Journal of Food Composition and Analysis 86 (2020) 103375

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Original Research Article

Investigation of biologically active amines in some selected edible T

mushrooms
Guilherme C.L. Reisa, Flávia B. Custódioa, Bruno G. Botelhob, Letícia R. Guidic,
Maria Beatriz A. Gloriaa,d,*
a
LBqA - Laboratório de Bioquímica de Alimentos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, 31270-
901, Brazil
b
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, 31270-901, Brazil
c
Faculdade de Engenharia Química, Universidade Federal de Uberlândia, Campus Avançado Patos de Minas, Rua Padre Pavoni 290, Patos de Minas, MG, CEP 38701-
002, Brazil
d
LCQ – Laboratório de Controle de Qualidade, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Antônio Carlos 6627, Belo Horizonte, MG, 31270-901,
Brazil

ARTICLE INFO ABSTRACT

Chemical compounds studied in this article: Mushrooms are highly valued due to nutritional and functional properties as well as small environmental
Spermidine (PubChem CID: 9539) footprint. However, scarce information is available regarding amines in commercial products. The objective of
Agmatine (PubChem CID: 2794990) this study was to investigate the levels of bioactive amines in eight fresh edible commercial mushrooms species.
Putrescine (PubChem CID: 9532) An ion-pair HPLC method with post-column derivatization with o-phthalaldehyde and fluorescence detection
Cadaverine (PubChem CID: 80282)
was fit for the purpose. Seven out of nine amines were present and levels varied among species. Spermidine was
Histamine (PubChem CID: 5818)
ubiquitous to mushrooms, with highest content in Black Shimeji (12.4 mg/100 g). The levels of spermidine in
Tyramine (PubChem CID: 66449)
Phenylethylamine (PubChem CID: 9075) mushrooms classify them as high polyamines sources, which is valued due to its association with growth, health
Tryptamine (PubChem CID: 67652) promotion and antioxidant properties. Agmatine was present in all Pleurotus. Tyramine, tryptamine and phe-
Serotonin (PubChem CID: 160436) nylethylamine were detected in some species; the levels of cadaverine and putrescine were discrete. A four-
principal component model explained 99.4% of the variance and it was able to separate Pleurotus spp. (White
Keywords: shimeji, Hiratake, Black shimeji and Salmon) from Agaricus bisporus (Champignon and Portobello) and Lentinula
Principal component analysis edodes (Shitake). Hierarchical cluster analysis confirmed the potential of using the occurrence and levels of
Hierarchical cluster analysis amines to separate some mushroom species.
Spermidine
Agmatine
Polyamines
Biogenic amines

1. Introduction environmental footprint, as they grow from agricultural and forest

Edible mushrooms are widely consumed in many countries due to
nutritional value, low calories and specific aroma and texture, a deli-
cacy (Kalač, 2013; Feeney et al., 2014). The consumption of mushrooms
has increased throughout the world. According to the Food and Agri-
culture Organization (FAO) of the United Nations, cultivated mushroom
worldwide production increased from 5.91 in 2007 to 10.24 million
tons in 2017, which represents an increment higher than 73 % in ten
years (FAOSTAT, 2019). Besides, several types of mushrooms are
available, both cultivated/commercial and wild noncommercial edible
species. In addition to the economic value, mushrooms have a small
wastes and require relatively little water or land. Furthermore, they can
be used as agents of environmental management, by using natural re-
sources in a less destructive way for the biosphere and by promoting
sustainable development in all ecosystems (Donnini et al., 2013; Feeney
et al., 2014). According to the Associação Nacional dos Produtores de
Cogumelos – ANPC (Mushrooms Producers National Association), it is
estimated that Brazil produces over 12 thousand tons of fresh mush-
rooms every year (AOAC (Association of Official Analytical Chemists,
2012; ANPC (Associação Nacional dos Produtores de Cogumelos),
2019)). The most popular edible mushroom species available are
Agaricus bisporus, Lentinula edodes and some Pleurotus species.

⁎
Corresponding author.
E-mail address: mbeatriz@ufmg.br (M.B.A. Gloria).

https://doi.org/10.1016/j.jfca.2019.103375
Received 6 July 2019; Received in revised form 3 November 2019; Accepted 11 November 2019
Available online 12 November 2019
0889-1575/ © 2019 Elsevier Inc. All rights reserved.
G.C.L. Reis, et al. Journal of Food Composition and Analysis 86 (2020) 103375

Table 1
Types of edible mushroom included in this study and their respective species, common names and illustrative photograph.
Specie/Mushroom Other common names Picture

Agaricus bisporus
Champignon (A) Button, Paris, Table or cultivated
mushroom
Portobello (B) Field mushroom
Lentinula edodes
Shiitake (C) Oat mushroom
Pleurotus spp.
Black Shimeji (D) Oyster mushroom
White Shimeji (E) Florida mushroom
Hiratake (F)
Salmon (G) Pink oyster mushroom
Eryngii (H)

