsaponifiable matter.

| Antiradical scavenging activity
Spectrophotometry was used to detect the antiradical scavenging activity of mushroom oil, which was evaluated comprehensively by The reducing ability of ferric ions in mushroom oil was measured according to the modified method of Zouirech et al. (2022).Briefly, a working solution was prepared by mixing sodium acetate trihydrate acetic acid solution (0.3 mol/L), TPTZ hydrochloric acid (10 mmol/L), and FeCl 3 solution (20 mmol/L) at ratio of 10:1:1.Similar to the operation of ABTS, the reducing ability of ferric ions in mushroom oil was calculated by the absorbance at 593 nm, after mixing 200 μL of working solution and 10 μL of oil methanol extract at 37°C for a 10 min reaction.

| Statistical analysis
All experiments were performed in triplicate, and the results were calculated as the means and standard deviations.SPSS Statistics software was applied to find differences in data between groups, and p-values <.05 were regarded as statistically significant.

| Oil yield and distribution of lipid category
As shown in Table 1, the oil yield of the four mushrooms ranged from 1.31 to 2.98%.Similar to glycerides, polar lipids containing glycolipids and phospholipids were also regarded as the main lipid composition of mushroom oil because of the 14.96-32.55%percentage, conforming to the low lipid content (Vidović et al., 2011) and high polar lipid proportion (Hanuš et al., 2008) in most mushrooms.Agrocybe cylindracea had the highest oil yield (2.98%), while Volvariella volvacea had the lowest oil content (1.31%).Even so, Volvariella volvacea oil containing 29.53% glycolipids and 3.02% phospholipids was still believed to have a better emulsifying property, which was reported to be positively correlated with the percentage of polar lipids in the oil.

| Composition of glycerides, glycolipids, and phospholipids
The exact lipid molecular compositions of the four mushroom oils were detected by UPLC/MS, 62 glycerides, 7 glycolipids, 7 phospholipids, and 3 sterols were identified, as shown in Table 3.The area normalization method was applied to quantify the different lipid components, and glycerides with more than 95% content, were thought to be the majority of four mushroom oils, which were found to represent a larger percentage of phospholipids than glycolipids.

| Unsaponifiable matters
Oil unsaponifiable matters are some substances that cannot be saponified by strong basic solutions in oil, with the sterol component as the main component, oil unsaponifiable matter has often been studied, as sterol is often reported to present strong activities of lowering blood LDL-cholesterol (Products and Allergies, 2014).
GC-MS was applied to detect the unsaponifiable matter in four mushroom oils, which were quantified by internal standard 5α-cholestane.According to the data in three indicators: DPPH radical scavenging activity, ABTS radical cation decolorization assay, and the reducing ability of ferric ions in this research.With Trolox methanol solutions at different concentrations as antioxidant standards, all antioxidant indicators of mushroom oil in vitro were quantified by Trolox.Measurement of DPPH radical scavenging activity was operated according to Nafis et al. (2019) with slight modification.Equal volumes of oil methanol extract (0.2 g/mL) and DPPH-methanol solution (0.4 mmol/L) were mixed in the dark for 30 min.With Trolox methanol solutions (10-180 μmol/L) as standards, the DPPH radical scavenging activity of the oil sample was measured by the absorbance at 517 nm.The ABTS radical cation decolorization assay of mushroom oil was performed based on a study by Huang et al. (2021) with a slight change.A working solution containing ABTS (3.5 mmol/L) and potassium persulfate (1.3 mmol/L) was stored at room temperature for 12-16 h before use.After adjusting the absorbance at 734 nm to 0.70, the working solution (200 μL) was mixed with oil methanol extract (0.2 g/mL, 10 μL) and reacted for 20 min at 30°C, and Trolox methanol solution (200-800 μmol/L) was also used to quantify the ABTS radical cation decolorization assay of the oil sample, which was detected at 734 nm.
sterol-esters were found to exist in most mushroom oils except for Volvariella volvacea oil, which presented glycolipids, phospholipids, and sterol-esters at similar proportions.Volvariella volvacea oil contained Cer-NS 21:3;2O/18:2 and CASE 28:1/18:2 as its main ingredients of phospholipids and sterols, which was different from other mushroom oils with the same main components of phospholipids and sterols, that is, Cer-BS 19:1;2O/16:2;(3OH) and BRSE 28:2/18:2.However, this situation changed when discussing the glycolipid content in mushroom oils, as MGDG O-9:0/18:3 was the main glycolipid of Agrocybe cylindracea and conventional L. edodes oils, while MGDG O-9:0/18:2 was main glycolipid of flower mushroom and Volvariella volvacea oils.On the whole, Volvariella volvacea oil obviously had a higher content and molecular composition of polar lipids (5.39%, 14 molecules) than the other oils (flower mushrooms, approximately 4%, 9 molecules at most), consistent with the results described in Section 3.1.3.4 | Trace components3.4.1 | Total phenol, flavone, and tocopherol contentsAlthough phenolic compounds, flavones, tocopherols, and sterols were poorly soluble in oil, they are still regarded as effective antioxidants responsible for the storage stability of high-PUFA oil.Tocopherol, a fat-soluble vitamin, has often been studied owing to its antioxidant activity; however, in four tested mushroom oils, α-tocopherol was identified as the only tocopherol analog.According to Figure1, the four mushroom oils were discovered to have greater total phenol and tocopherol contents than flavone, which varied less from mushroom species.Distinguished from two mushroom oils (conventional L. edodes and flower mushroom oils) containing flavone at 29.390 mg/100 g and 28.34 mg/100 g, Volvariella volvacea oil appeared to have the lowest flavone content (23.28 mg/100 g) but the highest amount of total phenol (124.081mg/100 g) and tocopherol (139.86 mg/100 g) among the four tested oils.Therefore, based on the large amount of antioxidants (total phenols and tocopherol), Volvariella volvacea oil would be bound to exhibit stronger free radical scavenging ability.

Table 2
cupied the dominant position.In spite of the same molecular composition, the content of fatty acids varied from different lipid fraction as well as the mushroom species.For example, glycerides are apt to possess more polyunsaturated fatty acids (PUFAs) than polar lipids, and saturated fatty acids are more easily enriched in phospholipids sections apart from flower mushroom oil.More specifically, Agrocybe cylindracea contained 66.64%, 59.63%, 40.35%, and 63.38% PUFAs in glyceride, glycolipid, phospholipid, and whole oil, respectively.

Table 4
Compositions of the unsaponifiable matter of four test mushroom oils (mg/kg).Note: Datas with different superscript letter mean a significant difference (p < .05)betwwen two samples, while that with same letter signifys no significant difference.