Rapid Determination of Total Tryptophan in Yoghurt
by Ultra High Performance Liquid Chromatography
with Fluorescence Detection
Mena Ritota and Pamela Manzi *
CREA-Centro di Ricerca Alimenti e Nutrizione, Via Ardeatina 546, 00178 Rome, Italy; email@example.com
Academic Editors: Daniel Cozzolino and Susy Piovesana
Received: 5 October 2020; Accepted: 27 October 2020; Published: 29 October 2020
Tryptophan (TRP) is an essential amino acid which cannot be synthesized by humans
and animals, but has to be supplied by exogenous sources, notably through the diet. The bulk
of dietary TRP ﬂows into the synthesis of body’s proteins, but the TRP metabolism also involves
several biochemical reactions (i.e., serotonin and kynurenine pathways). Defects in the TRP transport
mechanism or catabolism are related to a large number of clinical abnormalities. Therefore, dietary
TRP intake is necessary not only for the body’s growth but also for most of the body’s metabolic
functions. Among protein-based foods, milk proteins provide a relatively high amount of TRP. In this
paper, a rapid chromatographic method for TRP determination in yoghurt, by ultra high performance
liquid Chromatography on a reversed-phase column with fluorescence detection (280 nm Ex; 360 nm Em),
is provided. A linear gradient elution of acetonitrile in water allowed TRP analysis in 8.0 min. The limit
of detection and limit of quantiﬁcation of the method were 0.011 ng/
L and 0.029 ng/
using 5-methyl-l-tryptophan as the internal standard. The analytical method was successfully applied
to commercial yoghurts from diﬀerent animal species, and the TRP values ranged between 35.19 and
121.97 mg/100 g (goat and cow Greek type yoghurt, respectively).
Keywords: tryptophan; ultra high performance liquid chromatography; yoghurt
Tryptophan (TRP) is an essential amino acid needed for normal growth, and is involved in the
synthesis of diﬀerent bioactive compounds, such as nicotinamide (vitamin B6), melatonin, tryptamine,
kynurenine, 3-hydroxykynurenine, and quinolinic and xanthurenic acids .
In lower organisms, TRP is formed through the condensation of serine with an indole group by
the action of tryptophan synthase [
]. In humans and animals, though, TRP cannot be synthesized
because they are lacking in tryptophan synthase [
]. Therefore, TRP has to be supplied to the body by
exogenous sources, especially through the diet [
]. Besides participating in the formation of the body’s
proteins, TRP is involved in numerous chemical reactions.
TRP and its metabolites seem to have the potential to contribute to the therapy of autism,
multiple sclerosis, cardiovascular, chronic kidney and inﬂammatory bowel diseases, cognitive function,
depression, and microbial infections [
]. TRP transport through the cell membranes is competitively
inhibited by the other large neutral amino acids (NAA), such as valine, leucine, isoleucine, phenylalanine
and tyrosine .
From these premises, it is clear that TRP intake through the diet, or the intake of TRP-rich proteins,
is necessary not only for the body’s growth, but also to carry out most of the body’s metabolic functions.
However, the wide use of TRP as a dietary supplement for its potential health beneﬁts has raised the
Molecules 2020,25, 5025; doi:10.3390/molecules25215025 www.mdpi.com/journal/molecules
Molecules 2020,25, 5025 2 of 11
issue of assessing its safety [
], so much so that an upper limit of safe intake for diet-added tryptophan
of 4.5 g/day has been proposed in young adults .
Among food proteins introduced through the diet, cereals, and especially maize, are generally
poor in tryptophan content; as such, TRP may represent the nutritionally limiting amino acid in these
food items [
]. Egg, soy, beans, seafood and poultry proteins, instead, have been described as good
sources of tryptophan .