Due to their chemical composition, edible mushrooms constitute a
food of nutritional value. They have low calories (low fat contents) and
high contents of minerals (K), essential amino acids, vitamins (provi-
tamin D2, vitamin B12) and fiber (Roupas et al., 2012; Kalač, 2013;
Feeney et al., 2014; Manninen et al., 2018). They are important sources
of polysaccharides with immunomodulating properties. In addition,
mushrooms contain several natural phytochemicals with a wide range
of positive health effects and medicinal benefits (Roupas et al., 2012).
Some preclinical and clinical studies suggest positive impacts of
mushrooms on brain health and cognition, weight management, oral
health, constipation, diabetes, anti-arthritic and anti-viral agents
(Roupas et al., 2012; Feeney et al., 2014; Pop et al., 2018). Preliminary
evidence suggests that mushrooms may support healthy immune and
inflammatory responses through interaction with gut microbiota, en-
hancing the development of adaptive immunity and improved immune
cell functionality (Roupas et al., 2012; Feeney et al., 2014).
Many of the immunomodulation effects attributed to mushrooms
are due to polysaccharide, either β-glucans or polysaccharide-protein
complexes (Roupas et al., 2012). However, part of the therapeutic
properties is related to their inhibition of oxidative stress (Yang et al.,
2002; Elmastas et al., 2007; Silva and Jorge, 2014; Janjušević et al.,
2017; Pop et al., 2018). The antioxidant activity of mushrooms may be
attributed to the presence of several components, such as phenolics
(Janjušević et al., 2017; Pop et al., 2018) and bioactive amines (Rider
et al., 2007; Toro-Funes et al., 2013).
Reports on the occurrence and levels of bioactive amines in mush-
room are scarce, especially commercial ones. Turkish wild-growing
mushrooms were reported to be high sources of spermidine, followed
by putrescine, tyramine, tryptamine and phenylethylamine (Dadáková
et al., 2009). Commercial mushrooms have been analyzed mainly for
the presence of polyamines (Okamoto et al., 1997; Cipolla et al., 2007;
Nishibori et al., 2007). Only a few studies investigated additional
amines in commercial mushroom including putrescine (Okamoto et al.,
1997), putrescine and cadaverine (Yamamoto et al., 1982) and tryp-
tamine, 2-phenylethylamine, histamine and tyramine (Yen, 1992).
In this context the profile and levels of bioactive amines in eight
fresh edible mushrooms species commercialized in Brazil were analyzed
for the first time. A HPLC method for the determination of nine amines
in mushroom was validated. The potential functional properties of the
mushrooms due to bioactive amines were described. Furthermore, the
possibility of using amines as criterion for clustering different mush-
rooms species was investigated by multivariate analysis (principal
component analysis - PCA and hierarchical and clustering analysis -
HCA).
2. Material and methods
2.1. Reagents and solvents
The reagents used were of analytical grade, except HPLC solvents
(acetonitrile and methanol) which were chromatographic grade. For
HPLC analysis, the organic and aqueous solvents were filtered through
0.45 μm pore size HAWP and HVWP membranes, respectively
(Millipore Corp., Milford, MA, USA). Deionized water was obtained
from Milli-Q Plus System (Millipore Corp., Milford, MA, USA).
Spermine (SPM) tetrahydrochloride, spermidine (SPD) trihy-
drochloride, putrescine (PUT) dihydrochloride, cadaverine (CAD) di-
hydrochloride, tyramine (TYM) hydrochloride, histamine (HIM) dihy-
drochloride, agmatine (AGM) sulphate, serotonin (SRT) hydrochloride,
2-phenylethylamine (PHM) hydrochloride, tryptamine (TRM) free base,
and o-phthalaldehyde (OPA) were purchased from Sigma Chemical Co.
(St. Louis, MO, USA).
2.2. Samples
Fresh mushrooms were purchased at commercial maturity from
producers within the region of Belo Horizonte, state of Minas Gerais,
Brazil, among them, Pleurotus spp. – Black shimeji, White shimeji,
Hiratake, Salmon and Eryngii; Agaricus bisporus – Champignon and
Portobello; and Lentinula edodes – Shiitake (Table 1). Different lots of
each mushroom species (≥ 200 g) were obtained, except for Eryngii
which, because of its limited availability, had only two lots collected.
All the lots were obtained from the same producer, which is the major
provider for commercial use, immediately after harvest. The fresh
mushrooms were homogenized, grated and analyzed immediately for
moisture (AOAC, 2012) and bioactive amines contents.
2.3. Method for the determination of bioactive amines
Nine free bioactive amines (spermidine, agmatine, putrescine, ca-
daverine, tyramine, tryptamine, phenylethylamine, histamine and ser-
otonin) were determined according to the method described by
Bandeira et al. (2012). Briefly, the amines were extracted from 3 g fresh
mushrooms with 5 % trichloroacetic acid. The samples were agitated
for 5 min on a shaker (250 rpm) and centrifuged at 8422 g for 20 min at
4 °C. These steps were repeated two more times. The supernatants were
combined, filtered through qualitative filter paper and through HAWP
membrane (0.45 μm pore size, Millipore Corp., Milford, MA, USA) and
used for analysis.

