LWT - Food Science and Technology 156 (2022) 112908

Contents lists available at ScienceDirect

LWT
journal homepage: www.elsevier.com/locate/lwt

Determination of biogenic amines formation by autochthonous lactic acid

bacteria from ‘Refošk’ grapes using different analytical methods
Jelena Topić Božič a, Lorena Butinar b, Martina Bergant Marušič a, Dorota Korte a,
Branka Mozetič Vodopivec b, *
a
Laboratory for Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, Rožna Dolina, 5000 Nova Gorica, Slovenia
b
Wine Research Centre, University of Nova Gorica, Glavni trg 8, 5271 Vipava, Slovenia

A R T I C L E I N F O A B S T R A C T

Keywords: Different analytical methods were tested and optimized for the determination of four biogenic amines (BA)
Grapes histamine, putrescine, cadaverine and tyramine produced by grape-associated lactic acid bacteria (LAB).
Wine-like matrices Autochthonous LAB were isolated from ‘Refošk’ grapes belonging to Slovenian-Italian Karst region as they
Biogenic amines
represent a potential pool of beneficial LAB starter bacteria for improving the typicality and quality of wine. Six
Autochthonous lactic acid bacteria
isolated strains were screened by multiplex PCR, of which four strains were positive for BA-forming genes
Analytical methods
(MKBT-49, MKBT-282, MKBT-568, MKBT-570). The production of BA was evaluated by thin-layer chromatog­
raphy (TLC), high-performance liquid chromatography coupled to diode array detection (HPLC-DAD) and
enzymatic method. The HPLC-DAD results showed that strain MKBT-49 (tyrdc+) had high tyramine production
(386.6 ± 0.14 mg/L), which was confirmed by TLC. The ability to produce putrescine was confirmed in strain
MKBT-282 by PCR, HPLC-DAD (16.4 ± 1.72 mg/L), and TLC. Histamine-producing ability was detected in strain
MKBT-570, with a concentration below the limit of detection of the HPLC-DAD (<0.2 mg/L), while the other two
methods were not sensitive enough for confirmation. This study shows that the production of BA can detected in
native LAB and that relatively simple method such as TLC can be used effectively for the initial screening.

1. Introduction
Biogenic amines (BA) such as histamine, cadaverine, putrescine and
tyramine are low-molecular weight organic bases that play a role as
regulators in human metabolism. They are frequently found in fer­
mented foods and beverages such as cheese, fish and wine (De Las Rivas,
Marcobal, & Muñoz, 2005; Henríquez-Aedo, Durán, Garcia, Hengst, &
Aranda, 2016; Perestrelo, Bordiga, Locatelli, Silva, & Câmara, 2020;
Žurga, Vahčić, Pasković, Banović, & Staver, 2019). Consumption of
foods and beverages containing high levels of BA can adversely affect
human health by causing hypertension, headache, diarrhoea, flushing
and nausea. Although no legal limits have been set for BA in wines, some
European countries have recommended maximum levels for the pres­
ence of histamine in wines, including Germany (2 mg/L), France (8
mg/L) and Belgium (6 mg/L) (Konakovsky et al., 2011; Martuscelli,
Arfelli, Manetta, & Suzzi, 2013; Martuscelli & Mastrocola, 2019; Vanda,
Miranda, Leca, & Marques, 2017).
Some lactic acid bacteria (LAB) naturally present in grapes, musts
and wines are producers of histamine, tyramine and putrescine (Coton
et al., 2010; Henríquez-Aedo et al., 2016; Landete, Ferrer, & Pardo,
2007; Moreno-Arribas, Polo, Jorganes, & Muñoz, 2003; Rosi, Nannelli,
& Giovani, 2009; Soufleros, Barrios, & Bertrand, 1998) and any new LAB
isolate should first be tested for the BA-production. LAB are a minor part
of the grape microbiota and are responsible for malolactic fermentation
(Barata, Malfeito-Ferreira, & Loureiro, 2012). They have a significant
impact on wine deacidification, flavour and microbiological stability
(Guo, Yang, Peng, & Han, 2015; Henríquez-Aedo et al., 2016) and thus
represent a natural pool of potentially beneficial starters in wine
industry.
LAB in wines is thought to originate from grapes or contaminated
equipment used in wineries to process grape juice and must. Although
LAB have been frequently isolated from grape must and wine (Battistelli
et al., 2020; Nisiotou et al., 2015; Petri, Pfannebecker, Fröhlich, &
König, 2013), there are few studies available on isolation of LAB from
grape surfaces (Bae, Fleet, & Heard, 2006; Nisiotou et al., 2015; Renouf,
Claisse, & Lonvaud-Funel, 2005; Taroub et al., 2019). The use of

* Corresponding author.
E-mail address: branka.mozetic@ung.si (B. Mozetič Vodopivec).

