Towards an increased functionality in oyster (Pleurotus) mushrooms

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Agricultural and Environmental Chemistry

Towards an increased functionality in oyster (Pleurotus) mushrooms produced on grape marc or olive mill wastes serving as sources of bioactive compounds Georgios Koutrotsios, Nick Kalogeropoulos, Andriana C. Kaliora, and Georgios Zervakis J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01532 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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Journal of Agricultural and Food Chemistry

Towards an increased functionality in oyster (Pleurotus) mushrooms produced on grape marc or olive mill wastes serving as sources of bioactive compounds

Georgios Koutrotsiosa, Nick Kalogeropoulosb, Andriana C. Kaliorab, and Georgios I. Zervakisa,*

a

Agricultural University of Athens, Laboratory of General and Agricultural Microbiology,

Iera Odos 75, 11855 Athens, Greece b

Harokopio University, Department of Nutrition and Dietetics, Laboratory of Chemistry,

Biochemistry and Physical Chemistry of Foods, El. Venizelou 70, 17671 Kallithea, Greece

* Corresponding author: Georgios I. Zervakis; Tel: +302105294341, Fax: +302105294344, e-mail: [email protected]

1 ACS Paragon Plus Environment


1

Abstract:

2

Pleurotus ostreatus, P. eryngii and P. nebrodensis were cultivated on non-conventional

3

substrates containing grape marc (GMC) or olive mill by-products (OMB); wheat straw

4

(WHS) served as control. GMC-based media demonstrated equal/better mushroom

5

productivity than WHS for P. eryngii and P. nebrodensis, while the cultivation

6

performance of P. eryngii was improved in OMB-based media. Both GMC and OMB

7

substrates led to large increase of fruit-bodies content in phenolic acids, resveratrol,

8

triterpenic compounds and ergosterol; in particular, P. eryngii mushrooms presented

9

significantly more total phenolics and exhibited much higher antioxidant activity (2- to 8-

10

fold increase). Furthermore, substrates containing GMC or OMB presented up to 27%

11

increase in mushrooms β-glucans. Overall, Pleurotus species responded in a different and

12

mostly substrate-specific manner by selectively absorbing organic compounds. Phenolics

13

and squalene content of substrates correlated very well with mushrooms antioxidant

14

activity and ergosterol, respectively; the same was observed for triterpenics’ content of

15

substrates and mushrooms.

16 17

Key-words: Pleurotus; edible mushroom; phenolics; ergosterol; total serum oxidizability;

18

functional food; bioactive; alperujo; grape marc

19


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Introduction

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Nowadays, commercial edible mushroom production exceeds 27 million tons, a 25-fold

22

increase during the last 35 years, which is combined with a high rise in the respective per

23

capita consumption (> 4 kg/person/year compared to only about 1 kg/person/year in

24

1997).1 Cultivation of Pleurotus species (e.g. P. ostreatus, P. pulmonarius, P. eryngii, P.

25

djamor, P. citrinopileatus) corresponds to ca. 30% of the total mushroom production, and

26

constitutes the fastest growing and most profitable section for this particular market.

27

Pleurotus (oyster) mushrooms contain dietary fibers, vitamins, terpenics, statins,

28

essential amino acids, sterols, β-glucans and antioxidant microconstituents, which

29

demonstrate immunomodulatory, hypoglycaemic, hypocholesterolaemic, antimicrobial,

30

antithrombotic, antiatherogenic, anti-inflammatory, antihypertensive, antitumor and

31

prebiotic activities.2-9

32

The most popular among Pleurotus mushrooms is P. ostreatus, a cosmopolitan

33

species growing on dead wood of many broad-leaved and coniferous trees. Its cultivation is

34

widespread throughout the world on a large range of lignocellulosic substrates.10 On the

35

other hand, the Pleurotus eryngii complex comprises choice edible species including P.

36

eryngii and P. nebrodensis, which grow on roots and lower stem residues of Apiaceae

37

plants in Eurasia and north Africa;11 both exhibit excellent organoleptic properties, very

38

good texture and consistency of fruit-bodies, and long shelf life.12 In particular, P.