Milk proteins are particularly rich in TRP, which can be released by the proteolytic enzymes either
as a free amino acid or as a part of small peptides with functional activities [
]. Furthermore, it has
been shown that TRP released during
gastrointestinal digestion is one of the main factors
responsible for the radical scavenging activity of digested bovine milk [
]. TRP intake through the diet
considerably varies depending on the food protein type, since TRP distribution can signiﬁcantly diﬀer
among the various fractions [
]; in cow’s milk proteins, for example,
-lactalbumin contains around
6% TRP, whereas bovine serum albumin and
-casein are extremely poor in TRP [
the TRP bioavailability through dietary intake might be reduced during food processing or cooking,
mainly by oxidative degradation or cross-linking among proteins [
], as well as by decarboxylation
The nutritional and safety aspects of tryptophan emphasize the need for reliable analytical
methods for its determination in food. The ﬁrst rapid method for tryptophan determination dates back
to 1970, when Gaitonde and Dovey [
] proposed a colorimetric method, whereby TRP reacted with
acid ninhydrin giving a yellow product, which was spectrophotometrically revealed at
However, this method needed to be corrected for tyrosine absorption [
]. Later, Inglis [
a method for amino acid determination by preventing tryptophan degradation, which generally occurs
in the acid conditions of protein hydrolysis (HCl 6 N at 110
C for 22 or 24 h [
]), by protecting
TRP with tryptamine. Yamada et al. [
] improved this method by modifying the proteins with
vapor-phase S-pyridylethylation before the hydrolysis, and then treated the modiﬁed proteins with
mercaptoethanesulfonic acid at 176
C. However, all these early methods were applied to pure proteins,
without considering the matrix eﬀect.
When referring to a food product, one of the main concerns is certainly the release of the protein-bound
tryptophan from the matrix. Acid hydrolysis generally carried out for the total determination of most
amino acids cannot be performed for TRP analysis, because it is destroyed during acid hydrolysis [
Therefore, alkaline hydrolysis, as reported by Steven and Jorg as far back as 1989 [
], is currently the
pre-treatment method of choice for tryptophan determination in foods.
TRP detection and quantiﬁcation can be carried out with diﬀerent analytical techniques. Near
infrared spectroscopy (NIRS) was used to determine TRP, as well as other amino acids in dairy
products, and in particular to evaluate cheese ripening [
]. Even if these spectroscopy methods oﬀer
the advantages of a short time of analysis and a poor sample preparation, they need huge amounts
of samples for the calibration and validation of the models. Ion exchange chromatography (IEC)
with ﬂuorescence detection was also employed to determine TRP in pure proteins and feedstuffs ,
but IEC needed post-column derivatization with o-phthalaldehyde (OPA), thus resulting in further
time- and chemical-consuming steps. The determination of tryptophan in infant formula by high
performance liquid chromatography (HPLC) with UV detection (
=254 nm) needed a derivatization step,
with phenylisothiocyanate (PITC), in addition to the protein hydrolysis prior to the HPLC analysis [
Derivatization was necessary due to the poor absorbance of TRP in the UV spectral region, but this
could result in a time- and chemicals-consuming procedure. Furthermore, Tsopmo et al. [
of the use of the more sensitive tandem mass spectrometry (MS/MS) detector, derivatized TRP with
PITC, in order to evaluate the TRP in human milk, infant plasma and peptide fraction.
Fluorimetric detectors could be employed to increase the selectivity and sensibility of an HPLC
]. Furthermore, TRP exhibits a strong native ﬂuorescence [
], which allows one to avoid
the derivatization generally needed for most amino acids determined by HPLC.
Molecules 2020,25, 5025 3 of 11
Therefore, the aims of this paper were as follows: 1) to develop a robust, rapid and cost-eﬀective
method for tryptophan detection in yoghurt based on ultra high performance liquid chromatography
(UHPLC) by means of a ﬂuorescence detector; 2) to evaluate the levels of tryptophan in commercial
yoghurts from the milk of diﬀerent animal species. The analytical technique of choice in this study
was UHPLC, since this chromatography has the advantages of speed, enhancing resolution and peak
eﬃciency, as well as requiring smaller amounts of solvent compared to the traditional HPLC, so it can
be used for fast and eco-friendly analysis. To the best of our knowledge, this is the ﬁrst time that this
technique has been applied for the analysis of TRP in yoghurt samples.
2.1. Chromatographic Method Validation
The alkaline hydrolysis with NaOH, according to the method of Steven and Jorg [
carried out to extract TRP from the yoghurt samples. Partial disruption of TRP can occur during
hydrolysis: these losses can be corrected based upon the recovery of an internal standard [
and 5-methyl-l-tryptophan (M-TRP) has been revealed as the preferred internal standard for TRP
]. Furthermore, the presence of foreign substances in a matrix may cause a bias by increasing
or decreasing the signal attributed to the measurand [
]. Due to the absence of a suitable reference
material to estimate the potential inﬂuence of the interferences of the yoghurt matrix on the analysis of
TRP (the so called “matrix eﬀect”), the approach of recovery tests (using spiked samples) was used [
In the recovery value test, the original concentrations of TRP in the yogurt samples were determined
using the calibration curve described below. Each of the yoghurt samples was then spiked, prior to the
extraction, with a known concentration of TRP (at diﬀerent levels, ranging from 0.061 to 0.152
and the total TRP concentrations of the spiked samples were calculated using the same calibration
curve. The recovery values, adjusted for the value of M-TRP, ranged between 97.36% and 100.12%,
with a mean value of 97.27% (Table 1).
