a*

Abílio Guerreiro , Carimo Rassal b, Carlos M. Afonso c, Ludovina Galegod, Manuel Serrae, Maria A. Rodriguesf
a
Abílio Guerreiro – School of Management, Hospitality and Tourism, University of the Algarve, Campus of
Penha, 8005-139 Faro. Portugal.

b
Carimo Rassal - School of Management, Hospitality and Tourism, University of the Algarve, Campus of Penha,
8005-139 Faro. Portugal.
c
Carlos Miguel Afonso – School of Management, Hospitality and Tourism, University of the Algarve, Campus of
Penha, 8005-139 Faro. Portugal.
d
Ludovina Galego – Higher Institute of Engineering, University of the Algarve, Campus of Penha, 8005-139
Faro. Portugal.
e
Manuel Serra – School of Management, Hospitality and Tourism, University of the Algarve, Campus of Penha,
8005-139 Faro. Portugal.
f
Maria A. Rodrigues – MeditBio, Faculty of Science and Technology, University of the Algarve, Campus of
Gambelas, 8005-139 Faro. Portugal.

*Corresponding author
E-mail: ajguerreiro@ualg.pt

Tel: +351 917 778 771

Healthy, tasty and sustainable Mediterranean Food. UMAMI taste and polyphenols of twiggy glasswort
(Salicornia ramosissima)
RESUMO

Neste trabalho apresentamos resultados de uma avaliação preliminar dos compostos fenólicos maioritários
presentes em extratos aquosos de salicórnia (Salicornia ramosissima) e também uma avaliação da presença de
compostos que têm sido associados ao sabor UMAMI como o glutamato, o inosinato e o guanilato. Temos por
objetivo contribuir para o desenvolvimento de hábitos de alimentação saudável, atendendo aos princípios
básicos da Dieta Mediterrânica, que valorizam a utilização dos produtos autóctones e sazonais. O estudo foi
desenvolvido utilizando amostras de salicórnia em fresco, produto cultivado de forma sustentável pela
empresa RiaFresh. A identificação e quantificação dos compostos foi feita por LC-DAD-ESI-MS/MS. Os
compostos fenólicos maioritários presentes nos extratos aquosos são ésteres de ácido cafeico e ácido quínico.
Não foram detetadas quantidades significativas de compostos associados ao sabor UMAMI, nomeadamente do
glutamato, inosinato e guanilato. A ausência destes compostos em quantidades significativas não excluí a
presença do sabor UMAMI na salicórnia, já que a presença de NaCl, característica nesta planta, intensifica este
sabor. Estamos a desenvolver métodos analíticos para quantificação de glutamato, inosinato e guanilato em
amostras frescas e desidratadas desta planta.

ABSTRACT

In this work we present a preliminary evaluation of the main polyphenolic compounds present in water
extracts of twiggy glasswort (Salicornia ramosissima) and of the presence of compounds associated with
UMAMI taste, namely glutamate, inosinate and guanylate. Our main goal is to contribute to the development of
health eating routines using the basic principles of Mediterranean Diet and the use of local biological
resources. This study was developed using fresh twiggy glasswort samples grown by RiaFresh firm. The
identification and quantification of compounds was performed by LC-DAD-ESI-MS/MS. The major phenolic
compounds present in the aqueous extracts are esters of caffeic and quinic acids. In this preliminary study no
significant amounts of glutamate, inosinate and guanylate were detected in water extracts of fresh twiggy
glasswort. However, the presence of UMAMI taste cannot be exclude at this stage, as the high NaCl contents of
this plant potentiate this taste. We are currently developing analytical methods for quantification of UMAMI
related target compounds in fresh and dried samples.
Keywords: Healthy food, sustainable production, Salicornia, twiggy glasswort, UMAMI taste, polyphenols, LC-