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nebrodensis is endemic in Sicily and Greece, and is the only mushroom that has been

40

included in IUCN’s (International Union for Conservation of Nature) Top 50

41

Mediterranean Island Plants list of threatened species.13 Few individuals reach maturity

42

each year, and although its successful cultivation is considered of major importance for

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reducing the human pressure on wild populations due to harvesting, no information exists

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about its ex situ production.14



45

Although Pleurotus spp. are commonly cultivated on pasteurized wheat or rice

46

straw, they are particularly efficient at biodegrading a wide range of lignocellulosics, and

47

hence they play an important role in managing organic wastes whose disposal is

48

problematic, e.g. those deriving from olive-oil production (i.e., olive pruning residues,

49

olive mill waste sludge from two-phase decanters, olive leaves) and wineries (e.g. grape

50

marc).10,15-17

51

Several million cubic meters of two-phase olive-oil mill wastes (TPOMW or

52

‘alperujo’, a sludge-like effluent deriving from two-phase decanters) are generated

53

annually and are characterized by high organic load, toxicity and recalcitrance, which are

54

mainly attributed to their content in polyphenols and short-chain fatty acids.18 Grape

55

pomace or marc (GMC) is the main solid by-product deriving from winemaking; in Spain

56

alone, one million tons of GMC are produced within a three-month period. According to

57

the European Council Regulation 1493/1999, GMC should be further processed by alcohol

58

distilleries. However, most small wineries do not adhere to this regulation, hence GMC is

59

accumulated as a waste together with grape stalks.19 On the other hand, both olive mill

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residues and GMC are rich in organic compounds with renowned functional properties,

61

and are examined as primary sources for the production of food/feed of enhanced

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nutritional value.20,21

63

While some information exists about the bioconversion of certain major waste

64

streams (e.g. residues from olive mills and wineries) into value-added products by

65

macrofungi, no data are available on the impact such substrates exerts on mushrooms

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content in bioactive compounds, and consequently on their functional properties. Yet, since

67

mushrooms are renowned for their efficiency to absorb elements/nutrients from their

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growth environment, this study’s main objective was to verify whether the nature of

69

Pleurotus spp. production substrates could affect their cultivation performance, as well as


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mushrooms quality parameters, content in bioactive microconstituents and functionality.

71

For this purpose, different ratios of olive mill residues or grape marc were used in

72

combination (or not) with conventional wheat-straw substrate for producing and suitably

73

evaluating fruit-bodies of P. ostreatus, P. eryngii and P. nebrodensis. In addition,

74

correlations/interactions among selected compounds present in substrates and mushrooms

75

were assessed.

76 77

Materials and methods

78

Chemicals and reagents

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Ergosterol, squalene, p-hydroxybenzoic acid, gallic acid, p-coumaric acid, syringic acid,

80

protocatechuic acid, p-hydroxyphenylacetic acid, ferulic acid, caffeic acid, tyrosol,

81

resveratrol, ursolic acid, maslinic acid, uvaol and 2,4,6-tris (2- pyridyl)-s-triazine (TPTZ)

82

were obtained from Sigma (Steinheim, Germany). Folin-Ciocalteu reagent, bis-

83

(trimethylsilyl)-trifluoroacetamide

84

(DPPH•), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), homovanillic

85

alcohol, oleanolic acid, and 3-(4- hydroxyphenyl)-1-propanol were purchased from Aldrich

86

(Steinheim, Germany). Sinapic acid and erythrodiol were obtained from Fluka (Steinheim,

87

Germany); cholesterol and vanillic acid from Serva (Heidelberg, Germany). All the

88

solvents used were of HPLC grade and were purchased from Merck (Darmstadt, Germany)

89

or Aldrich (Steinheim, Germany).

90

Biological material

91

Three strains of Pleurotus ostreatus (P. ostreatus LGM 22, P. ostreatus LGAM 217 and P.

92

ostreatus LGAM 104) and three representing the Pleurotus eryngii complex (P. eryngii

93

LGAM 216, P. eryngii LGAM 170 and P. nebrodensis UPA 6) were examined. Strains

94

were cultivated and preserved on potato dextrose agar (PDA, Difco), and are maintained in

(BSTFA),

1,1- diphenyl-2-picrylhydrazyl


radical


95

the Culture Collection of the Agricultural University of Athens (Laboratory of General and

96

Agricultural Microbiology).

