Food Chemistry 123 (2010) 143150

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Impact of fermentation conditions and refrigerated storage on microbial quality

and biogenic amine content of sauerkraut
Elena Peas, Juana Frias, Beatriz Sidro, Concepcin Vidal-Valverde *
Instituto de Fermentaciones Industriales (CSIC), C/Juan de la Cierva 3, E-28006 Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history: White cabbage (Brassica oleracea L. var. capitata cv. Bronco) was fermented, at 0.5% and 1.5% NaCl, using
Received 24 July 2009 Lactobacillus plantarum or Leuconostoc mesenteroides as starter cultures and, subsequently, sauerkraut
Received in revised form 19 February 2010 was stored at 4 C for 3 months. Microbial populations and six biogenic amines (putrescine, cadaverine,
Accepted 13 April 2010
histamine, tyramine, spermine and spermidine) were investigated. Fermentation and storage increased
aerobic mesophilic bacteria and LAB populations in sauerkrauts, and this was accompanied by a rise in
biogenic amine content. L. plantarum sauerkrauts produced with 0.5% NaCl had the highest microbial
Keywords:
counts, whilst no differences between salt contents were found with L. mesenteroides. Total biogenic
Sauerkraut
Microbial quality
amine amount was lower at 0.5% NaCl than at 1.5% in both induced fermentations and L. mesenteroides
Biogenic amines produced a lower content than did L. plantarum. Spermidine was the major contributor to the total bio-
Starter culture genic amine content, followed by putrescine, whilst histamine was present at the lowest level. The indi-
vidual and total biogenic amine levels in the experimental sauerkrauts stored at 4 C for 3 months were
below the upper limits reported in the literature for fermented products, indicating good quality and
safety of the sauerkrauts. L. mesenteroides starter and 0.5% NaCl were the optimal fermentation conditions
for producing sauerkrauts with the lowest biogenic amine contents.
2010 Published by Elsevier Ltd.

1. Introduction ensures a uniform product quality by controlling the fermentation

Traditional foods are an expression of culture, history and life-
style and generally possess health qualities, since tradition rarely
favours foods which are not palatable and healthy. Sauerkraut, a
product obtained by the lactic acid fermentation of shredded and
salted white cabbage, is one of the best known traditional foods.
This fermented vegetable product is considered to be healthy since
it contains, not only high amounts of vitamins and minerals, but
also high levels of glucosinolate hydrolysis products, which present
important anticarcinogenic activity (Bonnesen, Eggleston, & Hayes,
2001; Martinez-Villalulenga et al., 2009; Verhoeven, Verhagen,
Goldbohm, van den Brandt, & van Poppel, 1997).
Spontaneous cabbage fermentation relies on the natural bacte-
rial population of fresh cabbage, and is initiated by heterofermen-
tative lactic acid bacteria (LAB), such as Leuconostoc mesenteroides,
which produce acetic and lactic acids. At a later stage of the pro-
cess, they are replaced by the more acid-tolerant homofermenta-
tive species, such as Lactobacillus plantarum, which produce lactic
acid almost exclusively (Holzapfel, Schillinger, & Buckenhskes,
2003). However, variations in cabbage microbiota can lead to a
great variability in sauerkraut quality. The use of starter cultures
* Corresponding author. Tel.: +34 91 5622900; fax: +34 91 5644853.
E-mail address: icv12@i.csic.es (C. Vidal-Valverde).
process (Wiander & Ryhnen, 2005).
Fermentation is a means for preventing cabbage deterioration
and extending its shelf life, since the organic acids released by
LAB inhibit the growth of undesirable microorganisms. However,
high microbial populations may produce measurable toxic metab-
olites such as biogenic amines. On the other hand, mild acidic sau-
erkrauts are preferred by consumers (Holzapfel et al., 2003) and
high LAB counts may lead to an excessively acidic product,
decreasing its acceptability. For these reasons, most of the sauer-
krauts manufactured in Europe are pasteurised when their pH
reaches values between 3.8 and 4.1. It is, therefore, of great impor-
tance to determine the microbial loads of sauerkrauts.
Sauerkraut usually contains between 0.6% and 2% NaCl
(Holzapfel et al., 2003). Health authorities recommend a reduction
of the salt content in food and nowadays consumers of industria-
lised countries demand low-salt foods for health reasons.
Biogenic amines are basic nitrogenous compounds, of low
molecular weight, endowed with biological activity. They are usu-
ally generated by decarboxylation of amino acids or by the amina-
tion and transamination of aldehydes and ketones as a result of
microbial, vegetable and animal metabolic processes (Silla-Santos,
1996; ten Brink, Damink, Joosten, & Huis int Veld, 1990). Biogenic
amines are found in relatively high amounts in some vegetable fer-
mented foods as a consequence of the activities of the fermenting