DAD-ESI-MS / MS

Introduction

Twiggy glasswort (Salicornia ramosissima) is a species of Salicornia genus, also known as sea beans or sea
asparagus. Salicornia species are halophyte plants (salt tolerant) (Glenn, et al. 1999; Parida, et al. 2005)
frequently found in saline areas of Algarve. Salicornia plants are rich in several acid and polyphenolic
compounds such as caffeic acid and quercetin, which are known by their antioxidant and anti-cancer
properties (Cho, et al., 2016; Rhee, et al., 2009). These compounds have been reported in Salicornia species,
such as S. european L., S. herbacea L., S. brachiate) (Kim, et al. 2011, Kim, et al. 2012). More recently
stigmastanol (Isca, et al. 2014) and the micronutrient selenium (Mishra et al. 2015) have been reported.
Stigma sterol is also known to possess health effects, namely reduction of cholesterol levels (Isca, et al. 2014).
Twiggy glasswort meets therefore the requirements to be included in the Mediterranean Diet has a healthy
component and takes advantage of the Algarve natural bio-resources.
Salicornia has been traditionally used in food or even for medical purposes (Ksouri, et al. 2012; Rhee, et al.,
2009). Regarding its food properties, this vegetable is slightly salty, crunchy, succulent and tasty (Patel, S.,
2016), which are the reasons why it has been progressively introduced at several cooking stages. Nowadays,
Algarve gourmet cuisine and other fine dining environments all over the world include this elegant vegetable.
Salicornia has been quite used in raw salads or cooked with meat, fish, shellfish and sushi (Rhee, et al. 2009). It
has also been used to prepare macerates using alcoholic or non-alcoholic drinks and vinegar (Patel, S., 2016).
Salicornia gives a special, refined and unique touch to dishes if it is used as seasoning or simply as garnish. The
possible UMAMI properties of this plant was a major motivation for the current study. UMAMI taste is the
savory taste, one of the five basic tastes human palate. This taste is associated with glutamate receptors and,
besides glutamate itself, it has also been related with 5′-ribonucleotides such as inosinate and guanylate
(Ninomiya, K., 2015) (see scheme 1). 5′-ribonucleotide compounds synergistically potentiate the UMAMI taste
(Karanewsky, et al. 2016). All these UMAMI related compounds are used as food additives to enhance food
flavor. Nevertheless, many foods consumed daily are naturally rich in these compounds. Glutamate can be
found in meat, fish and vegetables (Jinap, et al., 2010). Inosinate is mainly present in meat and fish, while large
amounts of guanylate can be found in dried mushroom. Recently, glutamate and others UMAMI compounds
have been detected in meat of Berkshire breed pigs by Subramaniyan, et al. (2016) using HPLC. The same and
other UMAMI compounds were also detected in tomato by Coulier, et al. (2011), using ion-pair LC-ESI-MS.
Barreira, et al. (2017), refers the use of Salicornia as succulent salty shoots in gourmet cuisine and emphasize
the nutritional profiles suitable for human consumption.
In this work, we present a preliminary study of major polyphenols compounds and glutamate, inosinate and
guanylate in aqueous extracts of twiggy glasswort produced in the RiaFresh production center, Faro, using a
LC-DAD-ESI-MS/MS system.

Scheme 1. Chemical structures of glutamate (1), di-sodium inosinate (2) and di-sodium guanylate (3),
compounds associated to UMAMI taste.

Experimental

Chemicals
Ultrapure water was obtained using a Milli-Q purification system (18 MΩ cm). Acetonitrile (HPLC super
gradient) was from VWR (Fontenary-Sous-Bois, France), methanol and formic acid (analytical grade) from
Merck (Darmstadt, Germany); glutamic acid, Ginkgo Biloba Flavonoids Mix and Green Tea Catechin Mix were
obtained from Sigma-Aldrich (Steinheim, Germany). Stock standard solutions were prepared in water.
Working solutions were prepared daily by diluting aliquots of their stock solutions. All materials were used
without further purification.

Plant material

The twiggy glasswort plants used in this study were supplied by RiaFresh, Lda., from RiaFresh Production
Center in Faro, Algarve. The plant material, grown under sustainable farming was harvested on-site in
February of 2017. In each sampling 500 g of the aerial part of the plant were taken. After harvesting, the
samples were refrigerated and transported to the laboratory. Fresh plants were processed within 2-3 hours or
stored at -20 ºC before analysis.

Extraction of polyphenols

Samples of twiggy glasswort were hand-macerated and submitted to water extraction. Five grams of plant
material were used per 25 mL of Milli-Q water. The mixture was hand-shaken for 3 min at room temperature
and filtered using paper filter Whatman nº 1 and then through 0.20 µm cellulose acetate membrane filters
Millipore. The follow-on extracts were kept at 4 ºC before LC-MS analysis.