2
G.C.L. Reis, et al. Journal of Food Composition and Analysis 86 (2020) 103375

Table 2
Fitness parameters (linearity, accuracy, precision and limits of detection and quantification) during method validation for the analysis of bioactive amines in
mushrooms.
Analytes Calibration curves Accuracy Precision (%) Limits (mg/100 g)

Slope Intercept R2 (%) CVr CVR LOD LOQ

Spermidine 3.41 × 105 −22669 0.9993 104.4 5.7 7.8 0.03 0.10
Agmatine 2.85 × 105 6009 0.9995 99.5 4.4 7.2 0.03 0.10
Putrescine 4.50 × 105 55661 0.9992 100.7 6.5 8.9 0.03 0.10
Cadaverine 5.41 × 105 45657 0.9995 94.9 6.3 8.7 0.03 0.10
Tyramine 2.02 × 105 −29496 0.9966 101.3 5.9 7.9 0.03 0.10
Phenylethylamine 3.26 × 105 20243 0.9990 101.3 4.7 6.7 0.03 0.11
Tryptamine 2.42 × 105 115250 0.9990 87.2 5.9 9.5 0.03 0.11
Histamine 2.99 × 105 −13880 0.9997 101.4 4.7 7.3 0.03 0.10
Serotonin 2.51 × 105 −12062 0.9975 70.7 5.8 15.3 0.03 0.10

CVr = intraday coeficient of variation - repeatability; CVR = interday coeficient of reproducibility.

The amines were separated by ion-pair HPLC and quantified, after
post column derivatization with o-phthalaldehyde, fluorometrically at
340 nm excitation and 450 nm emission. A Shimadzu LC-10AD with SIL-
20AHT automatic injector (Shimadzu, Kyoto, Japan) connected to a RF-
510 AXL spectrofluorometric detector and to a CBM-20A controller was
used. The column and pre-column used were Novapack® C18
(3.6 × 300 mm, 4 μm) (Waters, Milford, MA, USA). Two mobile phases
were used: (A) sodium acetate buffer (pH 4.9) with 15 mmol/L sodium
octanosulphonate and (B) acetonitrile, at a flow rate of 0.8 mL/min, in a
gradient elution: initial to 21.0 min/3–20 % B; 21.0–22.0 min/20–5 % B;
22.0–25.0 min/5 % B; 25.0–40.0 min/5-24 % B; 40.0–45.00 min/24 % B;
45.0–50.0 min/24–35 % B, 50.0–51.0 min/ 35.3 % B and further re-
equilibration at initial conditions for another 9.0 min, in a total cycle
time of 60.0 min until the next injection. The injection volume was 10 μL.
2.4. Intralaboratory validation of the method
The fitness of the method described by Bandeira et al. (2012) was
evaluated for the mushroom matrix according to the following para-
meters: specificity, linearity of calibration curves, accuracy, precision
and limits of detection and quantification (Thompson et al., 2002).
Agaricus bisporus mushrooms were chosen for the validation process.
The specificity of the method was determined by injection of 20 dif-
ferent mushroom extracts. The existence of any interference (possible
peaks) that could affect detection in the range of retention time of the
target analytes was investigated. The samples were spiked with nine
amines standards. The samples were also spiked with each single
standard to confirm the identity of each peak. The calibration curves
were constructed by using different concentrations of all amines (0.1,
0.2, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0 μg/mL) injected randomly. Re-
peatability and reproducibility were evaluated by analysis of spiked
samples (4 mg/100 g) in five replicates and by three different analysts.
Accuracy was calculated as the mean concentration found divided by
the fortification level and multiplied by 100. Repeatability was estab-
lished through determination of the coefficient of variation. The ana-
lyses were carried out at three different days with three different ana-
lysts to evaluate reproducibility. Mean concentration, standard
deviation and coefficient of variation were calculated for the spiked
samples of each analyst. The limit of detection was the lowest con-
centration of the analyte corresponding to three times the signal-to-
noise ratio. The limit of quantification was the lowest concentration of
the analyte that could be determined with acceptable accuracy and
precision. It was considered the first analyte concentration at the cali-
bration curve (Thompson et al., 2002).
2.5. Statistical analysis
The results were submitted to Shapiro Wilk test for normality and
Levene’s test for homoscedasticity. Then, the data was submitted to
analysis of variance and the means were compared by the Tukey test at
5 % probability (Granato et al., 2014) using the Past 3.19 software
(UIO, Oslo, Norway).
2.6. Multivariate analysis
Two multivariate exploratory techniques – Principal Component
Analysis (PCA) and Hierarchical Cluster Analysis (HCA) – were applied
for the characterization of mushrooms in relation to the profile and
level of bioactive amines (Granato et al., 2018a). PCA was used to
project multivariate data onto a smaller dimensional space, without
affecting the relation between samples. Consequently, relevant in-
formation was separated and amplified, making them more perceivable
by visual inspection, and allowing the discovery, visualization and in-
terpretation of differences between variables, the relation between
samples, and the identification of outliers. HCA was used as a tool for
dimensionality reduction, by clustering the objects by similarity,
minimizing intragroup variance and maximizing intergroup variance.
HCA results were presented in bidimensional plots (dendrograms), re-
presenting the hierarchical structure of the data, in which the length of
each branch represents similarity between samples (Otto, 2017;
Granato et al., 2018b). Both PCA and HCA models were built using PLS
ToolBox 6.5 (The Eigenvector Technologies, Manson, WA, USA) and
MATLAB software, version 2010a (The MathWorks, Natick, MA, USA).
3. Results and discussion
3.1. Method validation
The fitness of the method for the quantification of bioactive amine
in mushrooms is presented in Table 2. Regarding linearity, the cali-
bration curves had coefficients of determination (R2) equal or higher
than 0.9966 indicating a high correlation between the concentration
and the signal area. By visual inspection, good distribution of the re-
sidues was observed, which was confirmed by the tests of independence
(Durbin-Watson) (p-value > 0.05) and homoscedasticity (Breuch-
Pagan) (p-value > 0.05). Thus, the linear equations for the calibration
curves are valid for the quantification of each of the nine amines in
mushrooms in the range of 0.096–12.0 mg/100 g. The accuracy of the
method was evaluated by the recovery of the analytes in spiked sam-
ples, and ranged from 70.7 % (serotonin) to 104.4 % (spermidine). The
intraday precision (repeatability) of the method ranged from 4.7 %
(histamine) to 6.5 % (putrescine) and the interday precision (re-
producibility) ranged from 6.7 % (phenylethylamine) to 15.3 % (ser-
otonin). Therefore, the method was accurate and precise for the ana-
lysis of nine amines (spermidine, agmatine, putrescine, cadaverine,
tyramine, phenylethylamine and tryptamine) in mushrooms. The limits
of detection ranged from 0.029 to 0.032 mg/100 g and the limits of
quantification ranged from 0.096 to 0.105 mg/100 g (Table 2), which

