Autores
Vega-oliveros, C. (UNIVERSIDAD NACIONAL DE COLOMBIA BOGOTÁ) ; Sánchez-martínez, J.D. (UNIVERSIDAD AUTONOMA DE MADRID) ; Bueno, M. (UNIVERSIDAD AUTONOMA DE MADRID) ; Alvarez-rivera, G. (UNIVERSIDAD AUTONOMA DE MADRID) ; Morales, D. (UNIVERSIDAD AUTONOMA DE MADRID) ; Chegwin-angarita, C. (UNIVERSIDAD NACIONAL DE COLOMBIA BOGOTÁ) ; Ardila-barrantes, H. (UNIVERSIDAD NACIONAL DE COLOMBIA BOGOTÁ) ; Ibáñez, E. (UNIVERSIDAD AUTONOMA DE MADRID) ; Cifuentes, A. (UNIVERSIDAD AUTONOMA DE MADRID)
Resumo
Substrate variation can deeply affect the metabolites that fungus expresses. The
mushroom Lentinula edodes was obtained by submerged fermentation in two different
culture media. This work aims to explore the metabolite profile with high-
resolution chromatography. We cultured mycelium in two media and after that, we
analyzed extracts by fractioning them with different solvents. The analysis
employed UHPLC-qTOF-MS/MS. The annotation of metabolites allowed us to determine
that detected compounds had significant differences in the metabolite profile of
the cultured mycelia, and their correlation with the nutraceutical potential. We
found in the mycelia interesting compounds with nutraceutical reports. The results
constitute the first report of metabolomic analysis in mycelium of L. edodes
Palavras chaves
Shiitake; Nutraceutical; Metabolomics
Introdução
Among the challenges of working with fungi and yeasts is the choice of a culture
medium that favors their production and scaling and the biosynthesis of proteins
or metabolites, which are currently of great interest to the food and
pharmaceutical industries. (CHANG, 2009; GERBEC et al., 2015; HANSEN et al.,
2015). For macromycetes, substrate variation can radically affect the proteomic
or metabolic profile as a consequence of the ability of the fungus to adapt to
the environment in which it is cultivated (ARANGO et al., 2013; GREGORI;
ŠVAGELF; POHLEVEN, 2007). The mushroom Lentinula edodes was biotechnologically
obtained by submerged fermentation, or SF, in two culture media of different
compositions. In previous work, we determined that cultivation in the two media
evidenced significant differences in biomass yield and the proteomic profile.
Also, we found nutraceutical tendencies when in vitro analyses of the mycelium
extracts were applied. Such results were antioxidant capacity against oxygen
radicals (ROS) with ABTS and ORAC inhibition assays, and nitrogen radical
inhibition (RNS) with the nitroprusside assay, as well as inhibition of
Lipooxygenase (LOX) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR)
enzymes. The objective of this work is to explore the profile of medium and high
polarity metabolites in the mycelium obtained, to construct a database of
compounds that can be found in this vegetative tissue utilizing a non-targeted
search using liquid chromatography and high-resolution mass detectors, and
finally to statistically correlate the metabolites that could be involved with
the results obtained previously.
Material e métodos
Mycelium of L. edodes was cultured in submerged fermentation in two different
liquid culture media: bienestarina flour (BIE), which corresponds to a food
supplement type flour developed in Colombia containing wheat flour, soy flour,
corn starch, whole milk powder, linseed oil powder, vitamins, and minerals.