97

Experimental set-up – Preparation of spawn and mushroom cultivation substrates

98

For the purposes of this study, GMC from a winery in the Nemea area (northeast

99

Peloponnese, Greece) and TPOMW from an olive-oil mill in Kalamata (southwest

100

Peloponnese, Greece) were obtained. Experiments were organized in two phases. The

101

preliminary phase included the comparative examination of six Pleurotus strains in three

102

main media, i.e. TPOMW plus olive leaves (OLV) 1:1 w/w, GMC plus wheat straw

103

(WHS) 1:1 w/w, and plain wheat straw, for assessing the suitability of such substrates to

104

support mushroom production and provide satisfactory cultivation performance. Then, in

105

the main experimental phase, selected strains (one from each species examined) were

106

cultivated on eight substrates composed of different ratios of GMC or TPOMW to basal

107

raw materials (WHS or OLV respectively) as follows: GMC in 3:1, 1:1 and 1:3 ratios

108

(w/w) to WHS, and TPOMW in 3:1, 1:1 and 1:3 ratios (w/w) to OLV, in addition to plain

109

OLV and WHS (control treatments).

110

Substrates were prepared by milling all plant residues in particles of 2-3 cm and

111

soaking them in water for 24 h. After the surplus water was drained off (a moisture content

112

of 53-69% was obtained), materials were mixed with calcium carbonate and wheat bran

113

(2% w/w and 5% w/w respectively, in terms of dry weight), and each formulated substrate

114

was filled into autoclavable polypropylene bags (2 kg per bag), and sterilized twice for 1 h

115

(121 0C, 1.1 atm). Grain spawn was prepared as previously described22 and was used for

116

inoculating the sterilized substrates at a 4% w/w. Five replicates per strain and substrate

117

were used. For substrates colonization and mushroom production, environmental

118

conditions in cultivation rooms were regulated and maintained as described by Koutrotsios

119

et al. (2014).15


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Mushroom cultivation performance

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The following cultivation parameters were studied for assessing performance of each

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strain: (i) earliness (days), defined as the time elapsed from substrate inoculation to first

123

primordia appearance; (ii) duration of the crop cycle (days), including incubation,

124

formation of mushroom primordia and mushroom production; (iii) yield (g), corresponding

125

to the fresh weight of mushrooms harvested; (iv) average mushroom weight (g), defined as

126

the ratio of yield over the number of individual fruit-bodies obtained; (v) biological

127

efficiency (BE, %), defined as the ratio of mushrooms fresh weight over the substrate’s dry

128

weight; (vi) productivity, defined as the ratio of BE over the duration of the crop cycle.

129

Analyses of mushroom and substrate samples

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Five samples from each substrate prior to inoculation with Pleurotus spawn and five

131

mushroom samples from the first production flush of each Pleurotus strain were freeze-

132

dried (Telstar Cryodos apparatus), then grinded to fine powder, and stored in plastic bags

133

at -20 0C until analyzed.

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Phenolics extraction and total phenolic content

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Phenolics were isolated essentially as described by Kalogeropoulos et al. (2013).23 Briefly,

136

substrate and mushroom samples (0.5 g) were extracted with 10 mL methanol for 48 h in

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the dark at room temperature. After separation of extract by centrifugation, the extraction

138

was repeated with 2.5 mL methanol for 2 h. Extracts were combined, concentrated to 2 mL

139

by means of a centrifugal concentrator (Speed Vac, Labconco Corp.), and were stored in

140

GC vials in deep freeze.

141

Total phenolic content (TPC) of substrates and mushrooms methanolic extracts was

142

measured by the photometric Folin–Ciocalteu assay at 750 nm (U-2001 spectrophotometer;

143

Hitachi Instruments Inc., USA) by employing gallic acid as calibration standard.24 Results

144

were expressed as mg gallic acid equivalents (mg GAE) per 100 g sample dry weight.



145

Individual phenolic compounds and terpenics

146

Eleven simple phenols, the stilvenoid resveratrol, and five triterpenic acids and dialcohols

147

were detected and quantitated by selective ion monitoring GC–MS,23 by employing 3-(4-

148

hydroxyphenyl)-1-propanol as internal standard. The target and qualifier ions used were

149

obtained from commercial standards and are shown in Table S1 (Supporting Information).

150

Identification of chromatographic peaks was made by comparing the retention times and

151

ratios of two or three fragment ions of each polyphenolic and terpenic compound with

152

those of pure standards, while quantitation was carried out by using 3-(4-hydroxyphenyl)-

153

1-propanol as internal standard. Quantification of maslinic acid and the triterpenic

154

dialcohols uvaol and erythrodiol, was based on the response factors of oleanolic acid.