https://doi.org/10.1016/j.lwt.2021.112908
Received 23 July 2021; Received in revised form 29 September 2021; Accepted 30 November 2021
Available online 15 December 2021
0023-6438/© 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
J. Topić Božič et al.
autochthonous starter cultures could improve the typical characteristics
of wine varieties that might be lost when commercial starters are used
(Battistelli et al., 2020; Emer, Marques, Colla, & Reinehr, 2021).
BA determination methods can be generally divided into molecular
methods and phenotypic tests (Coton et al., 2010), providing either
qualitative or quantitative information. Molecular methods are pri­
marily based on polymerase chain reaction (PCR) (Coton & Coton, 2005;
Coton et al., 2010; Landete, De Las Rivas, Marcobal, & Muñoz, 2011;
Nannelli et al., 2008; Sebastian, Herr, Fischer, & König, 2011) and are
emerging as an alternative to traditional culture methods (Landete et al.,
2011). Phenotypic methods are primarily based on thin-layer chroma­
tography (TLC) and high-performance liquid chromatography (HPLC
methods) with a derivatization step using agents such as dansyl chloride
(Dns-Cl) or o-phthalaldehyde (Coton et al., 2010; Del Prete, Costantini,
Cecchini, Morassut, & Garcia-Moruno, 2009; García-Marino, Trigueros,
& Escribano-Bailón, 2010; Guo et al., 2015; Moreno-Arribas et al., 2003;
Rosi et al., 2009). HPLC and TLC methods are also acknowledged by the
International Organization of Vine and Wine (OIV) OIV 2009, 2010,
2015). A gas chromatography-mass spectrometry method (GS-MS) with
solid-phase microextraction was recently developed for the determina­
tion of BA in wines (Papageorgiou, Lambropoulou, Morrison,
Namieśnik, & Płotka-Wasylka, 2018). Enzymatic and immunoenzymatic
methods (Landete, Ferrer, & Pardo, 2004), along with development of
biosensors (Lange & Wittmann, 2002; Leonardo & Campàs, 2016), are
also gaining attention in the routine analysis of BA, as they are generally
considered low-cost methods that do not require specialized instru­
mentation and extensive sample preparation (Neofotistos, Tsagkaris,
Danezis, & Proestos, 2019; Papageorgiou, Lambropoulou, Morrison,
Kłodzińska, et al., 2018).
The main objective of this research was to develop and optimize
methods that can be used to confirm the BA-producing potential of LAB
in the traditional microbiology laboratory. Optimized in-house methods
such as multiplex PCR and analytical methods (TLC, HPLC-DAD were
evaluated for their ability to detect the BA-forming potential of LAB
strains. The second objective of the study was to isolate, characterize
and identify autochthonous LAB strains on ‘Refošk’ grapes from
Slovenian-Italian Karst region and to study their role in the formation of
BA. Strains that do not form BA could be utilized as starters to improve
typicality and quality of wines. To the best of our knowledge, this is the
first report on the BA-forming ability of LAB, isolated from ‘Refošk’
grapes from the Slovenian-Italian Karst region.
2. Materials and methods
2.1. Reference bacterial strains
Two reference bacterial strains, whose properties related to BA-
synthesis were known, were used as positive controls in this study and
were obtained from the University of Bordeaux, Institute for Vine and
Wine Science, France. Both control strains belonged to the genus
Lactobacillus. Strain IOEB 0006 (L. hilgardii) is a histamine producer
(Coton et al., 2010), while IOEB 9906 (L. brevis) is a putrescine producer
(Benkerroum, 2016; Romano et al., 2012).
2.2. Sample collection
Grapes of the ‘Refošk’ variety were sampled before the 2018 and
2019 harvests in vineyards in Slovenian and Italian Karst. The sampling
locations can be found in the Supplementary Information (Figure S2.1).
2.3. Isolation of LAB from grapes
Isolations followed Coton et al. (2010) with modifications regarding
to bacteria isolations from grapes. Therefore, 200 mL of sterile saline
solution (0.85% v/v NaCl) was added to the 80 grape berries. The grape
berries were shaken at 6 ◦ C and 250 rpm for 20 min. The suspension was
2
LWT 156 (2022) 112908
decanted and serially diluted. The dilutions were plated in duplicates on
the de Man, Rogosa and Sharpe (MRS) (Biolife, Milan, Italy) plates with
100 mg/L cycloheximide (Merck, Darmstadt, Germany) and 2% tomato
juice (Podravka, Koprivnica, Croatia) with the pH adjusted to 4.8.
Cycloheximide was added to prevent the growth of yeast and other
fungi. Petri dishes were incubated under anaerobic conditions at 28 ◦ C
for 3–5 days. After incubation LAB were counted and isolated.
Additionally, duplicates of LAB enrichment cultures were prepared
in the liquid MRS broth containing cycloheximide (100 mg/L), tomato
juice (2%), ethanol (5%) (VWR, Vienna, Austria) and pH 4.8. Ten mL of
the suspension was centrifuged (10 min, 6000 rpm) and the enrichment
MRS broth was added to the sediment. The culture was incubated under
anaerobic conditions at 28 ◦ C for 3–5 days. After incubation, cultures
were plated onto selective MRS plates and pure lactic acid cultures were
isolated. LAB isolates were stored as cryo-cultures in 30% (v/v) glycerol
at − 80 ◦ C. Details on origin of the isolated strains are provided in the
Supplementary Information (Table S2.2).
2.4. Isolation of genomic DNA
For the identification adnd genotypic characterization of the LAB
GeneElute DNA kit (Sigma-Aldrich, Steinheim, Germany) was used for
the extraction of DNA. Extraction was performed according to the
manufacturer’s instructions and samples were stored at − 20 ◦ C. LAB
identification was carried out using species-specific primers for PCR
assay. DNA sequencing of the purified PCR products was performed by
Eurofins Inc. (Hamburg, Germany). Detailed information on the primers
used, PCR conditions and identification of isolates is available in Sup­
plementary Information (Tables S2.3 and S2.4).
2.5. Genotypic characterization BA-forming potential of LAB
Multiplex PCR method by Coton et al. (2010) enabled simultaneous
detection of the histidine decarboxylase gene (hdc), the tyrosine decar­
boxylase gene (tyrdc), the ornithine decarboxylase gene and the agma­
tine decarboxylase gene (via odc or agdi). The method uses primer sets
(HDC3/HDC4, TD2/TD5, ODC1/ODC2 and AgD1/AgD2) associated
with the production of BA. A universal set of primers (BSF8/BSR1541)
for 16s rRNA was used for internal PCR control. PCR amplification was
performed by cyclic thermostat Robocycler® Gradient 96 temperature
cycler (Stratagene, USA) using the multiplex PCR program described in
Supplementary Information (Table S2.3). The sequences of the primers
used and the expected PCR product sizes are listed in the Supplementary
Information (Table S2.4).
The PCR products were visualized and their size was confirmed by
gel electrophoresis. Eighteen μL of the PCR product was applied to a
0.8% agarose gel containing ethidium bromide (Sigma-Aldrich, Stein­
heim, Germany) as a staining agent. Electrophoresis was run for 50 min
at 130 V in 1xTBE buffer (Tris-borate EDTA). The size and approximate
quantification of DNA fragments were assessed by comparison with the
DNA GeneRuler™ 1 Kb Plus DNA Ladder (ThermoFisher Scientific,
Waltham, Massachusetts, USA). Fragments were visualized using the
image capture system G-BOX for (Syngene, Cambridge, UK).
2.6. Phenotypic characterization of the BA-forming potential of LAB
2.6.1. Activation of microbial cultures
To promote enzyme induction prior to the screening, LAB strains
were sub-cultured in MRS broth with 10% tomato juice containing 0.1%
precursor amino acids (VWR, Vienna, Austria) and the co-factor pyri­
doxal-5-phosphate (0.005% (v/v) (Sigma-Aldrich, Steinheim, Germany)
(Bover-Cid & Holzapfel, 1999; Coton et al., 2010). The pre-cultures were
sub-cultured every two days into fresh activation medium and incubated
at 28 ◦ C. Sub-culturing was performed five times.