0308-8146/$ - see front matter 2010 Published by Elsevier Ltd.

doi:10.1016/j.foodchem.2010.04.021
144 E. Peas et al. / Food Chemistry 123 (2010) 143150
or contaminating microorganisms exhibiting amino acid decarbox-
ylase activity (Silla-Santos, 1996). Many factors may alter their
contents in food, e.g. temperature, pH, salt content, the microbial
load and the storage conditions (Bouchereau, Aziz, Larher, & Mar-
tin-Tanguy, 1999; Halsz, Brath, Simon-Sarkadi, & Holzapfel,
1994).
Among biogenic amines, it is known that histamine and tyra-
mine present psychoactive and vasoactive properties, which can
cause toxicological effects (Shalaby, 1996), especially in individuals
in whom the mechanisms for the catabolism of these amines are
inhibited or genetically decient (Bodmer, Imark, & Kneubhl,
1999). At a high concentration, histamine is known to cause head-
aches, hypotension, palpitation, skin diseases, vomiting and diar-
rhoea (Lehane & Olley, 2000), whilst tyramine can trigger
migraines and hypertension (Jansen, van Dusseldorp, Bottema, &
Dubois, 2003; Shalaby, 1996). Spermidine, spermine, their precur-
sor putrescine, and cadaverine, ubiquitous in all plant cells, are
essential for cell multiplication (Flores, Protacio, & Signs, 1989)
and can also protect against oxidative stress (Lavizzari et al.,
2007). Although not toxic in themselves, they can enhance the ad-
verse effects of histamine and tyramine, as they compete for some
of the mechanisms involved in their detoxication (Bardcz, 1995;
Straub, Kicherer, Schilcher, & Hammes, 1995). Thus, total biogenic
amine levels of 1000 mg/kg in food are considered harmful for hu-
man health (Taylor, 1986). For this reason, knowledge of the level
of biogenic amines in food is of particular interest and the produc-
tion of fermented foods with low levels of biogenic amines is a goal
that needs to be addressed by the food industry.
The present research was conducted to study the effect of dif-
ferent fermentation conditions (NaCl level and starter culture)
and storage at 4 C for 1, 2 and 3 months on the microbial status
and biogenic amine content of sauerkraut in order to obtain safe
and healthy sauerkraut products.
2. Materials and methods
2.1. Cabbage samples
Fresh white cabbages (Brassica oleracea L. var. capitata cv. Bron-
co) grown in winter 20072008 in Levante, Spain, were provided
by Bejo Iberica S.L. (Spain) and were stored for less than 5 days
at 4 C before fermentation. Raw shredded cabbage was freeze-
dried, milled and stored at 20 C for the determination of bio-
genic amine content.
2.2. Starter culture preparation
L. plantarum (CECT 748) and L. mesenteroides (CECT 219) strains
were obtained from the Spanish Type Culture Collection (CECT,
Valencia, Spain). They were propagated twice in MRS broth (Difco
Laboratories, Detroit, Mich.) and incubated overnight at 30 C.
After centrifugation (5000 rpm, 10 min), the microbial cells were
harvested and washed twice in sterile saline solution (0.9% NaCl)
before their inoculation.
2.3. Sauerkraut production
Cabbages were prepared by removing the rotten and dirty
leaves, as well as their central core. Subsequently, cabbages were
shredded to a 12 mm wide strip size, using a domestic shredder
(Moka Express, Barcelona, Spain). Different NaCl concentrations
(0.5% or 1.5%) were added to the shredded cabbage and mixed vig-
orously. Then, starter cultures were inoculated into shredded and
salted cabbage at 106 cfu/g of cabbage. Autochthonous microbiota
were not removed. Cabbage and brine were placed in autoclaved
polyethylene containers (8 l) and tightly pressed together to ex-
clude air. Three independent batches (5 kg per batch) were pre-
pared for each fermentation condition. Cabbages were fermented
for 7 days at room temperature (2225 C). On the third day of fer-
mentation, cabbages were pricked to remove air between particles.
2.4. Storage conditions
Three samples of sauerkraut, corresponding to each fermenta-
tion batch, were placed in sterile capped glass vessels (500 ml) at
the end of the fermentation process, simulating the packaging
and storage in households. They were, then, stored at 4 C for 1,
2, and 3 months (one sample for each storage time). Fermented
cabbages stored for 0, 1, 2 and 3 months were freeze-dried, milled
and stored at 20 C for the determination of biogenic amine
content.
2.5. Microbiological analyses
Microbiological analyses were carried out on raw shredded cab-
bage and sauerkraut samples taken at 0, 1, 2, and 3 months of
refrigerated storage. Ten grams of each replicate were aseptically