155

Antioxidant activity of mushroom extracts

156

Herein, the DPPH (AAR) and ferric reducing/antioxidant power (FRAP) assays were

157

applied to evaluate the radical scavenging activity and the reducing antioxidant potential

158

respectively of the methanolic extracts obtained from mushrooms.24 Results on DPPH and

159

FRAP are expressed as mmol Trolox equivalents (TEs) in mushroom dry weight (100 g).

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An assay of higher biological relevance is the inhibition of copper-induced lipid

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oxidation in total serum (TSO), lag-time being used as a criterion for antioxidative

162

potency.25 Serum was obtained following centrifugation (3000 rpm, for 10 min at 14 °C) of

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freshly obtained venous blood taken from healthy subjects. Copper-induced oxidation in

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serum was applied as has been previously described in methanolic extracts of mushrooms

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solubilized in PBS.6 Oxidation kinetics were assessed as lag-time (sec) prior to oxidation.

166

Squalene, ergosterol and glucans

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GC-MS was employed for the determination of squalene in substrates and ergosterol in

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mushrooms. Both analyses were performed after hot saponification of freeze dried samples

169

followed by silylation with BSTFA, as previously described26,27, employing cholesterol as


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internal standard. The identification of squalene, ergosterol and cholesterol was based on

171

the retention times of pure standards, and the comparison of their mass spectra to NIST 98

172

(NIST MS search v6.1d) mass spectra library. Squalene levels in substrates are of interest

173

due to its role as precursor of ergosterol biosynthesis.28 Total and α-glucans were measured

174

by means of a commercial kit (Megazyme Int., Ireland); β-glucans content was calculated

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by subtraction of α-glucans from total glucans.

176

Statistical analysis

177

Chemical analyses were performed in five replicates and results are presented as mean ±

178

standard deviation. Differences between means were established by conducting analysis of

179

variance and Duncan’s t-test (5% level of probability) (SPSS ver. 19). Relationships

180

between variables (at significance levels of 0.05 and 0.01) were determined by Pearson’s

181

correlation coefficient; relationships among mushroom microconstituents and assays were

182

established by principal component analysis (PCA).

183 184

Results and Discussion

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Evaluation of cultivation performance of various Pleurotus strains on olive mill and

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winery by-products

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Ιn the frame of this study, six Pleurotus strains (i.e. three P. ostreatus and three

188

representing the P. eryngii complex, i.e. P. eryngii and P. nebrodensis) were examined

189

following assessment of their mycelium growth on substrates consisting of wheat straw,

190

grape marc, olive leaves and two-phase olive mill waste in various ratios (‘race-tube’

191

experiments),29 and after the evaluation of previous results evidencing high variability

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among P. ostreatus strains in respect to cultivation performance and content in bioactive

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compounds.6



194

As regards the cultivation process performed in the preliminary phase, the

195

GMC+WHS substrate provided significantly lower/better earliness values (11-22 days) for

196

the P. ostreatus strains examined. Similar results were obtained for P. eryngii and P.

197

nebrodensis (26 to 35 days) although differences among treatments were -in this case- less

198

pronounced (Table 1). High concentrations in readily degradable organic constituents

199

(mainly simple sugars and hemicelluloses) and lack of inhibitory compounds in GMC

200

could explain the faster formation of mushroom primordia. In contrast, the use of

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OLV+TPOMW substrates resulted in a notable delay in P. ostreatus mushroom appearance

202

(i.e. up to 44 days), as it was previously reported when olive mill wastes were examined

203

for the production of Cyclocybe cylindracea and Hericium erinaceus.15,30 However, this

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retarding effect was attenuated by P. nebrodensis and especially by P. eryngii (earliness

205

values in OLV+TPOMW: 31 – 36 days vs. 26 – 37 days in other substrates), possibly due

206

to a faster and/or more efficient degradation of the toxic compounds present in olive mill

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by-products by these two species. Interestingly enough, the total duration of P. ostreatus

208

cropping period is not considerably affected by the nature of substrate. Therefore, with the

209

exception of P. nebrodensis which presented a shorter cropping period in WHS, Pleurotus

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strains did not show significant differences among substrates in respect to the cultivation

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time-length (Table 1).