Analyses of polyphenols

The polyphenol profiles were evaluated using a LC-DAD-ESI-MS/MS system. The system comprises a Agilent
Technologies 1200 Series LC equipped with a diode array detector (DAD), and a Bruker Esquire HCT ultra ion
trap mass spectrometer, equipped with an ESI source (Agilent). A GraceSmart RP-18 LC column (10.0 cm
length, 2.1 mm internal diameter, 3 μm), stabilized at 25 °C was used. The mobile phase comprises water (A)
and acetonitrile (B), both with 0.1 % (v/v) of formic acid. The gradient started with 85 % of A and 15 % of B,
there in 18 minutes increase to 40% of B. The mobile phase composition changes to 0 % of A, 100 % of B in 2
minutes and keeps this composition for additional 3 minutes. Finally, the system was allowed to recover the
initial composition of the mobile phase (75 % of A and 15 % of B) in 0.5 min and then stabilized for additional
5 minutes before the next run. The flow-rate was 0.3 mL/min and the injection volume 10 µL. LC-DAD traces
were obtained at 280, 350 and 370 nm. The LC-MS was running in negative mode alternating with positive
mode. Auto-MS mode was selected to obtain the fragmentation spectra of all compounds. Spray and ion optics
analysis conditions were the following: capillary voltage, 3.5 kV; nebulizer gas pressure, 40 psi; drying gas,
330°C; drying gas flow, 8.0 L/min; capillary exit voltage, -183.0 V; skimmer voltage, -60.3 V. The main
polyphenol compounds were identified by comparison of UV spectra and their fragmentation patterns with
published data in the literature. Quantification was performed against a calibration curve prepared with a
chlorogenic acid standard.

Detection of UMAMI compounds

Glutamate, inosinate and guanylate compounds were evaluated using the LC-DAD-ESI-MS/MS traces and
extracting the m/z values expected for these compounds under negative or positive ESI.

Results and discussion

Polyphenol profiles

We consider only the extraction with water as water extracts are the most representative from food/cooking
point of view. Figure 1 shows typical LC-DAD profiles of aqueous extracts of twiggy glasswort taken at 280 and
370 nm. The profile obtained at 370 nm is selective to flavonoid compounds, while the trace at 280 nm allows
the observation of compounds possessing a phenolic system. Quinic, p-coumaric and ferulic acids show very
low retention times (1). Esters of hydroxycinnamic acids were observed between 16 and 18 minutes (3 and 4)
while flavonols were mainly detected at 370 nm and can be observed between 16 and 20 minutes (5 and 6).
Hydroxycinnamic acids have been observed in cereals, fruits and seeds of fruits, vegetables and plants. These
compounds are products or intermediates of enzymatic processes and are also precursors of other compounds
widespread in plant materials such as stilbenes, chalcones, flavonoids, lignans, and anthocyanins (Ferreira, et
al. 2017).

Fig. 1. LC-DAD profiles of aqueous extracts of twiggy glasswort at 280 nm, a), and 370 nm, b). The inserts show
the structures of identified compounds.