Table 1. Accuracy of the method expressed as Recovery (%) in yoghurt samples.
Spiked Levels TRP Addition (µg/mL) Recovery (%)
1 0.061 97.36 ±0.70
2 0.091 98.90 ±1.58
3 0.122 92.79 ±1.09
4 0.152 100.12 ±1.16
The linearity range of the method was evaluated by injection, in triplicate, of the following
TRP standard solutions: 1.105
g/mL. The linearity range investigated covered the entire measurement range of the samples.
The calibration and quantiﬁcation of TRP in the yogurt samples were obtained by the standard
addition method. The calibration curve (y =502654x) was obtained with a correlation factor
with the error of curve equal to 1751. The limit of detection (LOD) and limit of quantiﬁcation (LOQ) of
the method were 0.011 ng/µL and 0.029 ng/µL, respectively.
The method’s precision was evaluated through repeatability and reproducibility measurements;
the method resulted in a good precision, and the intra- and inter-day relative standard deviation
(RSD %), on pure standards and on yoghurt samples, are shown in Tables 2and 3, respectively.
Molecules 2020,25, 5025 4 of 11
Table 2. Repeatability and reproducibility performances on pure standards.
Intra Day Inter Day
1.105 1.23 1.00 0.99 1.08
0.848 1.68 1.06 1.09 1.28
0.424 1.66 1.33 1.21 1.40
0.553 1.50 2.00 1.44 1.65
0.170 1.66 0.84 1.37 1.29
0.085 1.13 1.11 1.02 1.09
Table 3. Repeatability and reproducibility performances on yoghurt samples.
Intra Day Inter Day
121.97 2.59 2.87 1.79 2.59
62.96 1.11 1.14 1.18 1.71
35.19 1.36 1.55 1.27 1.76
Furthermore, the relative standard deviation in retention times was less than 0.2% and 0.3%,
for intra-day and inter-day, respectively.
The proposed chromatographic method allowed TRP determination in yoghurt samples in
a relatively short time; the total chromatographic running time was 8.0 min, including column
reconditioning, with a TRP retention time equal to 1.197 min and a 5-methyl-l-tryptophan (M-TRP)
retention time equal to 1.564 min (Figure 1).
Chromatographic proﬁle of a TRP and M-TRP standard solution and yoghurt samples
extracts, according to the chromatographic conditions reported in Section 4.
2.2. Tryptophan Levels in Commercial Yoghurts
The proposed method was applied to the analysis of the TRP contents in various commercial
yoghurts obtained from the milk of diﬀerent animal species. The results, reported in Table 4, showed a
Molecules 2020,25, 5025 5 of 11
Table 4. TRP contents (mg/100g) in commercial yoghurts from milk of diﬀerent animal species.
Yoghurt Sample Number Mean SD
Cow yoghurt (whole milk plain) 1. 41.94 0.37
2. 41.28 0.08
3. 41.44 0.20
4. 41.70 0.60
5. 40.78 0.26
6. 42.00 0.13
7. 45.48 0.12
8. 44.36 1.03
9. 48.82 0.38
10. 49.22 0.17
11. 48.91 0.06
12. 49.31 0.14
Ewe yoghurt (whole milk plain) 1. 62.91 1.20
2. 62.96 1.45
Goat yoghurt (whole milk plain) 1. 35.19 1.18
2. 37.61 1.33
Buﬀalo yoghurt (whole milk plain) 1. 53.01 1.23
2. 52.87 0.95
Cow yoghurt (lactose-free whole milk plain) 1. 47.93 0.47
2. 50.36 0.39
Cow yoghurt (Greek-type whole milk plain) 1. 121.97 2.69
2. 120.98 2.24
Among the whole yoghurt samples, there were signiﬁcant diﬀerences in the TRP levels (P<0.05):
ewe milk yoghurts showed the highest TRP concentration (on average 62.94 mg/100 g), while goat
milk yoghurts had the lowest one (36.40 mg/100 g). Furthermore, whole cow yoghurt samples showed
the greatest sample heterogeneity, with TRP levels ranging between 40.78 and 49.31 mg/100 g.