212

When the efficacy of different substrates to support mushrooms production was

213

examined, GMC+WHS performed best for P. eryngii and P. nebrodensis strains in respect

214

to biological efficiency (48 – 87%) and productivity (0.63 – 1.35) although values for the

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latter parameter were not significantly higher (Table 1). As regards P. ostreatus, both

216

GMC+WHS and plain WHS performed well (biological efficiency: 45 – 107% and

217

productivity: 0.83 – 2.02 respectively). On the other hand, substrates based on olive mill

218

by-products provided lower values for both P. ostreatus and P. nebrodensis, whereas P.


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eryngii demonstrated high biological efficiency and productivity values when cultivated in

220

TPOMW+OLV. Last, the evaluation of results related to the average size of fruit-bodies

221

did not reveal any notable effect which could be associated to the nature of substrates

222

(Table 1). The only noticeable difference in the quality of mushrooms produced was the

223

significant darker color of P. eryngii pilei derived from OLV media when compared to

224

those from WHS substrate (but within the range of variation observed in P. eryngii

225

mushrooms appearing in nature)11, whereas the morphology of P. ostreatus and P.

226

nebrodensis fruit-bodies were not affected by the nature of substrates examined (Fig. S1,

227

Supporting Information).

228

Previous pertinent studies reported biological efficiency values of 41 – 96% for P.

229

eryngii cultivated on various substrates,22,29,31,32 and of 50 – 137% for P. ostreatus grown

230

on a large range of -supplemented or not- lignocellulosic media including composted

231

TPOMW,15,22,29,33 while no data exist so far on productivity of P. nebrodensis. In general, a

232

high variability is noted in the values of mushroom cultivation parameters when olive mill

233

by-products are incorporated into substrates. This is attributed to the large variation in their

234

physicochemical properties which are considerably influenced by the olives processing

235

method, variety and cultivation regime.34,35

236

Pleurotus mushroom production on varying ratios of olive mill and winery by-products –

237

Assessment of selected organic compounds in cultivation substrates

238

Three selected strains (one per species examined, and qualified on the basis of their

239

productivity values, i.e. P. ostreatus LGM 22, P. eryngii LGAM 216 and P. nebrodensis

240

UPA6) were cultivated on varying ratios of GMC or TPOMW (1:3, 1:1 and 3:1, w/w) to

241

the basal raw materials (WHS and OLV respectively), and the substrates effect on

242

mushrooms antioxidant activity and content in bioactive compounds was assessed.



243

In this part, a detailed evaluation of cultivation parameters was out of scope.

244

However, pertinent results were in accordance with those already obtained from the

245

preliminary phase. In general, both early primordia formation, and high mushroom yields

246

and biological efficiencies (up to 82%) were observed in P. eryngii (for all ratios of GMC-

247

based substrates, and for OLV:TPOMW 3:1 and OLV:TPOMW 1:1 media) and P.

248

nebrodensis (for all ratios of GMC-based substrates, and for the plain OLV medium), thus

249

confirming the suitability of such by-products as cultivation substrates for these two

250

species (Fig. 1). In contrast, earliness, biological efficiency and productivity in P. ostreatus

251

was significantly and negatively affected by using medium to high ratios of GMC to WHS

252

and of TPOMW to OLV.

253

Prior to the analysis of mushroom extracts, all cultivation substrates were examined

254

in respect to their content in individual polyphenols, resveratrol, triterpenic compounds and

255

squalene, and the results are presented in Table 2 (Fig. S2, Supporting Information). In the

256

majority of cases, increase in concentrations of the aforementioned compounds was found

257

to be significantly associated with higher ratios of GMC and TPOMW, which was

258

anticipated due to the nature/properties of such by-products.20,21 This was particularly

259

notable in the cases of hydroxybenzoic acids, resveratrol, triterpenic compounds and

260

squalene, which were present at very low concentrations (or even not detected at all) in

261

WHS, whereas in GMC-supplemented substrates their content was found to be higher by

262

one to over three orders of magnitude (e.g. p-OH-benzoic acid and oleanolic acid in WHS

263

vs. WHS:GMC 1:3). As regards their content in the olive-based substrates, it was either

264

highly ascending or descending following TPOMW ratio increase, depending on the nature

265

of the compound examined. Hence, concentration of simple phenolics and squalene

266

increased by a factor of two to thirty when plain OLV was compared to OLV:TPOMW

267

1:3. In contrast, oleanolic acid and ursolic acid demonstrated the opposite trend, i.e. their


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increase was associated with increasing ratios of OLV in respect to TPOMW. Moreover,

269

sinapic acid, homovanillic alcohol and tyrosol were detected only in olive mill by-products

270

(or at very low concentrations in GMC-rich media), while resveratrol’s content did not

271

seem to be influenced by varying supplementation rates of TPOMW to OLV (Table 2).