3
G.C.L. Reis, et al. Journal of Food Composition and Analysis 86 (2020) 103375

Table 3 agmatine). However, White shimeji, Hiratake and Salmon had seven of
Occurrence and total levels of free bioactive amines in different types of edible the nine amines investigated.
mushroom. The occurrence of spermidine in mushroom, as well as in all living
Mushroom (n) Occurrence of amines* (%) Total levels cells, has been reported in the literature for wild mushrooms (Okamoto
(mg/100 g) et al., 1997; Dadáková et al., 2009; Kalač, 2013) and its presence is
SPD AGM PUT CAD TYM TRM PHM associated with several relevant roles on cellular metabolism and
growth. Agmatine was previously detected at very low levels in hon-
Agaricus
bisporus shimeji mushroom (Okamoto et al., 1997). Its presence in mushrooms
Champignon 100 7.7 23.1 9.97 ± 3.75c suggests an additional route for the synthesis of polyamines via arginine
(13) (Bandeira et al., 2012). Although putrescine is an obligatory precursor
Portobello 100 8.43 ± 2.00c
of polyamines, it was only detected in Pleurotus spp. Therefore, despite
(13)
Lentinus edodes
being relevant in the formation of polyamines, it does not seem to ac-
Shiitake (14) 100 7.1 7.1 7.1 7.77 ± 3.70c cumulate in some types of mushroom, or it is present at low con-
Pleurotus spp. centrations, below the limit of detection of the method. Unlike the
Black shimeji 100 100 21.4 20.3 ± 4.57b commercial mushrooms currently studied, putrescine was the pre-
(14)
dominant amine in wild-growing mushrooms (Dadáková et al., 2009).
White shimeji 100 100 53.8 23.1 30.8 100 84.6 20.3 ± 5.72b
(13) Tyramine, tryptamine and phenylethylamine may be inherent to some
Hiratake (12) 100 100 33.3 16.7 50 16.7 8.3 23.9 ± 4.34b mushroom species, as secondary metabolites, or they may result from
Salmon (14) 100 100 57.1 57.1 14.3 92.9 21.4 36.6 ± 8.49a the decarboxylation of the precursor amino acids (tyrosine, tryptophan
Eryngii (2) 100 100 10.2 ± 1.73c
and phenylalanine, respectively). They were also detected in wild-
growing mushrooms (Dadáková et al., 2009).
(n) = number of mushroom lots.
* SPD = spermidine; AGM = agmatine; PUT = putrescine; CAD = cadaverine; The presence of cadaverine in mushroom was reported by Okamoto
TYM = tyramine; TRM = tryptamine; PHM = phenylethylamine. et al. (1997); however, they failed to separate cadaverine from hista-
Histamine and serotonin were not detected in any mushroom. mine and quantified both of them (histamine/cadaverine) together.
Total levels of amines with different letters in the same column are significantly Histamine was not detected in the mushrooms analyzed in this work,
different (Tukey test, p ≤ 0.05). similar to the results found by Dadáková et al. (2009) for wild-growing
mushrooms.
confirms the good sensitivity of the method. These results indicate that
the method is fit for the analysis of amines in mushroom. The use of on
line derivatization and fluorescence detection improves selectivity and 3.3. Levels of amines in the mushrooms
sensitivity of the method, which certainly contributed to the success of
the method (Önal, 2013). The mean total levels of free bioactive amines in the mushrooms
varied from 7.77 up to 36.6 mg/100 g (Table 3), with the highest levels
found in Salmon. Lower contents were observed in Shiitake (L. edodes),
3.2. Occurrence of amines in the mushrooms Portobello and Champignon (≤10 mg/100 g) – A. bisporus, whereas
intermediate levels (from 10 to 25 mg/100 g) were found in Eryngii,
Among the nine amines investigated, seven were detected in dif- Black shimeji, White shimeji and Hiratake (Pleurotus spp.).
ferent mushroom species but in a distinct way (Table 3). Histamine and Spermidine contributed the most (≥ 98.6 %) in Portobello and
serotonin were not detected in any of the mushroom species studied. Champignon (Agaricus bisporus) and also in Shiitake (Lentinula edodes,
Spermidine was present in all mushroom species, suggesting that it is 92.8 %); however, in Pleurotus spp., the contribution varied from 31.4
inherent to mushrooms. Agmatine was detected in six out of the eight to 77%. Agmatine contributed with 23–57% to the total levels in
mushroom species; it was present in every Pleurotus spp. mushrooms Pleurotus spp. and with 6.3 % to the total levels in Shitake. Indeed, in
investigated, suggesting that agmatine, along with spermidine, can be some Pleurotus spp. mushrooms, the contribution of agmatine was
inherent to Pleurotus spp. Tryptamine was also present in 100% of the higher compared to spermidine, e.g. White shimeji, Hiratake and
samples of White shimeji, suggesting that it is ubiquitous to this Salmon. The other amines contributed with less than 5 %, except for
mushroom specie. putrescine in Salmon (6.8 %) and tryptamine in White shimeji (6.7 %).
It is interesting to observe that Portobello had only one amine The concentration of spermidine in the mushrooms ranged from
(spermidine), followed by Eryngii which had two (spermidine and 3.23 to 17.2 mg/100 g (Table 4). There was significant difference on the