J. Topić Božič et al.
2.6.2. Screening medium for determination of BA-producing LAB strains
The amino acid decarboxylase medium (AADM) was developed by
Bover-Cid and Holzapfel (Bover-Cid & Holzapfel, 1999). It contains
0.005% pyridoxal-5-phosphate, since its presence as a cofactor has an
enhancing effect on amino acid decarboxylase activity, and 1% of amino
acid precursors. As pH indicator, 0.006% bromocresol purple (Sig­
ma-Aldrich, Steinheim, Germany) was used. AADM was used to quali­
tatively asses the ability of LAB to produce BA. The positive result was
indicated by a colour change of the medium to purple in response to the
pH shift of the indicator (Supplementary Information Figure S2.5). The
pH change resulted from the higher alkalinity of BA compared to the
precursor amino acids (Bover-Cid & Holzapfel, 1999).
Based on the results of multiplex PCR and AADM screening, the
ability of LAB to form BA was confirmed by analytical methods.
Analytical methods (TLC and HPLC) were also used to determine the
ability of LAB to form cadaverine, which was not possible using the
multiplex PCR method.
For quantitative determination of BA, 150 μL of LAB pre-culture
grown in activation medium was inoculated into 1.6 mL of AADM me­
dium without indicator. Inoculations were performed in triplicate in
deep-well microtiter plates and incubated anaerobically at 28 ◦ C for 7
days. The supernatant was analysed for the presence of BA using HPLC
and TLC. The presence of histamine in the supernatant was determined
by an enzymatic method.
2.6.3. Thin layer chromatography (TLC) analysis
BA-forming capacity of LAB was confirmed by the TLC method for
the determination of BA in wines (Romano et al., 2012) and applied to
the analysis of AADM culture media. The proposed method uses dansyl
chloride (Dns-Cl) (Alfa Aeser, Kandel, Germany) (0.5% w/v in acetone)
as derivatizing agent. Two hundred μL of the sample was mixed with
200 μL of saturated sodium hydrogen carbonate (VWR, Vienna, Austria)
and 400 μL of Dns-Cl solution. The reaction was incubated for 1 h at
55 ◦ C in thermoblock (Techne Dri-block DB-2D, Cole-Palmer, Stafford­
shire, UK) in the dark. After derivatization, the samples were left at room
temperature for a few minutes and then 100 μL of saturated NaCl was
added. Two hundred μL of iso-hexane (Merck, Darmstadt, Germany) was
added to the mixture and the mixture was shaken (200 rpm, 5 min) by a
bench shaker (IKA-Vibrax-VXR, IKA®-Werke, Stauben, Germany).
Twenty μL of the organic phase was deposited on silica gel 60 TLC
plates without fluorescent indicator (Merck, Darmstadt, Germany).
Samples were deposited at a distance of 15 mm from the bottom of the
plates, and the distance between aliquots was 12 mm. The TLC plates
were developed in a saturated glass chamber. A mixture of chloroform
and trimethylamine (both Merck, Darmstadt, Germany) in a 4:1 ratio
was used as the eluent. The total migration distance was 17 cm. The
plates typically took 90 min to develope. Prior to detection, the plates
were allowed to dry briefly under a fume hood at room temperature. The
TLC plates were illuminated at a wavelength of 312 nm and images were
acquired using the G-BOX system for image acquisition.
2.6.4. HPLC analysis
2.6.4.1. Sample preparation. Determination of cadaverine, histamine,
putrescine and tyramine produced by LAB followed the protocol of Mitar
et al. (2018). 250 μL of sample was mixed with 70 μL of saturated so­
dium hydrogen carbonate and 65 μL of 0.1M potassium hydroxide (both
VWR, Vienna, Austria). 1 mL of Dns-Cl was added (0.5% w/v in acetone)
and the mixture was incubated at 40 ◦ C in a thermoblock (Techne
Dri-block DB-2D, Cole-Palmer, Staffordshire, UK) for 45 min. After in­
cubation, 100 μL of ammonia solution (28–30%) (Sigma-Aldrich.
Steinheim, Germany) was added. The mixture was incubated for addi­
tional 30 min at room temperature in the dark. The reaction mixture was
filled up to 5 mL with acetonitrile and filtered through a 0.45 μm PTFE
filter (VWR, Vienna, Austria) prior to HPLC analysis. BA standards were
3
LWT 156 (2022) 112908
prepared in AADM medium used for screening the BA-forming potential
of LAB.
2.6.4.2. Chromatographic conditions. Agilent 1100 HPLC system equip­
ped with quaternary pump and diode array detector (Agilent Technol­
ogies, Palo Alto, California, USA) was used. Two separation columns
were tested, Supelco Discovery C18 (250 × 4.6 mm, 5 μm i.d) (Sigma-
Aldrich, Steinheim, Germany) and Kinetex EVO C18 (250 × 4.6 mm, 5
μm i.d.) (Phenomenex, Torrance, USA). The injection volume was 20 μL,
the detection wavelength was 254 nm, and the column temperature was
25 ◦ C. Gradient elution at a constant flow rate of 1 mL/min of mobile
phase A (water) and mobile phase B (acetonitrile) was used. The con­
ditions for the detection of cadaverine, histamine, putrescine and tyra­
mine were the same as previously described in Mitar et al. (2018): a
linear gradient 0–0.5 min 40% B, 0.5–25 min 40–80% B, 25–30 min
80–95% B, 30–34 min 95% B, 34–35 min 95-40% B, 35–37 min equil­
ibration to initial conditions.
2.6.5. Enzymatic method for the histamine determination
The enzymatic assay for histamine determination followed the pro­
tocol for the microplate reader of Landete et al. (2004), with some ad­
aptations. The mechanism of the enzymatic assay is based on the
sequential activity of diamine oxidase (DAO) which catalyzes the
degradation of histamine into imidazole acetaldehyde, ammonia and
hydrogen peroxide. Horseradish peroxidase (HRP) reacts with hydrogen
peroxidase and causes a colour change in the chromogen 3,3-diamino­
benzimidine (DAB) (Landete et al., 2004). The final optimized condi­
tions are as follows: 28.6 μL DAB (Sigma-Aldrich, Steinheim, Germany)
solution (0.2% w/v in phosphate-buffered saline (PBS buffer) (Sig­
ma-Aldrich, Steinheim, Germany) at pH 6.8, 3.45 μL HRP (Sigma-Al­
drich, Steinheim, Germany) (1000 IU/mL), 14.3 μL DAO
(Sigma-Aldrich, Steinheim, Germany) (1 IU/mL), 40 μL sample (AADM)
and 114.3 μL PBS buffer at pH 6.8. The final volume of the reaction
mixture was 200 μL. The mixture was incubated at 37 ◦ C with occasional
shaking in a microplate reader (Tecan Infinite F200, Tecan,
Switzerland). Absorbance was measured at 405 nm. The optimized in­
cubation time was 6 h.
2.6.6. Statistical analysis
Means and standard deviation were calculated for the enzymatic
assays and HPLC results. One-way ANOVA and Tukey’s (HSD) multiple
comparison tests were applied to assess statistical differences in the
enzymatic assay. The results of the statistical analyses were considered
as significant at p < 0.05. Statistical analyses were performed using
Addinsoft XLStat software (Addinsoft, New York, USA). R-squared
values were calculated with Microsoft Excel (Redmond, Washington,
United States). Microsoft Excel (Redmond, Washington, United States)
and OriginLab (Northampton, Massachusetts, USA) software was used
for data calculation, tabulation and graphical representations.
3. Results and discussion
3.1. HPLC determination of BA
Fig. 1 shows the chromatograms of 100 mg/L mixture of four BA
using the Supelco Discovery C18 column (Fig. 1A) and the Kinetex EVO
C18 (column (Fig. 1B) tested for the separation of cadaverine, histamine,
putrescine and tyramine. The separation order of the BA was the same
for both columns. With the Supelco Discovery C18 column, peak front­
ing was observed for all four BA. Complete resolution of cadaverine and
histamine was not achieved even when other gradients were applied.
Poor resolution of cadaverine and histamine was also observed in a
previous study when Restek Ultra IBD C18 column (250 × 4.6 mm, 5 μm
i.d.) was used with Ultra IBD guard column (10 × 4 mm, 5 μm i.d.)
(Restek, Bellefonte, PA, USA) (Mitar et al., 2018). The use of the Kinetex
J. Topić Božič et al. LWT 156 (2022) 112908