272

Assessment of antioxidant activity and bioactive compounds in Pleurotus mushrooms

273

Phenolic compounds are among the most widely distributed plant secondary metabolites

274

and act as potent antioxidants. Literature data are limited as concerns the presence of

275

individual phenolic compounds in cultivated mushrooms;36,37 in the present work, their

276

content in mushrooms produced on different substrates was for the first time assessed.

277

Samples were taken from the pileus of the fully developed fruit-body (the stipe was

278

omitted from pertinent analyses) in order to exclude the possibility of cross-contamination

279

with substrate material. Three phenolic acids, the stilbene resveratrol, two triterpenic acids

280

and two triterpenic dialcohols were detected and quantified in extracts of Pleurotus fruit-

281

bodies (Table 3; Figs. S3, S4, S5, Supporting Information). Among phenolic acids, p-OH-

282

benzoic and p-OH-phenylacetic acids were the most abundant with concentrations of up to

283

177.6 and 159.6 µg/100 g d.w. respectively; maximum values for both of them were noted

284

in P. nebrodensis, especially in GMC-based media. On the other hand, protocatechuic acid

285

content increased in mushrooms of all three species from substrates which received high

286

TPOMW supplementation.

287

The stilbenoid resveratrol was found in significantly higher concentrations in P.

288

eryngii and P. nebrodensis mushrooms. Especially as regards the latter species, resveratrol

289

content was up to 15 times higher in comparison to P. ostreatus irrespectively of the

290

substrate used (Table 3). In general, resveratrol concentrations in mushrooms ranged from

291

0 to 151 µg/100g d.w. and were disproportionately lower than the values measured in the

292

respective substrates (the GMC-based media presented up to sixtyfold higher



293

concentrations than the olive-based media, reaching values up to 596 µg/100g d.w.; Table

294

2). This observation is indicative of a species/strain specific absorption mechanism (P.

295

nebrodensis seems to be more efficient than the other two species) by which the

296

mushrooms accumulate resveratrol irrespectively of the substrate’s pertinent content.

297

Among the triterpenic acids determined in substrates, namely oleanolic, ursolic and

298

maslinic, only the first two were detected in mushrooms. Their accumulation demonstrated

299

a species-specific mode in mushrooms deriving from olive-based substrates; i.e.

300

concentrations of both compounds increased in P. eryngii fruit-bodies when higher ratios

301

of OLV was used, whereas no significant differences were detected in P. ostreatus (Table

302

3). Moreover, oleanolic acid and ursolic acid concentrations in Pleurotus mushrooms

303

exhibited a similar trend when cultivated on GMC-based substrates; hence, their

304

concentrations were significantly higher when GMC supplementation increased. Similarly,

305

erythrodiol content in mushrooms of all species deriving from GMC-based substrates

306

increased as GMC ratios got higher. On the other hand, Pleurotus mushroom content in

307

uvaol was significantly affected only when olive by-products were used (Table 3).

308

Overall, it seems that increasing ratios of GMC and TPOMW to WHS and OLV

309

respectively, had a marked effect on the content of simple phenolics, resveratrol and

310

triterpenic compounds in Pleurotus mushrooms; this effect was particularly pronounced for

311

p-OH-phenylacetic acid, oleanolic acid and erythrodiol in all treatments. In addition,

312

especially in P. eryngii mushrooms, p-OH-benzoic acid, resveratrol and ursolic acid values

313

were significantly affected by GMC and TPOMW addition to the basal substrates.

314

As regards total phenolic content (TPC) and antioxidant activity, a high variability

315

was determined in Pleurotus fruit-bodies produced on eight cultivation media (Table 4).