placed in a sterile Stomacher bag, diluted in buffered peptone
water (Scharlau Chemie, Barcelona, Spain) and homogenised for
1 min at medium speed in a Stomacher laboratory blender (IUL
Masticator, Barcelona, Spain). Decimal dilutions were prepared
and inoculated using the pour plate technique, into the corre-
sponding media as follows: total aerobic mesophilic bacteria were
enumerated on Tryptone Soya Agar (TSA) (Scharlau Chemie, Barce-
lona, Spain) after incubation at 30 C for 72 h; total and faecal col-
iforms on Violet Red Bile Agar (VRBA) (Scharlau Chemie, Barcelona,
Spain) containing lactose as carbohydrate source, after incubation
at 37 and 44 C, respectively, for 24 h; moulds and yeasts on Sab-
ouraudChloramphenicol Agar (Scharlau Chemie, Barcelona,
Spain), after incubation at 23 C for 96 h; LAB on MRS Agar (Schar-
lau Chemie, Barcelona, Spain) after incubation under anaerobic
conditions at 30 C for 2448 h.
2.6. Determination of biogenic amines
Biogenic amine contents of raw cabbage and sauerkraut sam-
ples were determined by acid extraction, derivatisation with dan-
syl chloride and HPLC quantication according to Frias et al.
(2007) with some modications. Briey, 1 g of each freeze-dried
sample was extracted with 20 ml of 0.1 M HCl in an Ultra-Turrax
T25 homogenizer (Ika Werke GMBH & Co. KG, Staufen, Germany)
for 2 min. The resulting slurry was centrifuged at 12,000 rpm for
20 min at 4 C. The supernatant was collected and the remaining
residue was re-extracted under the same conditions. Both extracts
were combined, ltered through a Whatman No. 1 lter paper and
the volume was made up to 100 ml with 0.1 M HCl. Then, 1 ml of
the diluted extract was mixed with 0.5 ml of saturated NaHCO3
and 1 ml of dansyl chloride (Sigma, Steinheim, Germany) solution
(20 mg/ml in acetone) and the mixture was stirred in darkness at
40 C for 1 h. The residual dansyl chloride was precipitated by add-
ing 200 ll of a proline solution (100 mg/ml), vortexing for 1 min
and leaving to react in darkness at room temperature for 15 min.
Subsequently, the sample was extracted twice with 1 ml of diethyl
ether. Both extracts were combined and dried under a stream of
nitrogen. The dry residue was dissolved in 1 ml of HPLC grade ace-
tonitrile (LAB-SCAN, Gliwice, Poland) and ltered through a PVDF
Millipore lter (pore size 0.45 lm) before HPLC analysis.
Biogenic amine standards (putrescine dihydrochloride, cadaver-
ine dihydrochloride, histamine dihydrochloride, tyramine, spermi-
dine trihydrochloride and spermine tetrahydrochloride) were
purchased from Fluka (Steinheim, Germany). A stock standard
 E. Peas et al. / Food Chemistry 123 (2010) 143150
aqueous solution of amines was prepared by placing an accurately
weighed amount of each standard (ca. 50 mg) in a 25 ml volumet-
ric ask. Standards were processed and derivatised as described
previously for samples.
Biogenic amine quantication was performed by HPLC, using an
Alliance Separation Module 2695 (Waters, Milford, USA), a Photo-
diode Array detector 996 at 254 nm (Waters, Milford, USA) and a
personal computer running the Empower 2 for Microsoft Windows
chromatographic software (Waters). 20 ll of sample were injected
into a C18 Kromasil 250  4.6 mm i.d., 5 lm size column (Symta)
equipped with C18 guard column (Symta), at 30 C. Chromato-
graphic separations were carried out by using a binary gradient
elution with bidistilled water (solvent A) and acetonitrile (solvent
B). The elution programme started with 65% B for 1 min, ramped at
80% (10 min), 90% (12 min), 100% of B (16 min) and held until the
end of the run (23 min) with a ow rate of 0.8 ml/min. Calibration
curves were obtained for standard amines and the regression coef-
cient r was always above 0.99.
2.7. Statistical analysis
Data were subjected to multi-factor analysis of variance to
determine signicant differences between samples using the Stat-
graphics 5.0 Program (Statistical Graphic, Rockville, MD, USA) for
Windows. A value of P 6 0.05 was used to indicate signicant
differences.
3. Results
3.1. Inuence of fermentation conditions and refrigerated storage on
microbial quality of sauerkraut
Changes in microbial counts of raw cabbage and sauerkraut, ob-
tained at 0.5% and 1.5% NaCl, by using L. plantarum and L. mesen-
teroides stored in refrigeration for 0, 1, 2 and 3 months are
shown in Tables 1 and 2. Aerobic mesophilic bacteria were present
in the largest load (5.0 log cfu/g), followed by LAB (2.6
2.8 log cfu/g) and total coliforms (1.31.8 log cfu/g), whilst faecal