Table 2
Calibration parameters for biogenic amines detected by HPLC-DAD method.
Biogenic amine PUTRESCINE CADAVERINE HISTAMINE TYRAMINE

Retention time 16.7 17.8 18.2 23.0

[min]
Linearity range 0.5–500 0.5–1000 0.5–1000 5–500
[mg/L]
Slope [k] 2.3561 2.4342 3.4505 0.0967
Intercept [n] 22.993 51.457 47.009 2.0609
R2 0.9960 0.9938 0.9957 0.9951
LOD [mg/L] 0.2 0.2 0.2 0.8
LOQ [mg/L] 0.7 0.7 0.6 2.9
System precision 5.3 3.2 3.7 4.2
[RSD - % using
standard
solutions of 1
mg/L]

− 20 ◦ C (B) is shown in the Supplementary Information (Figure S3.3).

3.2. TLC determination of BA

Mixtures of BA and solutions containing individual BA were spotted

Fig. 1. Chromatograms of 100 mg/L mixture of biogenic amines. Fig. 1A shows
chromatogram obtained when Supelco Discovery C18 column was used for separa­
tion. Fig. 1B represent separation with Kinetex EVO C18 column. Numbers 1 – 4
correspond to biogenic amines; 1 – putrescine, 2 – cadaverine, 3 – histamine, 4 –
tyramine. Chromatograms with single biogenic amines and blanks are shown in the
Supplementary Information (S3.3).
EVO C18 column resulted in better peak resolution as the cadaverine
and histamine peaks were separated. Furthermore, the use of the Kinetex
EVO C18 column resulted in higher peak heights and thus improved
sensitivity of the method (Table 1). The increase in peak height was
more than 100% for putrescine, cadaverine and histamine and almost
70% for tyramine. Due to its better performance, the Kinetex EVO C18
column was selected for further analyses.
The calibration parameters for the tested BA are listed in Table 2. The
method is similarly sensitive for putrescine, cadaverine and histamine
(LODs 0.2 mg/L), although the linearity range is wider for cadaverine
and histamine.The method is least sensitive for tyramine, with LOD and
LOQs at least four times higher compared to other three tested BA. The
method could be further improved by adjusting the gradient conditions
to improve the sensitivity of the method for tyramine.
Stability of the stored samples was also tested to determine whether
the samples could be derivatized in advance and stored at − 20 ◦ C until
analysis. This is of great importance as equipment is not always readily
available to researchers. It was observed that the difference in peak
height between the sample analysed on the day of derivatization (t = 0)
and after 14 days of storage at − 20 ◦ C (t = 14) was less than 1.1% for all
four BA, confirming the assumption that the use of Dns-Cl as a deriva­
tizing agent produces stable derivatives (Ordóñez, Troncoso, García-­
Parrilla, & Callejón, 2016; Peña-Gallego, Hernández-Orte, Cacho, &
Ferreira, 2012). Chromatogram of the 1000 mg/L mixture of BA ana­
lysed on the day of derivatization (A) and after 14 days of storage at
onto the TLC plate to determine the retention factors (Rf; the ratio be­
tween the distance that the spot of the compound travels and the dis­
tance the eluent travels) of histamine, cadaverine, putrescine and
tyramine. Plate A in Fig. 2 shows the spots identified as histamine,
cadaverine, putrescine and tyramine. The Rf value increased from
putrescine < tyramine < cadaverine < histamine, which is also an
indication of the increasing polarity of the compounds. Com­
pounds with higher polarity adhere more strongly to the adsor­
bent, resulting in lower Rf value.
TLC plates from 5–2500 mg/L are shown in Fig. 2 (B–D). The
method is least sensitive for tyramine as the TLC spot is visible at a
concentration of 50 mg/L. Spots of cadaverine and putrescine are visible
already at 5 mg/L, while histamine is visible at 10 mg/L. Both the HPLC
and TLC methods are least sensitive for the determination of tyramine.
On the other hand, the TLC method is most sensitive for cadaverine and
putrescine, while the HPLC method is most sensitive for the determi­
nation of histamine. Latorre-Moratalla, Bover-Cid, Veciana-Nogués, and
Vidal-Carou (2009) developed a semi-quantitative TLC method to
determine the BA-forming potential of known LAB isolates. Compared
with our method, the time required for dansylation is shorter (18 min),
at a higher temperature (100 ◦ C) and a lower detection limit (1 mg/L).
The TLC results were in agreement with those obtained by the HPLC
method (Latorre-Moratalla et al., 2009). Based on the results reported by
Latorre-Moratalla et al. (2009), the proposed TLC method should be
further investigated to possibly improve its performance (Latorre-Mor­
atalla et al., 2009).
Samples were reapplied to the TLC plate after 14 days of storage at
− 20 ◦ C to determine if the derivatives were stable and could be analysed
later (Figure S3.4 in the Supplementary Information). The results show
that the BA peaks are still visible after 14 days. This suggests that the
method can also be used with stored samples, which is particularly
useful when equipment is not readily available, or in collaboration with
other institutions where samples can be prepared at one location and
analysed at another.

Table 1
Comparison of Supelco Discovery C18 and Kinetex EVO C18 columns for the HPLC analysis (mean ± SD; n = 3).
Biogenic amine PUTRESCINE CADAVERINE HISTAMINE TYRAMINE

Supelco Discovery C18 Retention time [min] 20.3 ± 0.03 21.5 ± 0.01 21.8 ± 0.04 26.9 ± 0.01
Average Peak Height of 100 mg/L solution 17.4 ± 0.41 26.3 ± 2.62 24.3 ± 1.61 0.6 ± 0.10
[mAU]
Kinetex EVO C18 Retention time [min] 16.7 ± 0.01 17.8 ± 0.01 18.2 ± 0.03 23.0 ± 0.04
Average Peak Height of 100 mg/L solution [mAU] 38.5 ± 0.42 44.5 ± 4.53 49.9 ± 2.91 1.3 ± 0.07
Increase in peak height [%] 121.3 69.4 105.1 105.4

4
J. Topić Božič et al. LWT 156 (2022) 112908

Fig. 2. TLC plate A is spotted with 1000 mg/L concentration of biogenic amines. Numbers 1–6 correspond to different samples; 1 – blank (AADM medium), 2–1000
mg/L histamine solution, 3–1000 mg/L cadaverine solution, 4–1000 mg/L putrescine solution, 5–1000 mg/L tyramine solution, 6–1000 mg/L mixture solution
(histamine, cadaverine, putrescine, tyramine). TLC plates of the mixtures of biogenic amines in the range of 5–2500 mg/L are shown in the plates B-D. Tested samples
and the blank sample (derivatized AADM medium) were spotted on TLC plates in triplicates.