316

Particularly pronounced was the species-specific response to supplementation -or

317

substitution- of WHS by other cultivation media which was exhibited by P. eryngii LGAM


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216 (also presented by P. eryngii LGAM 70 during the preliminary experiments, data not

319

shown). For example, when values obtained in WHS and OLV:TPOMW 1:3 were

320

compared, then TPC, AAR and FRAP increased approx. eightfold, fivefold and fourfold

321

respectively (intermediate values were detected in the other tested substrates). In general,

322

increased ratios of GMC to WHS resulted at progressively higher values for TPC (up to

323

88% increase), AAR and FRAP (up to 80 and 92% increase respectively, albeit not always

324

with significant differences between treatments) for all species examined (Table 4; Fig. S6,

325

Supporting Information). A similar substrate-dependent effect was noted for TPC and

326

antioxidant activities when TPOMW was added to OLV for P. eryngii. In contrast, AAR

327

and FRAP values from P. nebrodensis and P. ostreatus fruit-bodies were not equally

328

affected in this particular type of substrate despite the increased values of TPC observed at

329

high TPOMW ratios. Elongation of total serum oxidisability lag time was also positively

330

affected by GMC or TPOMW supplementation to the basal substrates, mainly in the cases

331

of P. eryngii (up to 37% increase in respect to the WHS control) and P. ostreatus (up to

332

31% increase) although no significant differences were observed among treatments (Table

333

4). Such notable effect on antioxidant activities (i.e. oxygen radical absorbance capacity)

334

was only reported in methanolic extracts deriving from P. ostreatus mushrooms cultivated

335

on selenium-enriched media.38

336

Previous studies reported either no significant differences in TPC of P. ostreatus

337

and P. pulmonarius mushrooms produced on substrates containing up to 90% TPOMW,33

338

or significantly higher TPC values in Hericium erinaceus fruit-bodies cultivated on olive

339

pruning residues.30 In the present study, TPC and antioxidant activities of P. eryngii and P.

340

nebrodensis mushrooms were markedly influenced by the type of substrate used for their

341

production, in contrast to what was observed for P. ostreatus. Therefore, such type of

342

interaction seems to mainly depend on the fungal species/strain examined. This hypothesis



343

seems to be supported by results of TPC measured in methanolic extracts of other

344

mushrooms, e.g. Agaricus species mainly originating from commercial cultivation (133 to

345

1155 mg GAE/100 g d.w.),36 or various wild and cultivated edible species (ca. 80 to 550

346

mg GAE/100 g d.w., including P. ostreatus at ca. 140 mg GAE/100 g d.w.).39

347

Ergosterol (provitamin D2) constitutes a key component of fungal and yeast cell

348

membrane, which does not exist in animal or plant cells, and is of high interest since it

349

presents hypocholesterolaemic effects and its deficiency is linked to several chronic

350

diseases.40,41 The outcome of this study evidenced a combined substrate/species dependent

351

effect for ergosterol content in mushrooms. Hence, enrichment of WHS with GMC in P.

352

ostreatus and P. eryngii, or its complete substitution with olive mill by-products in P.

353

eryngii only, resulted in significant enhancement of ergosterol, i.e. from 107 to 570

354

mg/100 g d.w. in the case of the former, and from 26 to 627 mg/100 g d.w. for the latter

355

species (Table 4). Similar effects albeit less pronounced were observed for P. nebrodensis

356

(from 338 to 583 mg/100 g d.w. in GMC-based substrates). It is noteworthy that WHS

357

supplementation by higher ratios of GMC resulted in a progressive/consistent increase of

358

ergosterol in all mushrooms, whereas the use of different ratios of olive mill by-products

359

led to high ergosterol content in mushrooms but in a strain(species)-specific manner (i.e.

360

no effect for P. eryngii, increasing and decreasing values for P. ostreatus and P.

361

nebrodensis respectively as TPOMW supplementation got higher). Ergosterol’s content in

362

commercially cultivated P. ostreatus was previously reported at 419 – 440 mg/100 g

363

d.w.,42,43 which lies within the range of values determined in this study.

364

Squalene is a polyunsaturated triterpene which functions as a precursor in the

365

metabolic pathway for the biosynthesis of ergosterol in fungal cells.43,44 Interestingly

366

enough, despite that OLV+TPOMW substrates contain much higher amounts of squalene

367

than WHS+GMC (up to 8 times more by comparing the highest respective concentrations;


Page 16 of 38

Page 17 of 38


368

Table 2) and although higher TPOMW ratios resulted at up to thirteenfold increase in

369

substrates squalene content, the ergosterol content in Pleurotus mushrooms originating

370

from those substrates did not present an analogous increase. Hence, ergosterol exhibited up

371

to twofold increase in P. ostreatus, no increase in P. eryngii and a decrease in P.

372

nebrodensis mushrooms. The mechanism by which squalene content in cultivation

373

substrates influences the concentration of ergosterol in Pleurotus fruit-bodies is not fully

374

understood. However, it seems that each species presents a different response (e.g. P.