Among the cow yoghurts, the highest TRP content was observed in the Greek type samples
(on average 121.47 mg/100 g), followed by lactose free and whole cow yoghurts (49.15 mg/100 g and
44.60 mg/100 g, respectively). The highest TRP value reported in the Greek type samples is justiﬁed by
their high protein contents (about 9 g/100 g), due to the particular technological process whereby the
liquid whey is removed, obtaining a thick and creamy product.
3.1. Method Validation
Due to the presence of the indole ring in the tryptophan structure, which is degraded under the
acid conditions generally used in the protein hydrolysis, TRP cannot be analyzed by the standard
method for amino acid analysis [
]. Many attempts have been made over the years, from enzymatic
to modiﬁed acid hydrolysis, but all these procedures suﬀer from incomplete TRP recovery [
Therefore, alkaline hydrolysis is the common procedure for TRP analysis in foods. Among the diﬀerent
alkalis used for protein hydrolysis [
], NaOH does not suﬀer for the inconvenience of solubility in
water, unlike Ba(OH)
and LiOH, and it avoids the precipitation/adsorption problems associated with
the use of Ba(OH)
]. Therefore in this study, an alkaline hydrolysis with NaOH 4.2 M, according to
the method of Steven and Jorg , was performed.
While many scientiﬁc attempts have been made for TRP determinations in biological and
pharmaceutical samples [
], as well as in non-dairy food items [
], very few works have
been reported in the literature about TRP determinations in dairy products, and in particular in yoghurt.
The limit of detection (LOD) of this method (0.011 ng/
L) is comparable to that reported
by Yılmaz and Gökmen (0.0165 ng/
L in yoghurt) [
], who even used a more sensitive detector
(tandem mass spectrometry) to determine TRP and its derivatives in foods. Furthermore, our LOD
Molecules 2020,25, 5025 6 of 11
is lower compared to that obtained by Liu and Xu for TRP analysis in milk [
], who employed a
selective electrochemical sensor; the method proposed, in fact, showed a limit of detection of 6
corresponding to 1.226 ng/
L. Only the methods proposed by Wang et al. [
], who developed a
modiﬁed electrochemical sensor to determine TRP in milk, and by Baytak and Aslanoglu, who had to
resort to a nanosensor for TRP determination in cow’s milk [
], showed lower LOD values (0.0035 ng/
and 0.00135 ng/
L, respectively). Even though the electrochemical sensors generally allow a simple and
rapid determination of the analytes in food matrices without any sample pretreatment, TRP analysis
requires a modiﬁcation procedure of the electrodes, since under the traditional working electrode
conditions TRP oxidation suﬀers from high overpotential and sluggish kinetics [
]. These electrode
modiﬁcations may result in chemical reagent consumption. Furthermore, electrochemical sensors are
still little used in the laboratories for routine analysis.
The proposed chromatographic method allowed TRP determination in yoghurt samples in a
relatively short time (total chromatographic running time =8.0 min). This was possible thanks to the
high speed of the UHPLC technique. Furthermore, Delgado-Andrade et al. [
] proposed a liquid
chromatography-based method with ﬂuorescence detection for TRP analysis in milk-based ingredients,
but the total chromatographic time, even if not speciﬁed by the authors in the text, was at least 10 min.
The running chromatographic time of this proposed method was even shorter than that proposed by
La Cour et al. 
with UHPLC–single quadrupole mass spectrometry, who reported a total analysis
time of 11.5 min for TRP analysis in plant materials and dog foods, even if the author stated a possibility
of shortening the running time.
The results reported in this study show that the proposed chromatographic method can be used
for the routine analysis of TRP in foods; it is low in terms of time and chemical consumption, it ensures
a low sensitivity, and the analytical equipment is easy to access and much less expensive than the more
sensitive mass spectrometry.