(ICBF, 2020) at 30 g/L concentration (CHEGWIN-ANGARITA; NIETO-RAMÍREZ, 2014) and
a second medium containing glucose (20 g/L), peptone and yeast extract. (0.5 g/L
each one) (GPY) (SUÁREZ-ARANGO, 2012). The mycelium obtained from these two
media was lyophilized and macerated in liquid nitrogen for subsequent extraction
by fractionating the metabolites present in the mycelium, using solvents of
different polarity such as dichloromethane (DCM), ethanol (EtOH), and water. The
extracts were subjected to the study of their composition using UHPLC-qTOF-MS/MS
according to the conditions described in (BALLESTEROS VIVAS, 2020). For the
identification, a general database was constructed, and the specific databases
for L. edodes were cited in (FUKUSHIMA-SAKUNO, 2020; KWIECIEN et al., 2015; LI
et al., 2019; TANG et al., 2020; ZHANG et al., 2020). Monoisotopic masses and
MS/MS fragmentations were reviewed and compared with those reported in NIST,
HMDB, Metlin, and MassBanck. Finally, determinations were made in triplicate and
for metabolomic studies supervised and unsupervised (p <0.05), univariate and
multivariate analyses were performed. Data obtained from LC-MS/MS metabolites
were normalized to signal abundance following a sample-centered approach by
dividing the area of each relative intensity by the total area obtained from the
sample, according to (DOWLE; WILSON; THOMAS, 2016). This study considered the
compounds present in 2 of the three biological replicates. Univariate (ANOVA)
and unsupervised multivariate (PCA) tests with supervised analysis (PLS-DA) of
the data obtained were applied to determine differences between samples and
correlations between biological activity and metabolites detected in the
analyses. Statistical analyses were performed using the online application
Metaboanalyst (https://www.metaboanalyst.ca/).
Resultado e discussão
We confirm that metabolite composition variety is determined by the polarity of
every extract analyzed and the nature of each liquid media due to the annotation
of the metabolites. We could make a tentative identification of a total of 183
signs corresponding to compounds and their adducts for the extracts from the
mycelium grown in the two media. We established by PCA analysis statistical
differences between the compounds detected for the three different fractions
analyzed from the mycelium obtained in the two substrates. In this first part,
we detected sulfur-type compounds as compounds strongly related to this fungal
specie, but in smaller amounts than what has been reported for extracts of
fructifications (mushrooms) of L. edodes. Also, we detected phenolic-type
compounds, a flavone, fatty acids, sterols, monosaccharides, and amino acids
previously reported, and potentially lactone-type compounds that have not been
reported. Differentially, the metabolites detected along with adducts in the
mycelium in GPY were 181 and BIE 190, and a greater variety of fatty acid-type
compounds were detected in the mycelium obtained in BIE as well as a higher
detection of sterols in the mycelium obtained in GPY.
According to previous results, possible correlations were established between
some of the detected compounds with biological activities studied in the same
fungus extracts, according to the correlation studies performed by PLS-DA. In
the antioxidant activity (inhibition of ROS and RNS) the correlated compounds
were nitrophenols, benzenoids, polyols, tricarboxylic acid derivatives, some
amminoacids, secondary alcohols, dipeptides, pyrimidine nucleosides, fatty
acids, flavonoids, vitamin D derivative, 1,2-dithiol-3-thiones, hydroxy acids,
polyketides, naphthofurans, phenylpropanes, butyrolactones, shikimic acid, amino
alcohols, glycosphingolipids, fatty acylglycosides and Laurolactam-type
compound. Enzyme inhibition involved various compounds in LOX inhibition related
to fatty acyls, o-glycosyl compounds, prostaglandins, hydroxyeicosatrienoic
acids, alpha-, omega-dicarboxylic acid, fatty amides, and sterol lipids. HMGCR
inhibition correlated with amino acids and derivatives, diglycerides, carboxylic
acids, fatty acids, tricarboxylic acids and derivatives, fatty acylglycosides,
secondary alcohols, ketoacids, ketone alcohols, again the laurolactam type
compound and vitamin D and derivatives. Some of them shared with those
correlated with antioxidant activity.
On the other hand, the annotation of the detected compounds allowed to establish
significant differences between the compounds present in the mycelium that may
be related to or influence the nutraceutical potential of this biomass, finding
compounds previously reported to have biological activity in this species, as
well as others not reported but that have been reported as compounds with
nutraceutical character, mainly in the mycelium obtained in GPY (VEGA OLIVEROS,
2021).
Conclusões
The results reported in this work constitute the first report of metabolomic
analysis in vegetative tissue (mycelium) of L. edodes obtained in submerged
fermentation and can be used to perform statistical correlations to investigate
groups of compounds that may have nutraceutical potential.
Agradecimentos
Minciencias – Conv. 785 Doctorados Nacionales 2017, FoodOmics lab – PhD. Alejandro
Cifuentes and Department of Production and Characterization of Novel Foods – PhD.
Cristina Soler-Rivas, CIAL, Universidad Autónoma de Madrid
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