375

nebrodensis shows limited response to pertinent increases and is negatively affected when

376

concentrations surpass a plateau), and further experimentation is needed to draw safe

377

conclusions which would help to optimize the levels of ergosterol (and consequently of

378

vitamin D content) in cultivated mushrooms.

379

In the past, polysaccharides from Pleurotus mushrooms (especially β-D-glucans

380

such as pleuran) were identified, characterized and evaluated in respect to their potential

381

prebiotic, antioxidant, antimicrobial and anticancer properties.5 However, to the best of our

382

knowledge, it is the first time that glucans content is quantified in Pleurotus spp. and then

383

comparatively evaluated among mushrooms produced on different substrates. Results

384

revealed that β-glucans accounted for the most part of total glucans, i.e. 80 – 91% in P.

385

ostreatus and P. eryngii, and 70 – 77% in P. nebrodensis depending on the substrate (Table

386

4; Fig. S6, Supporting Information). On the other hand, P. eryngii presented the highest

387

content of β-glucans among the species examined in this work (32.76 – 42.43% d.w.), the

388

respective values being among the highest ever recorded in Pleurotus or other edible

389

(cultivated and wild) mushrooms.10,45

390

As regards the effect of cultivation substrates, α-glucans content in mushrooms did

391

not present any significant difference among various substrate combinations. In contrast,

392

the addition of GMC to WHS (especially at a ratio of 1:3, w/w) led to a notable increase of



393

β-glucans content (8-23% depending on the mushroom species). A similar increase in β-

394

glucans (up to 24%) was previously reported in H. erinaceus mushrooms cultivated on

395

substrates consisting of olive pruning residues when compared to a conventional sawdust-

396

based substrate.30 On the other hand, the use of olive-mill by-products resulted at an

397

increase of up to 27% (OLV:TPOMW 3:1 vs. WHS for P. ostreatus) in β-glucans content;

398

however, in most cases the pertinent values were not significantly different. It is

399

noteworthy that under adverse conditions, fungi preserve their cell wall integrity through

400

activation of β-glucan synthase;46,47 this could explain the increased content of β-glucans in

401

Pleurotus mushrooms deriving from TPOMW/OLV and GMC-based cultivation

402

substrates, i.e. as a response of the fungus to the stress (toxicity) induced by reactive

403

oxygen species which are abundant in such plant by-products. In fact, the high content of

404

olive mill wastewater in these compounds was considered the key-factor for detecting high

405

β-glucan synthase activity in Lentinula edodes and P. ostreatus (P. eryngii was not

406

affected) mycelium obtained from this particular growth medium.48

407

In total, and as an indication of the effect that the nature of substrate could have on

408

mushroom properties, it is worth mentioning that P. eryngii fruit-bodies exhibited ca. 24

409

times higher content in ergosterol, 3 to 67 times higher content in individual phenolic

410

acids, 3 to 4 higher content in triterpenic dialcohols, 18 times higher content in oleanolic

411

acid, 8 times higher TPC, and 2 to 5 times higher antioxidant activity (assessed by three

412

different assays, AAR, FRAP and TSO) when obtained from OLV:TPOMW 1:3 medium

413

in comparison to the conventional WHS substrate. In addition, resveratrol and ursolic acid

414

were not detected in WHS-derived mushrooms whereas they were measured in appreciable

415

amount in mushrooms grown on olive mill by-products.

416

Correlations between substrate composition and mushroom properties


Page 18 of 38

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417

All cultivation media used in the main experiment were analyzed in respect to their content

418

in selected constituents (phenolic compounds, resveratrol, triterpenic compounds and

419

squalene; Table 2) to allow determination of correlations versus the respective content in

420

mushrooms and antioxidant activity (Tables 2 and 3) as well as any other possible

421

associations between precursor compounds in substrates and mushroom ingredients.

422

Hence, high correlations were assessed for TPC in substrates vs. TPC, AAR and FRAP of

423

P. eryngii and P. nebrodensis mushrooms (r = 0.919 and r = 0.965, r = 0.959 and r = 0.927,

424

and r = 0.868 and r = 0.941, p < 0.01, respectively). However, significant correlations in P.

425

ostreatus were detected only when olive mill by-products and GMC-based substrates were

426

statistically analysed in separate datasets. In this case, high correlations were also obtained

427

between TPC in substrates and TSO of Pleurotus mushrooms (r = 0.958 to 0.978, p

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