3.2. Nutritional Evaluation of Eryptophan Levels in Commercial Yoghurts
Regarding the TRP levels in the yoghurt samples, the results obtained in this study are in agreement
with those previously reported by Gambelli et al. [
] in commercial yoghurt samples, through a similar
analytical method, but using an HPLC. The TRP levels reported in this study are also of the same order
of magnitude as those reported by Posati and Orr [
] on similar commercial samples. Furthermore,
az et al. [
] evaluated the total TRP content in commercial yoghurts, but they reported
slightly higher levels (374 mg/kg) by using a completely diﬀerent method based on heavy atom-induced
room temperature phosphorescence.
On the contrary, our results are very high compared to those reported by Bertazzo et al. [
similar food items (TRP =0.7
g/mL in yoghurt samples); the main diﬀerence is due to the fact that the
] did not hydrolyze the commercial yoghurt samples, but carried out the HPLC analysis
on the supernatants directly after centrifugation, so their results referred to the “free” TRP content of
yoghurts. Furthermore, Yılmaz and Gökmen  reported similar results for free TRP in commercial
yoghurts, with values ranging between 3.2 and 13.4 mg/kg dry weight. The diﬀerences in the free amino
acid contents observed by the authors [
] could be due to the diﬀerent microorganisms involved
in the production of the yoghurts, since free amino acid content has been shown to be inﬂuenced by
the interactions between the microorganisms involved in the yoghurt fermentation [
], and by the
diﬀerent strains of the microorganisms employed .
Diﬀerences in the total TRP levels of yoghurt samples from the milk of diﬀerent animal species
are essentially due to the diﬀerent protein contents [
]. However, Yılmaz and Gökmen [
the presence of TRP, together with its derivatives, in the kynurenine pathway in commercial yoghurts,
suggesting a fermentation eﬀect on the level of TRP and its derivatives. Similar ﬁndings were reported
also by Bertazzo et al. [
], who observed TRP and its derivatives in both the serotonin and kynurenine
pathways in milk and fermented dairy products. The authors also reported an increase in the free
Molecules 2020,25, 5025 7 of 11
TRP levels with increasing fermentation, due to the proteolytic action of the added cultures, thus
corroborating the hypothesis of Yılmaz and Gökmen .
The highest TRP content of yoghurt from ewe milk could be due to the higher general TRP content
in ewe milk compared to milk from other animal species [
]. Similar values were reported for TRP in
cow and buﬀalo milk [
], while in some cases slightly lower levels of TRP were reported in goat milk
compared to other animal species [
]. Furthermore, the nutrient composition of yoghurts has been
shown to be highly dependent on the technological process .
The typical recommended daily intake for tryptophan has been set by FAO/WHO at 4 mg/kg of
body weight per day for adults , that is to say, 280 mg/day for a 70 kg adult .
According to the data reported in this study, three daily recommended servings of cow yoghurt
(125 grams for a serving [
]) supply almost 60.6% of the recommended TRP daily intake for adults,
while 84.3%, 48.8% and 70.9% of the recommended TRP daily intake is supplied by three servings of
ewe, goat and buﬀalo yoghurt, respectively. It is worth noting that only two Greek yoghurt servings
are enough for achieving the entire TRP recommended daily intake (108.5%), thanks to its high
TRP levels in yoghurts are generally of the same order of magnitude in milk [
], so a yoghurt
serving (with the exception of Greek yoghurt, due to its typical production process) provides almost the
same TRP intake of a milk serving. However, yoghurt’s shelf life is longer than milk’s, and yoghurt can also
be consumed by lactose-intolerant people. Furthermore, according to the study of
Bertazzo et al. 
the TRP content in yoghurt does not decrease during the storage, so the TRP intake is guaranteed until
the expiration date of the yoghurt. For all these reasons, yoghurt can be considered a good source of
TRP, and its consumption should be encouraged, not only for ensuring the recommended daily intake
of TRP, but also for its overall nutritive value, above all the probiotic properties and the calcium intake.
4. Materials and Methods
4.1. Samples Preparation
In total, 22 commercial brands of yoghurt from diﬀerent species were purchased in the local markets:
-12 diﬀerent brands of whole cow yoghurt;
-2 diﬀerent brands of whole ewe yoghurt;
-2 diﬀerent brands of whole goat yoghurt;
-2 diﬀerent brands of whole buﬀalo yoghurt;
-2 diﬀerent brands of whole cow yoghurt, lactose-free;
-2 diﬀerent brands of whole cow Greek yoghurt.
All samples were stored at 4 ◦C, as indicated on the label, prior to testing.