3.3. Enzymatic determination of histamine
Fig. 3A shows regression curves of the histamine standard solutions
in AADM as a function of incubation time. The absorbance of the blank
(non-spiked AADM) was subtracted from the histamine-spiked AADM.
An incubation time of 6 h gave the best R2 in the linear regression
between histamine concentration and absorbance of the medium, sug­
gesting that the enzyme occupied all available active sites. Shorter in­
cubation times (two and 4 h) resulted in a decrease of R2 and lower
sensitivity of the method, as a lower R2 of the histamine calibration
curve is observed with shorter incubation times.
An 8-h incubation also showed a decrease in R2 compared to a 6-h
incubation, suggesting colour saturation at higher histamine concen­
trations. Similarly, Landete et al. (2004) showed that R2 of histamine
calibration curve was dependent on incubation time (Landete et al.,
2004). The LOD was calculated as 25 mg/L because at this concentration
the difference between sample and blank was higher than 0.1. The
concentration ranges where the method is applicable are between 25

Fig. 3. R2 of the histamine calibration curve in relation to the incubation time (Fig. 3A) and influence of increased histamine concentration on the absorbance and linearity of
the curve (Fig. 3B).

5
J. Topić Božič et al.
and 300 mg/L. Above 300 mg/L, the relationship between concentration
and absorbance is no longer linear (Fig. 3B). Nevertheless, the positive
reaction, i.e. the presence of histamine, can be detected but not
quantified.
The AADM medium was spiked with 100 mg/L of single BA and a
mixture of four BA at a concentration of 100 mg/L to test the applica­
bility of the method for the potential determination of multiple BA
(Fig. 4). The absorbance values of AADM spiked with a mixture of four
BA and AADM spiked with histamine were significantly different from
the absorbance values of AADM spiked with cadaverine, putrescine or
tyramine (Tukey’s multiple comparison test), while there was no sta­
tistical difference between AADM spiked with histamine and a mixture
of BAs. The results suggest that the method is only suitable for the
determination of histamine.
3.4. Comparison of the HPLC, TLC and enzymatic method
The comparison of the methods is shown in Table 3. The HPLC
method has been shown to be the most sensitive method, allowing
multiple BA detections. One of the main advantages of HPLC is auto­
mation, with the possibility of running samples overnight, thus short­
ening the analysis time. Disadvantages include high solvent
consumption and the need for skilled technicians to operate the equip­
ment. It is also the most expensive method and not always available in
microbiology laboratories. Although the enzymatic method is the
simplest and the greenest according to the AGREE metric approach
(Pena-Pereira, Wojnowski, & Tobiszewski, 2020) thanks to its sample
size and preparation, minituarization, solvent consumption and auto­
mation, the major disadvantage of the method is that it is only suitable
for the determination of histamine and has low sensitivity (LOD 25
mg/L). Further studies could be conducted to test different sources and
combinations of DAO enzymes to extend the applicability of the enzy­
matic method for the determination of several BA (Verma, Hooda,
Gahlaut, Gothwal, & Hooda, 2020). TLC is relatively sensitive, does not
require specialized equipment and is suitable for use in conventional
microbiological laboratories. Depending on the type of TLC chamber
used, a larger number of plates can be developed simultaneously (e.g. 5)
thus shortening analysis time and solvent consumption. Additionally,
automation and shorter analysis times are possible by using automated
TLC samplers. TLC can also be used to visually confirm the produced BA,
which does not require special and time-consuming processing of the
Fig. 4. Absorbance values of AADM spiked with single biogenic amines and a mixture of four biogenic amines. Abbreviations HIS, PUT, CAD, and TYR stand for histamine,
putrescine, cadaverine and tyramine respectively. Values with the same letter are not statistically different (p < 0.05).
6
LWT 156 (2022) 112908
result. The TLC method can be used to analyse a large number of samples
in less time than the HPLC method (Latorre-Moratalla et al., 2009).
Although the TLC method itself is a semi-quantitative method, it can
become a quantitative method with densitometry. Romano et al. (2012)
successfully developed a TLC-densitometry procedure for the quantita­
tive analysis of putrescine, cadaverine, histamine, and tyramine in wine
(Romano et al., 2012).
3.5. Determination of LAB ability to produce BA
An important goal of this research was to develop and optimize
methods for evaluating BA production in LAB strains. The selected
methods were first tested on two known BA-producing LAB strains. The
results are presented in Table 4. Strains IOEB 0006 and IOEB 9906
produce histamine and putrescine, respectively, at excessive concen­
trations under desirable conditions (Coton et al., 2010; Romano et al.,
2012). The production of histamine in IOEB 0006 strain was confirmed
by all three methods used. IOEB 0006 produced histamine at an exces­
sive concentration (>1000 mg/L), which was above the linear range for
both HPLC-DAD and enzymatic methods. A large spot also appeared on
the TLC plate confirming the production of histamine. Similarly, IOEB
9906 produced putrescine at a concentration above 1000 mg/L. The TLC
and HPLC-DAD methods were successfully applied to detect putrescine,
although quantification was not possible because the concentrations
were above the linearity range. Both the TLC and enzymatic methods are
suitable as no special equipment is required and sample preparation is
straightforward, with the TLC method having higher sensitivity. The
next step in the BA analysis was to test isolated autochthonous LAB
strains for their BA-producting ability.
3.6. Determination of BA-producing capabilities of LAB grape isolates
The developed methods were tested on six LAB grape isolates from
the Slovenian-Italy Karst region (Table 4). There is not much data on
grape-associated LAB in Slovenia, although the grape microbiome may
represent a pool of potential LAB starters for the food industry. The
search for microorganisms from indigenous flora is important from the
quality and differentiation aspects. Autochthonous starter cultures can
not only ensure the safety of wines, but also the quality and uniqueness,
i.e. regional typicality of wines (Emer et al., 2021). This was demon­
strated in the study by Battistelli et al. (2020), where an indigenous LAB
J. Topić Božič et al. LWT 156 (2022) 112908

Table 3
Comparison of the HPLC-DAD, TLC and enzymatic method for detection of biogenic amines. Abbreviations HIS, PUT, CAD, and TYR stand for histamine, putrescine,
cadaverine and tyramine respectively.
METHOD HPLC-DAD TLC ENZYMATIC