coliforms and moulds/yeasts were not detected (<1 log cfu/g).
During L. plantarum fermentation at both salt concentrations,
aerobic mesophilic bacteria and LAB populations increased by 3
and 5 log cfu/g, respectively, whilst total coliforms dropped to
undetectable levels (Table 1). During fermentation, 0.5% NaCl re-
sulted in signicantly higher (P 6 0.05) aerobic mesophilic bacteria
and LAB loads than did 1.5% NaCl. During refrigerated storage, both
aerobic mesophilic bacteria and LAB counts increased gradually,
reaching the highest populations after 3 months (8.48.6 log cfu/
g) and 0.5% NaCl caused signicantly (P 6 0.05) larger aerobic mes-
Table 1
Effect of storage at 4 C on the microbiological status of cabbage fermented with L. plantarum at different levels of NaCl.A
Cabbage Aerobic mesophilic bacteria
Fermentation with 0.5% NaCl
Raw cabbage 5.10 0.08a
0 time storage 8.18 0.06b
1 month storage 8.29 0.05c
2 month storage 8.43 0.07
3 month storage 8.60 0.06
Fermentation with 1.5% NaCl
Raw cabbage 5.01 0.04a
0 time storage 8.09 0.03
1 month storage 8.19 0.04b
2 month storage 8.31 0.04c
3 month storage 8.51 0.08
A
Mean values (log cfu/g) SD of six determinations. The same superscript in the same column means no signicant difference (P 6 0.05).
145
ophilic bacteria and LAB counts than did 1.5% NaCl in the same per-
iod of time. In contrast, no changes in total coliforms population
were observed during storage.
Table 2 summarises the microbial evolution in sauerkraut dur-
ing fermentation with L. mesenteroides at 0.5% and 1.5% NaCl and
subsequent storage at 4 C for 1, 2 and 3 months. During cabbage
fermentation with L. mesenteroides, aerobic mesophilic bacteria
and LAB underwent a signicant (P 6 0.05) proliferation, reaching
populations of 7.9 and 7.7 log cfu/g, respectively, irrespective of
the level of salt used in the process. Total coliforms decreased to
levels below 1 log cfu/g at both salt concentrations (Table 2). These
populations demonstrated behaviours similar to those of sauer-
kraut produced by L. plantarum fermentation (Table 1), although
they achieved slightly lower counts than those recorded in Table
1. Refrigerated storage resulted in a signicant increase of aerobic
mesophilic and LAB populations, which reached loads of >8 log cfu/
g at the end of the storage period, whilst it had no inuence on the
total coliform population. Increases in salt level did not cause sig-
nicant differences in microbial counts during L. mesenteroides fer-
mentations and subsequent storage (Table 2).
3.2. Inuence of fermentation conditions and storage on the prole and
content of biogenic amines in sauerkraut
Tables 3 and 4 give an overview of the individual biogenic
amine content in raw cabbage and sauerkraut obtained at 0.5%
and 1.5% NaCl, by using L. plantarum and L. mesenteroides as starter
cultures, respectively. Fig. 1 shows the inuence of fermentation
and storage on the total level of biogenic amines in cabbage. The
results are expressed in mg/kg of dry matter (DM). Contributions
of each biogenic amine to total levels in raw and fermented cab-
bage stored for 0, 1, 2, and 3 months are indicated in Fig. 2.
Raw cabbage showed a total biogenic amine concentration of
66 mg/kg DM (Fig. 1), and spermidine (22 mg/kg DM) was the ma-
jor amine found in this vegetable, followed by tyramine (19 mg/kg
DM). Putrescine, cadaverine and spermine were also present at
concentrations of 12 mg/kg DM, 8 mg/kg DM and 5 mg/kg DM,
respectively, whilst histamine (1 mg/kg DM) was the minor con-
tributor to the total amine content of cabbage (Fig. 2). Fermenta-
tion caused a signicant (P 6 0.05) increase in the individual and
total biogenic amine contents in both the L. plantarum and the L.
mesenteroides processes (Tables 3 and 4).
In L. plantarum sauerkrauts, the total concentration of biogenic
amines reached levels of 187 mg/kg DM in fermentations per-
formed at 0.5% NaCl and 223 mg/kg DM in those at 1.5% NaCl
(Fig. 1). Putrescine and spermidine were present in the highest
amounts in L. plantarum sauerkrauts (4244 and 89102 mg/kg
DM, respectively), levels four times larger than those observed in
raw cabbage. Cadaverine, tyramine, histamine and spermine
Lactic acid bacteria Total coliforms Faecal coliforms Yeasts and moulds
2.84 0.03a 1.77 0.11 <1a <1a
8.14 0.05b <1a <1a <1a
8.23 0.05c <1a <1a <1a
8.37 0.06d <1a <1a <1a
8.50 0.07 <1a <1a <1a
2.74 0.07a 1.36 0.17
8.02 0.11 <1a <1a <1a
8.14 0.05b <1a <1a <1a
8.26 0.03c <1a <1a <1a
8.40 0.12d <1a <1a <1a
146 E. Peas et al. / Food Chemistry 123 (2010) 143150