Table 4
Levels of free bioactive amines in different types of fresh edible mushrooms.
Mushrooms Dry matter (g/100 g) Range (mean levels) of amines (mg/100 g)

Spermidine Agmatine Putrescine Cadaverine Tyramine Tryptamine Phenyl-ethylamine

A. bisporus
Champignon 9.68 4.41-16.5 (9.83)ab nd (0.00)c nd (0.00)b nd (0.00)b nd-0.10 (0.01)a nd-0.77 (0.13)c nd (0.00)b
Portobello 8.98 6.24-12.6 (9.72)ab nd (0.00)c nd (0.00)b nd (0.00)b nd (0.00) a nd (0.00)c nd (0.00)b
L. edodes
Shitake 9.54 5.18-12.6 (6.26)b nd-6.86 (0.49)c nd (0.00)b nd (0.00)b nd-0.28 (0.02)a nd-0.63 (0.05)c nd (0.00)b
Pleurotus spp.
Black Shimeji 9.40 6.90-15.7 (12.4)a 1.41-13.4 (7.77)b nd (0.00)b nd (0.00)b nd (0.00)a nd-0.63 (0.08)c nd (0.00)b
White Shimeji 9.66 3.23-11.5 (7.00)b 2.78-13.7 (9.91)b nd-4.53 (0.93)ab nd-0.83 (0.13)ab nd-0.70 (0.14)a 0.36-2.73 (1.37)a nd-1.91 (0.84)a
Hiratake 10.14 4.13-14.2 (9.21)ab 6.15-15.9 (12.7)b nd-3.74 (0.62)b nd-2.16 (0.30)ab nd-5.98 (0.88)a nd-1.23 (0.18)c nd-0.64 (0.05)b
Salmon 9.66 5.68-17.2 (11.5)a 12.6-34.1 (20.9)a nd-10.4 (2.48)a nd-2.72 (0.59)a nd-2.09 (0.29)a 0.32-1.85 (0.82)b nd-0.39 (0.06)b
Eryngii 11.57 7.13-8.62 (7.87)ab 1.87-2.84 (2.36)c nd (0.00) nd (0.00)b nd (0.00)a nd (0.00) nd (0.00)b

Mean values were calculated using zero as nd (not detected, < 0.03 mg/100 g).
Mean values with different letters in the same column are significantly different (Tukey test, p ≤ 0.05).