SENSITIVITY LOD LOD LOD

<1 mg/L for PUT, TYR, CAD, HIS 5 mg/L PUT, CAD, 25 mg/L HIS
10 mg/L HIS
50 mg/L TYR
SAMPLE PREPARATION app. 2h app. 1.5h app. 15 min
TIME OF ANALYSIS 37 min per sample app. app. 90 min per sample 6h per sample
2 h per triplicate app. 90 min per triplicate 6h per triplicate
4 samples in a triplicate per 90 min 6h for 32 samples in a
triplicate
SOLVENT CONSUMPTION HIGHEST MIDDLE LOWEST
37 mL per sample 30 mL per sample 0.16 mL per sample
Larger than 100 mL for triplicate (30 mL per triplicate) (0.48 per triplicate)
12 samples per TLC plate
STABILITY OF PREPARED POSSIBLE REINJECTION OF DERIVATES (after 14 days, POSSIBLE RESPOTTING OF DERIVATES (after 14 days, NO
SAMPLES storage at − 20 ◦ C) storage at − 20 ◦ C)
- No significant change in the peak area and height - Similarly large spots appear
DETECTION OF BIOGENIC PUT, CAD, TYR, HIS PUT, CAD, TYR, HIS HIS
AMINES

starter culture was used for the production of Montepulciano d’Abruzzo
wine. The wines produced retained the typical characteristics and were
also histamine-free (Battistelli et al., 2020).
The results obtained by PCR showed that four of the six isolates
possessed BA-producing genes. The results agreed well with the HPLC
and TLC identification and quantification. Production of BA was not
detected in the strains MKBT-572 and MKBT-555, which were both
negative in PCR analysis. The absence of BA was confirmed with TLC,
HPLC and enzymatic method for histamine. HPLC results showed that
strain MKBT-49 showed high tyramine production, 386.6 ± 0.14 mg/L,
which was additionally confirmed by semi-quantitativeTLC as tyramine
spot was visible. Due to the largeness of the spot, it can be concluded
that the concentration was above 250 mg/L (Table 4). Although the hdc
+ gene was found in strain MKBT-570, the histamine concentration was
below LOD of the HPLC method (0.2 mg/L). TLC and enzymatic methods
were not sensitive enough to confirm histamine production. The odc +
gene was confirmed also in strains MKBT-568 and MKBT-282. In MKBT-
568 strain, the concentration of putrescine was below LOQ of the HPLC
method (<0.7 mg/L), while the TLC method was not sensitive enough
for the confirmation of putrescine production ability. MKBT-282 strain
produced putrescine at a concentration of 16.4 ± 1.72 mg/L. The pro­
duction was also confirmed with semi-quantitative TLC method as the
size of the spot was bigger than the one for 10 mg/L.
Most of the LAB research in wine science focuses on the strains iso­
lated from grape juice, grape must and wine (Battistelli et al., 2020;
Henríquez-Aedo et al., 2016; Sebastian et al., 2011). Very few research
efforts have focused on LAB isolated directly from grapes, which has
been the primary focus of our research. Bae et al. (2006) isolated LAB
from grapes of five red varieties (Cabernet Sauvignon, Merlot, Pinot
Noir, Shiraz, Tyrian) and three white grape varieties (Chardonnay,
Sauvignon Blanc, Semillon) from Australian vineyards, but no data on
BA production were reported (Bae et al., 2006). Taroub et al. (2019)
isolated 18 LAB strains from two red table grape varieties, Red Globe
and Cardinal. No data were reported on possible BA production by the
isolated strains (Taroub et al., 2019). Nisiotou et al. (2015) isolated LAB
from Greek grape varieties (“Vilana” (white), “Mandilaria”, “Kotsifali”,
“Agiorgitiko” (red)) and wines. The ability of BA production has been
demonstrated only in a few LAB strains isolated from a winery tank, but
not in the grape-associated LAB strains (Nisiotou et al., 2015). Data on
the ability of LAB to produce BA are important when isolates are
considered as potential starters in wine production or as
bio-preservatives or probiotics and should be considered when
researching LAB strains.
3.7. Conclusion and future trends
Three methods were developed and optimized for the confirmation
of BA-production ability of autochthonous LAB isolates. Although the
HPLC-DAD method is already the most sensitive, it can be further
improved, particularly for tyramine detection. The TLC method proved
to be sensitive enough to be used as a complementary tool to traditional
PCR and culture methods, especially in microbiological laboratories,
since minimal equipment is required and the results are easy to process.
Compared to a single sample run with HPLC, the TLC method can pro­
cess a large number of samples in a shorter time. The main disadvantage
of the enzymatic method is that it is only suitable for the determination
of histamine and has a relatively low sensitivity, which makes it less
suitable for broad BA detection.
The developed methods were successfully applied to determine the
BA-forming potential of six LAB isolates from “Refošk” grapes from the
Karst region, a well-known wine region. The environment from which
LAB were isolated could be extended to isolates from grape must and
wine, expanding the pool of potential starters for malolactic fermenta­
tion. The search for autochthonous LAB starters is important to ensure
the wine differentiation in terms of origin, to maintain its quality and to
guarantee the safety of the wine.
Our study shows that indigenous LAB strains have the ability to
produce BA. For this reason, any potential LAB starter should be first
tested for BA production. For initial fast and low cost screening that can
be performed in conventional microbiological laboratories the TLC
method should be a method of choice as it does not require special
instrumentation and sensitivity is satisfactory. HPLC could be used as a
confirmatory method where concentration of BA produced by LAB
would be below the LOD of the TLC method.
CRediT authorship contribution statement
Jelena Topić Božič: Investigation, Formal analysis, Validation,
Visualization, Writing – original draft. Lorena Butinar: Conceptuali­
zation, Methodology, Resources, Funding acquisition. Martina Bergant
Marušič: Resources, Methodology, Writing – review & editing. Dorota
Korte: Writing – review & editing, Supervision. Branka Mozetič
Vodopivec: Conceptualization, Methodology, Writing – review & edit­
ing, Supervision, Funding acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence

7
J. Topić Božič et al. LWT 156 (2022) 112908

Table 4
Biogenic amine-forming ability of control strains (IOE 0006, IOEB9906) and native LAB strains isolated from grapes tested with PCR, HPLC, TLC and enzymatic
method. Abbreviations TYR, PUT and HIS stand for tyramine, putrescine and histamine, respectively.
STRAIN PCR HPLC TLC Enzymatic

IOEB0006 hdc+ Positive reaction:

15
Absorbance at 405 nm after subtracted
blank:
0.66 ± 0.01

IOEB9906 odc+ Not applicable

34,35

MKBT-49 tyrdc+ Not applicable

(continued on next page)

8
J. Topić Božič et al. LWT 156 (2022) 112908

Table 4 (continued )
STRAIN PCR HPLC TLC Enzymatic

MKBT- odc+ Not applicable

282

MKBT- n.d. HIS: not detected

555

MKBT- odc+ Not applicable

568
(continued on next page)