Table 2
Effect of storage at 4 C on the microbiological status of cabbage fermented with L. mesenteroides at different levels of NaCl.A

Cabbage Aerobic mesophilic bacteria Lactic acid bacteria Total coliforms Faecal coliforms Yeast and moulds
Fermented cabbage with 0.5% NaCl
Raw cabbage 5.05 0.05a 2.71 0.04a 1.31 0.17 <1a <1a
0 time storage 7.87 0.05b 7.71 0.12b <1a <1a <1a
1 month storage 8.08 0.05c 7.96 0.13c <1a <1a <1a
2 month storage 8.29 0.04d 8.23 0.05d <1a <1a <1a
3 month storage 8.47 0.08e 8.36 0.13e <1a <1a <1a
Fermented cabbage with 1.5% NaCl
Raw cabbage 5.06 0.11a 2.63 0.05a 1.61 0.17
0 time storage 7.85 0.26b 7.67 0.17b <1a <1a <1a
1 month storage 8.06 0.05c 7.91 0.17c <1a <1a <1a
2 month storage 8.24 0.04d 8.17 0.08d <1a <1a <1a
3 month storage 8.45 0.08e 8.30 0.19e <1a <1a <1a
A
Mean values (log cfu/g) SD of six determinations. The same superscript in the same column means no signicant difference (P 6 0.05).