4
G.C.L. Reis, et al.
Fig. 1. Scatter plot [PC1 and PC2 (a), PC3 and PC4 (b)] and PC loadings [PC1-PC2 (c), and PC3-PC4 (d)] obtained for the mean levels of bioactive amines of each
edible mushroom by Principal Component Analysis.
levels of spermidine among mushrooms, with the highest levels in Black
himeji (12.4 mg/100 g) and the lowest in White shimeji (7.00 mg/
100 g). Based on the classification proposed by Kalač (2014), the
mushrooms analyzed in this study can be considered as high (> 1 mg/
100 g) or very high (> 10 mg/100 g) sources of polyamines. Such
polyamines levels can provide various health benefits, including pro-
motion of intestinal health (Kalač, 2014; Ramani et al., 2014; Rogers
et al., 2015), development of the immune system, wound healing, anti-
inflammatory and antioxidant agents and cardioprotective effects
(Kalač, 2014; Ramani et al., 2014; De Cabo and Navas, 2016; Handa
et al., 2018; Sharma et al., 2018). In spite of this, polyamines should be
avoided by individuals with cancer as they may increase tumor growth
(Kalač, 2014; Ramani et al., 2014).
Higher mean levels of agmatine (20.9 mg/100 g) were found in
Salmon; followed by the other Pleurotus spp. mushrooms; and, them, by
Shitake. Agmatine is formed from the decarboxylation of arginine.
Recently, agmatine has been associated with beneficial health effects,
among them, neuroprotection in the central nervous system, in mental
illness, in depression and schizophrenia (Laube and Bernstein, 2017).
With respect to putrescine, even though it is an obligatory precursor
of the polyamines, it was not detected in Agaricus bisporus and in
Lentinula edodes. It was found only in three of the five Pleurotus spp.
mushrooms. Higher mean levels were found in Salmon and White shi-
meji. These levels are lower compared to the values reported for wild-
growing mushrooms (Dadáková et al., 2009). In a similar way, cada-
verine levels were low (≤ 2.72 mg/100 g) and it was only detected in
the same mushrooms which contained putrescine. It is well known that
both putrescine and cadaverine can contribute with putrefactive flavor
to foods; however, the levels found are 78 % lower than the threshold
values reported for putrescine and cadaverine – 109 mg/kg (Wang
et al., 1975).
Tyramine was detected in all mushroom species, except Portobello
and Black shimeji. It was not present in all the lots of mushrooms
analyzed, which suggests that its formation is affected by extrinsic
factors during production, including stress and substrate composition.
No significant difference was found among mean levels for different
5
Journal of Food Composition and Analysis 86 (2020) 103375
mushroom species. Tryptamine was detected in most mushroom spe-
cies, except for Portobelo and Eryngii. Higher mean levels (p < 0.05)
were found in White shimeji (1.37 mg/100 g), followed by Salmon
(0.82 mg/100 g) and, then, by the other types (0.05 – 0.18 mg/100 g).
All analyzed lots of White shimeji and Salmon contained tryptamine,
which could suggest that this amine is inherent to these mushrooms.
Phenylethylamine was detected only in Pleurotus spp., except for Black
shimeji and Eryngii. Levels were lower than 1.91 mg/100 g, with
highest levels in White shimeji. Tyramine, tryptamine and pheny-
lethylamine were also detected at similarly low levels in wild-growing
mushrooms. They were detected in commercial mushrooms for the first
time.
Tyramine and phenylethylamine are neuroactive compounds. At
low concentrations, they can exert important roles such as neuromo-
dulation and mood-lifting effects. However, at high concentrations,
tyramine and tryptamine can cause adverse effects to human health and
are, therefore, of food safety concern. Hypertension and symptoms such
as headache, vomiting, perspiration, pupil dilatation and migraine have
been described (EFSA, 2011). There is still limited information on the
safe levels of tryptamine. As for tyramine, the levels in mushrooms were
≤ 5.98 mg/100 g, which are below the no adverse health effect level
established for healthy individuals (600 mg per meal); but the con-
sumption of Hiratake could cause adverse effects to individuals taking
classical monoamine oxidase inhibitor (MAOI) drugs (NOAEL =6 mg
per meal) (EFSA, 2011).
3.4. Multivariate analysis
Multivariate analysis of autoscaled data indicated that a four-prin-
cipal component (PC) model explained 99.4 % of the variance. PC1
explained 59.4 % of the variance (Fig. 1a) and it separated White
Shimeji, Hiratake and Salmon mushrooms (positive PC1 values) from
the other mushrooms. According to PC1 loadings (Fig. 1c), all of the
amines contributed to this differentiation (positive PC1 values), but
with various intensities. Putrescine, cadaverine, agmatine and total
amines content had a higher impact than tyramine, spermidine and
G.C.L. Reis, et al.
Fig. 2. Dendrogram obtained by Classical Cluster Analysis for the mean of
bioactive amines for each edible mushroom.
phenylethylamine. These three mushrooms (White Shimeji, Hiratake
and Salmon) belong to the same genus – Pleurotus spp. Eryngii and Black
shimeji also belong to this genus, but they differed from the others as
most of the biogenic amines were not detected.