Caffeoylquinic acid and other hydroxycinamic acid derivatives were already reported in S. herbacea and S.
europea (Lee, et al., 2004; Kim, et al. 2011; Cho, et al. 2016). Flavonoids isorhamnetin and isorhamnetin
glucose were also previously identify in S. ramosissima extracts (Surget et al., 2015). The presence of phenyl
propenoid feruloyl-caffeoyl glycerol and isorhamnetin-7-O-(6-O-malonyl)-glucoside in simple non-processes
twiggy glasswort aqueous extract is reported here for the first time. The identifications were performed after
analysis of the LC-MS traces correspondent to profiles of Figure 1 obtained under AutoMS mode. The UV
spectra taken with the DAD detector confirmed the assignments.
Figure 2 presents representative data demonstrating how the identification of flavonoid Isorhamnetin-7-O-(6-
O-malonyl)-glucoside was performed. In this case the assignment was made taking into account the UV
spectrum and parameters obtained under negative and positive ESI, namely the m/z values, isotope
distributions and fragmentation patterns. The assignment was performed by comparing these data with the
correspondent published in the literature (Surget et al., 2015). The UV-spectrum of compound 6 shows an
absorption maximum at 354 nm (Figure 2 b) which is an important identification element. However, the full
scan spectrum taken under negative ESI (Fig. 3. c), together with its fragmentation pattern (Figure 3 d), were
very sharp in clarifying the structure. The ion at m/z 562.92 ([M–H]-) releases fragments at m/z 518.97, m/z
504.20 and m/z 314.91. The fragment at m/z 518.97 corresponds to a loss of CO2, [M-H-44]-, and m/z 504.20
corresponds to a loss of CH3CO2, [M-H-59]-, the latter a malonyl-glucose unit [M-H-malonyl-glucose]-. The
signal at m/z 314.91 is due to the isorhamnetin aglycone. This assignment was in agreement with the m/z
value (Figure 3 e)) and fragmentation pattern and isotope distributions (Figure 3 f)) observed under positive
ESI.
Figure 2. Parameters used in the assignment of the structure of compound 6 (isorhamnetin-7-O-(6-O-
malonyl)-glucoside). a) detail of the LC-DAD trace at 370 nm; b) UV spectrum at 19.80 minutes; c), full scan
spectrum at 19.80 minutes under negative polarity; d) MS/MS spectrum of m/z 562.92 (negative mode); e),
full scan spectrum at 19.80 minutes under positive polarity; f) MS/MS spectrum of m/z 565.10 (positive
mode) and experimental and simulated isotope patterns.

Glutamate, inosinate and guanylate (see scheme 1) were evaluated in aqueous extracts of twiggy glasswort by
searching for the m/z values expected for these compounds under both, positive and negative ESI. As an
example, single ion traces at m/z 347 (a), m/z 349 (b) and m/z 371 (c), correspondent to 5'-guanylic acid
under negative ESI, 5'-guanylic acid under positive ESI and sodiated 5'-guanylic acid under positive ESI,
respectively, are shown in Figure 3.

Fig. 3. Extracted ion chromatogram at m/z 347 (a), m/z 349 (b) and m/z 371 (c), correspondent to 5'-guanylic
acid under negative ESI, 5'-guanylic acid under positive ESI and sodiated 5'-guanylic acid under positive ESI,
respectively
The guanylate, as well as the other UMAMI compounds are polar and expected to show low retention times
under the used chromatographic conditions. The absence of strong signals of UMAMI compounds might be due
to the presence of salts in the same time window, which are known to quench the ionization process. It should
be noted that the apparent low signals of glutamate, inosinate and guanylate do not necessarily exclude their
UMAMI effect in twiggy glasswort. The presence of NaCl, a feature of this plant, is known to potentiate this
taste, and therefore it is expected that UMAMI flavor detection by glutamate receptors is reached at much
lower concentrations of UMAMI compounds. We are currently developing and validating analytical methods
for the quantification of UMAMI compounds in fresh and dehydrated twiggy glasswort extracts.

Conclusion

Twiggy glasswort aqueous extracts contain quinic acid, p-coumaric and ferulic acid, 3-O-caffeoylquinic acid,
feruloyl-caffeoylglycerol, a feruloyl-caffeoylglycerol isomer, quercetin-3-O-glucoside and Isorhamnetin-7-O-(6-
O-malonyl)-glucoside. The presence of phenyl propenoid feruloyl-caffeoyl glycerol and flavonoid
isorhamnetin-7-O-(6-O-malonyl)-glucoside are reported for the first time. Glutamate, inosinate and guanylate
UMAMI compounds were not detected in significant amounts. Analytical methods to specifically quantify these
compounds in extracts need to be performed in order to properly evaluate the contribution of this plant to
UMAMI taste.

The results of this study can also be complemented by future researches to apply the use of Salicornia in
different stages of the cooking process as well as to improve its use in raw as a specific ingredient for salads or
garnishing dishes, enhancing textures and flavor intensity. Considering the main goal of developing healthy
eating habits, attending to the main principals of mediterranean diet, it is intended to understand if the
responsible components for the UMAMI flavor naturally intensify the salty taste, with lower values of Na than
those that exists in the salt.

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