9
J. Topić Božič et al. LWT 156 (2022) 112908

Table 4 (continued )
STRAIN PCR HPLC TLC Enzymatic

PUT: not detected

MKBT- his+ HIS: not detected

570

HIS: not detected

MKBT- n.d. HIS: not detected

572

10
J. Topić Božič et al.
the work reported in this paper.
Acknowledgment
Authors acknowledge Slovenian Research Agency (ARRS) (Grant
number: ARRS-MR-LP-2017/393 and Agrotur/Karst agrotourism proj­
ect, implemented within the Cross-Border Cooperation Programme Ita­
ly–Slovenia 2014–2020, funded by European Regional Development
Fund and national funds for financial support.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.lwt.2021.112908.
References
Bae, S., Fleet, G. H., & Heard, G. M. (2006). Lactic acid bacteria associated with wine
grapes from several Australian vineyards. Journal of Applied Microbiology, 100(4),
712–727. https://doi.org/10.1111/j.1365-2672.2006.02890.x
Barata, A., Malfeito-Ferreira, M., & Loureiro, V. (2012). The microbial ecology of wine
grape berries. International Journal of Food Microbiology, 153(3), 243–259. https://
doi.org/10.1016/j.ijfoodmicro.2011.11.025
Battistelli, N., Perpetuini, G., Perla, C., Arfelli, G., Zulli, C., Rossetti, A. P., et al. (2020).
Characterization of natural Oenococcus oeni strains for Montepulciano d’Abruzzo
organic wine production. European Food Research and Technology, 246(5),
1031–1039. https://doi.org/10.1007/s00217-020-03466-3
Benkerroum, N. (2016). Biogenic amines in dairy products: Origin, incidence, and
control means. Comprehensive Reviews in Food Science and Food Safety, 15(4),
801–826. https://doi.org/10.1111/1541-4337.12212
Bover-Cid, S., & Holzapfel, W. H. (1999). Improved screening procedure for biogenic
amine production by lactic acid bacteria. International Journal of Food Microbiology,
53(1), 33–41. https://doi.org/10.1016/S0168-1605(99)00152-X
Coton, E., & Coton, M. (2005). Multiplex PCR for colony direct detection of Gram-
positive histamine- and tyramine-producing bacteria. Journal of Microbiological
Methods, 63(3), 296–304. https://doi.org/10.1016/j.mimet.2005.04.001
Coton, M., Romano, A., Spano, G., Ziegler, K., Vetrana, C., Desmarais, C., et al. (2010).
Occurrence of biogenic amine-forming lactic acid bacteria in wine and cider. Food
Microbiology, 27(8), 1078–1085. https://doi.org/10.1016/j.fm.2010.07.012
De Las Rivas, B., Marcobal, Á., & Muñoz, R. (2005). Improved multiplex-PCR method for
the simultaneous detection of food bacteria producing biogenic amines. FEMS
Microbiology Letters, 244(2), 367–372. https://doi.org/10.1016/j.
femsle.2005.02.012
Del Prete, V., Costantini, A., Cecchini, F., Morassut, M., & Garcia-Moruno, E. (2009).
Occurrence of biogenic amines in wine: The role of grapes. Food Chemistry, 112(2),
474–481. https://doi.org/10.1016/j.foodchem.2008.05.102
Emer, C. D., Marques, S., Colla, L. M., & Reinehr, C. O. (2021). Biogenic amines and the
importance of starter cultures for malolactic fermentation. Australian Journal of
Grape and Wine Research, 27(1), 26–33. https://doi.org/10.1111/ajgw.12462
García-Marino, M., Trigueros, Á., & Escribano-Bailón Teresa, T. (2010). Influence of
oenological practices on the formation of biogenic amines in quality red wines.
Journal of Food Composition and Analysis, 23(5), 455–462. https://doi.org/10.1016/j.
jfca.2010.02.003
Guo, Y. Y., Yang, Y. P., Peng, Q., & Han, Y. (2015). Biogenic amines in wine: A review.
International Journal of Food Science and Technology, 50(7), 1523–1532. https://doi.
org/10.1111/ijfs.12833
Henríquez-Aedo, K., Durán, D., Garcia, A., Hengst, M. B., & Aranda, M. (2016).
Identification of biogenic amines-producing lactic acid bacteria isolated from
spontaneous malolactic fermentation of Chilean red wines. Lebensmittel-Wissenschaft
und -Technologie- Food Science and Technology, 68, 183–189. https://doi.org/
10.1016/j.lwt.2015.12.003
International Organization of Wine and Vine (OIV). (2009). Analysis of biogenic amines
in musts and wines using HPLC. Compendium of International Methods of Analysis:
Method OIV-MA-AS315-18, 1–12. Retrieved from http://www.oiv.int/. (Accessed 26
June 2021).
International Organization of Wine and Vine (OIV). (2010). Qualitative method for
detection of biogenic amines produced by lactic acid bacteria by thin-layer
chromatography: Oeno 348/2010. International Oenological Codex, 1–3. Retrieved
from http://www.oiv.int/. (Accessed 26 June 2021).
International Organization of Wine and Vine (OIV). (2015). Method of determination of
biogenic amines in wine by high-performance liquid chromatography with
photodiode array detection. Compendium of International Methods of Analysis: Method
OIV-MA-AS315-26, 1–16. Retrieved from http://www.oiv.int/. (Accessed 26 June
2021).
Konakovsky, V., Focke, M., Hoffmann-Sommergruber, K., Schmid, R., Scheiner, O.,
Moser, P., et al. (2011). Levels of histamine and other biogenic amines in high-
quality red wines. Food Additives & Contaminants Part A Chemistry, Analysis, Control,
Exposure and Risk Assessment, 28(4), 408–416. https://doi.org/10.1080/
19440049.2010.551421
11
LWT 156 (2022) 112908
Landete, J. M., De Las Rivas, B., Marcobal, A., & Muñoz, R. (2011). PCR methods for the
detection of biogenic amine-producing bacteria on wine. Annals of Microbiology, 61,
159–166. https://doi.org/10.1007/s13213-010-0068-6
Landete, J. M., Ferrer, S., & Pardo, I. (2004). Improved enzymatic method for the rapid
determination of histamine in wine. Food Additives & Contaminants, 21(12),
1149–1154. https://doi.org/10.1080/02652030400019737
Landete, J. M. M., Ferrer, S., & Pardo, I. (2007). Biogenic amine production by lactic acid
bacteria, acetic bacteria and yeast isolated from wine. Food Control, 18(12),
1569–1574. https://doi.org/10.1016/j.foodcont.2006.12.008
Lange, J., & Wittmann, C. (2002). Enzyme sensor array for the determination of biogenic
amines in food samples. Analytical and Bioanalytical Chemistry, 372(2), 276–283.
https://doi.org/10.1007/s00216-001-1130-9
Latorre-Moratalla, M. L., Bover-Cid, S., Veciana-Nogués, T., & Vidal-Carou, M. C. (2009).
Thin-layer chromatography for the identification and semi-quantification of biogenic
amines produced by bacteria. Journal of Chromatography A, 1216(18), 4128–4132.