Tryptophan (TRP) was extracted by alkaline hydrolysis according to the method of Steven and
]. In brief, 8 mL of NaOH 4.2 M was added to 0.5 g of the yoghurt samples. An appropriate
amount of the internal standard (5-methyl-l-tryptophan) was added, then the oxygen was removed to
avoid the oxidative degradation of TRP. The hydrolysis was carried out at 110
C for 20 h. Afterwards,
the samples were cooled in an ice bath and neutralized with HCl, added with EtOH and ﬁlled to the
mark with phosphate buﬀer 0.2 M. The samples were ﬁltered (0.2 µm) prior to the UHPLC analysis.
All the 22 commercial brands of yoghurt were analyzed in triplicate.
l-tryptophan (TRP) and 5-methyl-triptophan (M-TRP) were purchased from Sigma (Sigma-Aldrich
Co., St. Louis, MO, USA).
The hydrochloric acid, sodium hydroxide and acetonitrile of HPLC grade were from Merck
(Darmstadt, Germany). All the other chemicals used were of analytical purity. All solvents were
ﬁltered through 0.2 µm membrane ﬁlters (Phenomenex Inc., Torrance, CA, USA).
Molecules 2020,25, 5025 8 of 11
4.3. UHPLC Equipment and Conditions
A Nexera UHPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with two LC-30AD
pumps, an RF-20A ﬂuorimetric detector and an SIL 30-AC autosampler, was employed for the
chromatographic analyses. A Shim-Pak ODS II column (2.2
m; 75 mm
2 mm i.d., Shimadzu
Corporation, Kyoto, Japan), maintained at 25
C during the analysis, was used for the separation.
The selected elution system, for a total of running time equal to 8.0 min, is reported in Table 5. The ﬂow
rate was set at 0.4 mL/min, while the injection volume was set at 1
L. Tryptophan and 5-methyl-
tryptophan were detected at 280 and 360 nm for excitation and emission wavelengths.
Table 5. Gradient elution system.
Time (Min) CH3CN % H2O % mL/min
0.0 10 90 0.4
3.0 80 20 0.4
4.0 10 90 0.6
7.9 10 90 0.6
8.0 10 90 0.4
4.4. Method Validation
Standard stock solution of TRP (21.2 mg in 25 mL of deionized water) was prepared. Individual
working standard solutions were prepared at six diﬀerent levels by dilution in deionized water
of the standard stock solution (0.085
Quantities of 100
L of the internal standard 5-methyl-l-tryptophan (856
g/mL in NaOH 0.05 M)
were added before hydrolyzing all the samples.
The method’s performance parameters were evaluated according to the EURACHEM Guide 2014 [
In more detail, the method precision was evaluated through the measurements of repeatability (intra-day
precision) and reproducibility (inter-day precision) upon direct injection of TRP standard solutions
at six levels, respectively, on three replicates and on three non-consecutive days. The repeatability
and reproducibility on yogurt samples at three levels, respectively, on three replicates and on three
non-consecutive days, were considered as well to evaluate the variation due to the entire analytical
procedure. Precision was expressed as relative standard deviation (RSD %).
The limit of detection (LOD) and limit of quantiﬁcation (LOQ) for TRP were calculated according
to the following equations: LOD =X
, LOQ =X
, where X
was the blank mean
value (n=10) and SD
the blank standard deviation [
]. Procedural blanks [
], where deionized
water was used in place of the yoghurt matrix, were used for determining LOD and LOQ.
The recovery was calculated by means of spiked samples, and expressed as relative spike recovery,
according to the following equation: R (%) =[(x
100. Here, x
is the mean value of the
spiked sample, x is the mean value of the non-spiked sample and x
is the added concentration [
All the analyses were performed in triplicate. Data were reported as mean value with standard
deviation (SD). Mean values were subjected to one-way analysis of variance (ANOVA), coupled with the
Tukey’s post hoc test. Statistical analysis was performed using the PAST Software (2.17c version) [
Conceptualization and methodology, P.M.; writing—original draft preparation, M.R.;
writing—review and editing, M.R. and P.M. All authors have read and agreed to the published version of
This research was funded by the Italian Ministry “Ministero delle Politiche Agricole, Alimentari e
Forestali (MiPAAF)” within the Project: QUALIFU “QualitàAlimentare e Funzionale”, D.M. 24292/7303/14.
Conﬂicts of Interest: The authors declare no conﬂict of interest.
Molecules 2020,25, 5025 9 of 11
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