Table 3
Effect of storage at 4 C on biogenic amine contents of cabbage fermented with L. plantarum at different levels of NaCl.A

Cabbage Putrescine Cadaverine Histamine Tyramine Spermidine Spermine

Raw cabbage 11.6 0.75 7.59 0.51 1.00 0.09 18.6 0.88 22.3 1.50 4.69 036
Fermentation with 0.5% NaCl
0 time storage 42.1 2.88 17.1 1.09a 2.87 0.14a 26.5 1.13 89.0 3.09 9.49 0.43a
1 month storage 58.8 2.57 23.7 0.78b 3.40 0.20b 32.5 0.75 141 1.04 10.2 0.36a
2 month storage 101 1.71 31.5 1.81c 8.75 0.16 74.9 0.86 174 1.45 13.4 0.86b
3 month storage 122 2.83 32.7 1.37c 11.3 0.50 94.4 1.29a 214 5.10 21.6 1.10
Fermentation with 1.5% NaCl
0 time storage 44.3 1.39 18.5 1.00 3.08 0.19ab 41.5 1.37 102 5.59 13.8 0.47b
1 month storage 51.2 1.77 24.1 1.13b 4.06 0.48 51.5 1.28 169 3.78 14.9 0.79
2 month storage 77.1 1.81 32.1 2.55c 5.24 0.38 81.0 2.49 219 2.26 17.4 0.69
3 month storage 102 1.99 32.3 3.29c 7.75 0.28 95.6 1.94a 276 6.17 11.0 0.85
A
Mean values (mg/kg DM) SD of eight determinations. The same superscript in the same column means no signicant difference (P 6 0.05).

Table 4
Effect of storage at 4 C on biogenic amine content of cabbage fermented with L. mesenteroides at different levels of NaCl.A

Cabbage Putrescine Cadaverine Histamine Tyramine Spermidine Spermine

Raw cabbage 11.6 0.75 7.59 0.51 1.00 0.09 18.6 0.88 22.3 1.50 4.69 036
Fermentation with 0.5% NaCl
a
0 time storage 32.1.1.07 19.1 0.87 2.14 0.26 26.4 1.62 75.5 1.60 9.72 0.45
1 month storage 40.5 1.52 27.1 2.65 3.54 0.49 32.9 1.72a 97.0 1.54 17.9 0.56a
2 month storage 86.1 2.64 41.1 1.30a 6.09 0.41b 58.9 1.61b 166 1.50 21.5 0.97b
3 month storage 92.1 3.01 40.9 1.41a 7.12 0.36 56.6 1.85 198 3.14a 21.6 1.50b
Fermentation with 1.5% NaCl
0 time storage 43.0 1.61 20.3 0.68 2.47 0.14a 33.1 0.90a 84.5 2.86 10.8 0.39
1 month storage 52.9 0.83 24.2 0.91 4.07 0.34 40.6 1.48 123 2.60 15.7 1.11
2 month storage 101 2.37 32.2 1.54 6.13 0.52b 51.2 1.10 143 2.54 18.5 0.71a
3 month storage 108 2.30 39.4 1.17 6.59 0.42 60.1 2.37b 198 5.45a 21.0 0.86b
A
Mean values (mg/kg DM) SD of eight determinations. The same superscript in the same column means no signicant difference (P 6 0.05).