https://doi.org/10.1016/j.chroma.2009.02.045
Leonardo, S., & Campàs, M. (2016). Electrochemical enzyme sensor arrays for the
detection of the biogenic amines histamine, putrescine and cadaverine using
magnetic beads as immobilisation supports. Microchimica Acta, 183(6), 1881–1890.
https://doi.org/10.1007/s00604-016-1821-8
Martuscelli, M., Arfelli, G., Manetta, A. C., & Suzzi, G. (2013). Biogenic amines content as
a measure of the quality of wines of Abruzzo (Italy). Food Chemistry, 140(3),
590–597. https://doi.org/10.1016/j.foodchem.2013.01.008
Martuscelli, M., & Mastrocola, D. (2019). Biogenic amines: A claim for wines. In
C. Proestos (Ed.), Biogenic amines. IntechOpen. https://doi.org/10.5772/
intechopen.80362
Mitar, I., Ljubenkov, I., Rohtek, N., Prkíc, A., Andelíc, I., & Vuletíc, N. (2018). The
content of biogenic amines in Croatian wines of different geographical origins.
Molecules, 23(10). https://doi.org/10.3390/molecules23102570
Moreno-Arribas, M. V., Polo, M. C., Jorganes, F., & Muñoz, R. (2003). Screening of
biogenic amine production by lactic acid bacteria isolated from grape must and
wine. International Journal of Food Microbiology, 84(1), 117–123. https://doi.org/
10.1016/S0168-1605(02)00391-4
Nannelli, F., Claisse, O., Gindreau, E., De Revel, G., Lonvaud-Funel, A., & Lucas, P. M.
(2008). Determination of lactic acid bacteria producing biogenic amines in wine by
quantitative PCR methods. Letters in Applied Microbiology, 47(6), 594–599. https://
doi.org/10.1111/j.1472-765X.2008.02472.x
Neofotistos, G. A.-D., Tsagkaris, S. A., Danezis, P. G., & Proestos, C. (2019). Emerging
trends in biogenic amines analysis. In C. Proestos (Ed.), Biogenic amines. IntechOpen.
https://doi.org/10.5772/intechopen.81274
Nisiotou, A. A., Dourou, D., Filippousi, M. E., Diamantea, E., Fragkoulis, P., Tassou, C.,
et al. (2015). Genetic and technological characterisation of vineyard- and winery-
associated lactic acid bacteria, 2015 BioMed Research International, 1–8. https://doi.
org/10.1155/2015/508254. Article ID 508254.
Ordóñez, J. L., Troncoso, A. M., García-Parrilla, M. D. C., & Callejón, R. M. (2016).
Recent trends in the determination of biogenic amines in fermented beverages – a
review. Analytica Chimica Acta, 939, 10–25. https://doi.org/10.1016/j.
aca.2016.07.045
Papageorgiou, M., Lambropoulou, D., Morrison, C., Kłodzińska, E., Namieśnik, J., &
Płotka-Wasylka, J. (2018a). Literature update of analytical methods for biogenic
amines determination in food and beverages. TRAC Trends in Analytical Chemistry,
98, 128–142. https://doi.org/10.1016/j.trac.2017.11.001
Papageorgiou, M., Lambropoulou, D., Morrison, C., Namieśnik, J., & Płotka-Wasylka, J.
(2018b). Direct solid phase microextraction combined with gas chromatography –
mass spectrometry for the determination of biogenic amines in wine. Talanta, 183,
276–282. https://doi.org/10.1016/j.talanta.2018.02.006
Peña-Gallego, A., Hernández-Orte, P., Cacho, J., & Ferreira, V. (2012). High-performance
liquid chromatography analysis of amines in must and wine: A review. Food Reviews
International, 28(1), 71–96. https://doi.org/10.1080/87559129.2011.594973
Pena-Pereira, F., Wojnowski, W., & Tobiszewski, M. (2020). AGREE—analytical
GREEnness metric approach and software. Analytical Chemistry, 92(14),
10076–10082. https://doi.org/10.1021/ACS.ANALCHEM.0C01887
Perestrelo, R., Bordiga, M., Locatelli, M., Silva, C., & Câmara, J. S. (2020). Polyphenols,
biogenic amines and amino acids patterns in Verdelho wines according to vintage.
Microchemical Journal, 153, Article 104383. https://doi.org/10.1016/j.
microc.2019.104383
Petri, A., Pfannebecker, J., Fröhlich, J., & König, H. (2013). Fast identification of wine
related lactic acid bacteria by multiplex PCR. Food Microbiology, 33(1), 48–54.
https://doi.org/10.1016/j.fm.2012.08.011
Renouf, V., Claisse, O., & Lonvaud-Funel, A. (2005). Understanding the microbial
ecosystem on the grape berry surface through numeration and identification of yeast
and bacteria. Australian Journal of Grape and Wine Research, 11(3), 316–327. https://
doi.org/10.1111/j.1755-0238.2005.tb00031.x
Romano, A., Klebanowski, H., La Guerche, S., Beneduce, L., Spano, G., Murat, M. L., et al.
(2012). Determination of biogenic amines in wine by thin-layer chromatography/
densitometry. Food Chemistry, 135(3), 1392–1396. https://doi.org/10.1016/j.
foodchem.2012.06.022
Rosi, I., Nannelli, F., & Giovani, G. (2009). Biogenic amine production by Oenococcus oeni
during malolactic fermentation of wines obtained using different strains of
Saccharomyces cerevisiae. Lebensmittel-Wissenschaft und -Technologie- Food Science and
Technology, 42(2), 525–530. https://doi.org/10.1016/j.lwt.2008.08.004
Sebastian, P., Herr, P., Fischer, U., & König, H. (2011). Molecular identification of lactic
acid bacteria occurring in must and wine. South African Journal of Enology and
Viticulture, 32(2), 300–309. https://doi.org/10.21548/32-2-1390
J. Topić Božič et al.
Soufleros, E., Barrios, M. L., & Bertrand, A. (1998). Correlation between the content of
biogenic amines and other wine compounds. American Journal of Enology and
Viticulture, 49(3), 266–278.
Taroub, B., Salma, L., Manel, Z., Ouzari, H. I., Hamdi, Z., & Moktar, H. (2019). Isolation
of lactic acid bacteria from grape fruit: Antifungal activities, probiotic properties,
and in vitro detoxification of ochratoxin A. Annals of Microbiology, 69(1), 17–27.
https://doi.org/10.1007/s13213-018-1359-6
Vanda, P., Miranda, A., Leca, M. J., & Marques, C. J. (2017). Analytical methodologies
for the determination of biogenic amines in wines: An overview of the recent trends.
12
LWT 156 (2022) 112908
Journal of Analytical, Bioanalytical and Separation Techniques, 2(1), 52–57. https://
doi.org/10.15436/2476-1869.17.1296
Verma, N., Hooda, V., Gahlaut, A., Gothwal, A., & Hooda, V. (2020). Enzymatic
biosensors for the quantification of biogenic amines: A literature update. Critical
Reviews in Biotechnology, 40, 1–14. https://doi.org/10.1080/
07388551.2019.1680600
Žurga, P., Vahčić, N., Pasković, I., Banović, M., & Staver, M. M. (2019). Occurrence of
ochratoxin A and biogenic amines in Croatian commercial red wines. Foods, 8(8).
https://doi.org/10.3390/foods8080348