PC2 explained 24.4 % of the variance and it basically separated
White Shimeji (positive values) from all the other types of mushrooms
(Fig. 1a), mainly because of the concentrations of phenylethylamine
and tryptamine – positive values, and spermidine – negative values
(Fig. 1c). In fact, according to Table 2, White Shimeji had the highest
concentrations of phenylethylamine and tryptamine (1.37 and 0.84 mg/
100 g, respectively) and the lowest spermidine concentration (7.00 mg/
100 g).
PC3 explained 11.0 % of the variance and it separated Hiratake
from all the other mushrooms (Fig. 1b), as it had the highest con-
centration of tyramine (0.88 mg/100 g) (Fig. 1d). PC4 explained 4.55 %
of the variance, but no relevant information could be obtained from it
(Figs. 1b and 1d).
The HCA dendrogram (Fig. 2), built using the Ward’s method, se-
parated into clusters the different mushroom species. Portobello and
Champignon, both from Agaricus bisporus showed no distance between
its cluster’s centers, which indicates that their amines’ profiles and
contents are very similar., Therefore, it is an efficient tool for the
characterization of this specie. White Shimeji, Salmon, Black Shimeji
and Hiratake mushrooms from Pleurotus spp. were also clustered to-
gether, but with a higher weighted distance, which indicates that this
group presented a great internal variability in amines’ profile and
content, as detailed in Tables 3 and 4. Even though, the HCA was
capable of clustering this Pleurotus species together. Eryngii, another
member of the Pleurotus genus, was similar to Shitake, that belongs to
Lentinula edodes. This clustering is not surprising, once both mushrooms
presented only SPD and AGM at detectable levels for most samples. But,
since the number of Eryngii lots analyzed was too small, further studies
are needed. Therefore, multivariate analysis using bioactive amines’
contents can be a potential tool in the discrimination and authenticity
determination of mushroom species.
4. Conclusions
The ion pair reverse phase liquid chromatographic method followed
by post column derivatization with o-phtalaldehyde and fluorescence
detection was fit for the analysis of bioactive amines in mushroom.
Biologically active amines were detected in fresh edible mushrooms
species analyzed, among them spermidine, agmatine, putrescine, ca-
daverine, tyramine, tryptamine and phenylethylamine. Spermidine was
the prevalent amine followed by agmatine, tryptamine and tyramine.
Putrescine, cadaverine and phenylethylamine were found only in a few
Pleurotus spp mushrooms. Histamine and serotonin were not detected in
6
Journal of Food Composition and Analysis 86 (2020) 103375
any analyzed mushroom. Spermidine was ubiquitous to the mush-
rooms, which is relevant due to its association with growth and health
promotion and antioxidant properties. Agmatine was inherent to
Pleurotus spp. and its presence is relevant as another route for the for-
mation of polyamines. Furthermore, agmatine has been associated with
beneficial health effects. The levels of spermidine found in mushrooms
classify them as high sources of polyamines. The aromatic vasocon-
strictor amines tyramine, tryptamine and phenylethylamine were also
detected in some mushrooms. The levels of cadaverine and putrescine
were discrete. Multivariate analysis based on bioactive amines contents
was effective in showing differences among different types of mush-
rooms. Thus, the chemometric approach in the analysis of bioactive
amines is a promising tool for mushrooms identity.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
The authors acknowledge Coordenação de Pessoal de Nível Superior
– CAPES, Conselho Nacional de Desenvolvimento Científico e
Tecnológico – CNPq (Brasília, DF, Brazil) and Fundação de Amparo a
Pesquisa do Estado de Minas Gerais – FAPEMIG (Belo Horizonte, MG,
Brazil) for the financial support.
References
ANPC (Associação Nacional dos Produtores de Cogumelos) (2019). Retrieved May 14,
2019 from: https://www.anpccogumelos.org/cogumelos.
AOAC (Association of Official Analytical Chemists), 2012. 19th ed. Official Methods of
Analysis v. 1. AOAC, Maryland, pp. 3.1 meth. 931.04.
Bandeira, C.M., Evangelista, W.P., Gloria, M.B.A., 2012. Bioactive amines in fresh, canned
and dried sweet corn, embryo and endosperm and germinated corn. Food Chem. 131,
1355–1359.
Cipolla, B.G., Havouis, R., Moulinoux, J.P., 2007. Polyamine contents in current foods: a
basis for polyamine reduced diet and a study of its long-term observance and toler-
ance in prostate carcinoma patients. Amino Acids 33, 203–212.
Dadáková, E., Nova, T.P., Kalač, P., 2009. Content of biogenic amines and polyamines in
some species of European wild-growing edible mushrooms. Eur. Food Res. Technol.
230, 163–171.
De Cabo, R., Navas, P., 2016. Spermidine to the rescue for an aging heart. Nat. Med. 22
(12), 1389–1390.
Donnini, D., Gargano, M.L., Perini, C., Savino, E., Murat, C., Di Piazza, S., et al., 2013.
Wild and cultivated mushrooms as a model of sustainable development. Plant Biosyst.
14 (1), 226–236.
EFSA (European Food Safety Authority), 2011. Scientific Opinion on risk based control of
biogenic amine formation in fermented foods. EFSA J. 9, 1–93.
Elmastas, M., Isildak, O., Turkekul, I., Temur, N., 2007. Determination of antioxidant
activity and antioxidant compounds in wild edible mushrooms. J. Food Compos.
Anal. 20 (3–4), 337–345.
FAOSTAT. Food and Agriculture Organization of the United Nations. (2019). Retrieved
May 14, 2019 from: http://www.fao.org/faostat/en/#data/QC.
Feeney, M.J., Dwyer, J., Hasler-Lewis, C.M., Milner, J.A., Noakes, M., Rowe, S., Wu, D.,
2014. Mushrooms and health summit proceedings. J. Nutr. 144 (7), 1128–1136.
Granato, D., Calado, V.M.A., Jarvis, B., 2014. Observations on the use of statistical
methods in Food Science and Technology. Food Res. Int. 55, 137–149.
Granato, D., Putnik, P., Kovačevič, D.B., Santos, J.S., Calado, V., Rocha, R.S.,
Pomerantsev, A., 2018a. Trends in chemometrics: food authentication, microbiology,
and effects of processing. Compr. Rev. Food Sci. Food Saf. 17, 663–673.
Granato, D., Santos, J.S., Escher, G.B., Ferreira, B.L., Maggio, R.M., 2018b. Use of prin-
cipal component analysis (PCA) and hierarchical cluster analysis (HCA) for multi-
variate association between bioactive compounds and functional properties in foods:
a critical perspective. Trends Food Sci. Technol. 72, 83–90.
Handa, A.K., Fatima, T., Mattoo, A.K., 2018. Polyamines: bio-molecules with diverse
functions in plant and human health and disease. Front. Chem. 6, 1–18.
Janjušević, L., Karaman, M., Šibul, F., Tommonaro, G., Iodice, C., Jakovljević, D., Pejin,
B., 2017. The lignicolous fungus Trametes versicolor (L.) Lloyd (1920): a promising
natural source of antiradical and AChE inhibitory agents. J. Enzyme Inhib. Med.
Chem. 32, 355–362.
Kalač, P., 2013. Chemical composition and nutritional value of European species of wild
growing mushrooms: a review. Food Chem. 113, 9–16.
Kalač, P., 2014. Health effects and occurrence of dietary polyamines: a review for the
period 2005-mid 2013. Food Chem. 161, 27–39.
Laube, G., Bernstein, H., 2017. Agmatine: multifunctional arginine metabolite and magic
bullet in clinical neuroscience? Biochem. J. 474, 2619–2640.
Manninen, H., Rotola-Pukkila, M., Aisala, H., Hopia, A., Laaksonen, T., 2018. Free amino
G.C.L. Reis, et al.
acids and 5′-nucleotides in Finnish forest mushrooms. Food Chem. 247, 23–28.
Nishibori, N., Fujihara, S., Akatuki, T., 2007. Amounts of polyamines in foods in Japan
and intake by Japanese. Food Chem. 100, 491–497.
Okamoto, A., Sugi, E., Koizumi, Y., Yanagida, F., Udaka, S., 1997. Polyamine content of
ordinary foodstuffs and various fermented foods. Biosci. Biotechnol. Biochem. 61,
1582–1584.
Önal, C., 2013. A review of the liquid chromatographic methods for the determination of
biogenic amines in foods. Food Chem. 138 (1), 509–515.
Otto, M., 2017. Chemometrics – Statistics and Computer Application in Analytical
Chemistry, 3rd ed. Wiley-VCH, Weinheim, Germany.
Pop, R.M., Puia, I.C., Puia, A., Chedea, V.S., Leopold, N., Bocsan, I.C., Buzoianu, A.D.,
2018. Characterization of Trametes versicolor: Medicinal mushroom with important
health benefits. Not. Bot. Horti Agrobot. Cluj. 46 (2), 343–349.
Ramani, D., De Bandt, J.P., Cynober, L., 2014. Aliphatic polyamines in physiology and
diseases. Clin. Nutr. 33 (1), 14–22.
Rider, J.E., Hacker, A., Mackintosh, C.A., Pegg, A.E., Woster, P.M., Casero, R.A., 2007.
Spermine and spermidine mediate protection against oxidative damage caused by
hydrogen peroxide. Amino Acids 33 (2), 231–240.
Rogers, A.C., McDermott, F.D., Mohan, H.M., O’Connell, P.R., Winter, D.C., Baird, A.W.,
2015. The effects of polyamines on human colonic mucosal function. Eur. J.
Pharmacol. 764, 157–163.
Roupas, P., Keogh, J., Noakes, M., Margetts, C., Taylor, P., 2012. The role of edible
Journal of Food Composition and Analysis 86 (2020) 103375
mushrooms in health: evaluation of the evidence. J. Funct. Foods 4, 687–709.
Sharma, S., Kumar, P., Deshmukh, R., 2018. Neuroprotective potential of spermidine
against rotenone induced Parkinson’s disease in rats. Neurochem. Int. 116, 104–111.
Silva, A.C., Jorge, N., 2014. Influence of Lentinus edodes and Agaricus blazei extracts on the
prevention of oxidation and retention of tocopherols in soybean oil in an accelerated
storage test. J. Food Sci. Technol. 51 (6), 1208–1212.
Thompson, M., Ellison, S.L.R., Wood, R., 2002. Harmonized guidelines for single la-
boratory validation of methods of analysis (IUPAC Technical Report). Pure Appl.
Chem. 74, 835–855.
Toro-Funes, N., Bosch-Fusté, J., Veciana-Nogués, M.T., Izquierdo-Pulido, M., Vidal-Carou,
M.C., 2013. In vitro antioxidant activity of dietary polyamines. Food Res. Int. 51,
141–147.
Wang, L.C., Thomas, B.W., Warner, K., Wolf, W.J., Kwolek, W.E., 1975. Apparent odor
thresholds of polyamines in water and 2 % soybean flour dispersions. J. Food Sci. 40,
274–276.
Yamamoto, S., Itano, H., Kataoka, H., Makita, M., 1982. Gas-liquid chromatographic
method for analysis of di- and polyamines in foods. J. Agric. Food Chem. 30,
435–439.
Yang, J.H., Linb, H.C., Mau, J.L., 2002. Antioxidant properties of several commercial
mushrooms. Food Chem. 77, 229–235.
Yen, G.C., 1992. Effects of heat treatment and storage temperature on the biogenic amine
contents of straw mushroom (Volvariella volvacea). J. Sci. Food Agric. 58, 59–61.