showed amounts of 1718, 2742, 3 and 1014 mg/kg DM, respec-
tively, which were two or threefold higher than those found in
white cabbage (Table 3). Signicantly (P 6 0.05) lower individual
and total biogenic amine levels were found in sauerkrauts manu-
factured with 0.5% than with 1.5% NaCl, with the exception of his-
tamine content, in which the differences between the salt
concentrations were not statistically (P 6 0.05) signicant.
Total biogenic amine contents of sauerkrauts obtained with L.
mesenteroides were 165 mg/kg DM at 0.5% NaCl and 194 mg/kg
DM at 1.5% NaCl (Fig. 1). All the individual biogenic amines showed
a sharp rise after L. mesenteroides fermentation, reaching levels of
3243 mg/kg DM for putrescine, 1920 mg/kg DM for cadaverine,
23 mg/kg DM for histamine, 2633 mg/kg DM for tyramine, 75
84 mg/kg DM for spermidine and 1011 mg/kg DM for spermine,
depending on the salt content used during fermentation (Table
4). These increments were around two or threefold those detected
in unprocessed cabbage. Sauerkraut produced with the lowest salt
concentration (0.5% NaCl) presented a signicantly (P 6 0.05) low-
er content of individual bioactive amines than did those obtained
at higher salt concentration (1.5% NaCl), with the exception of his-
tamine, whereas no signicant (P 6 0.05) differences between the
salt levels were found (Table 4), similar to the situation observed
in L. plantarum fermentations (Table 3).
The effect of refrigerated storage on both L. plantarum and
L. mesenteroides sauerkrauts, at both salt levels, is also shown in
Tables 3 and 4. In general, the individual amine levels underwent
a gradual increase during storage, which was slight during the rst
month, and more pronounced during the second and third months.
Total and individual biogenic amine contents were two- or three-
fold higher at the end of the storage period than those recorded
 E. Peas et al. / Food Chemistry 123 (2010) 143150 147

(mg/Kg DM)
600

400

200

0
L. plantarum L. plantarum L. mesenteroides L. mesenteroides
0.5% NaCl 1.5% NaCl 0.5% NaCl 1.5% NaCl

Raw cabbage Sauerkraut (0 month storage) Sauerkraut (1 month storage)

Sauerkraut (2 month storage) Sauerkraut (3 month storage)

Fig. 1. Total biogenic amine levels in raw cabbage and sauerkrauts.

L. plantarum 0.5% NaCl L. plantarum 1.5% NaCl

Sauerkraut (3 month storage) Sauerkraut (3 month storage)

Sauerkraut (2 month storage) Sauerkraut (2 month storage)

Sauerkraut (1 month storage) Sauerkraut (1 month storage)

Sauerkraut (0 month storage) Sauerkraut (0 month storage)

Raw cabbage Raw cabbage

0% 20% 40% 60% 80% 100% 0% 20% 40% 60% 80% 100%

L. mesenteroides 0.5% NaCl L. mesenteroides 1.5% NaCl

Sauerkraut (3 month storage) Sauerkraut (3 month storage)

Sauerkraut (2 month storage) Sauerkraut (2 month storage)

Sauerkraut (1 month storage) Sauerkraut (1 month storage)

Sauerkraut (0 month storage)
Sauerkraut (0 month storage)
Raw cabbage
Raw cabbage
0% 20% 40% 60% 80% 100%
0% 20% 40% 60% 80% 100%

PUT CAD HIS TYR SPD SPM

Fig. 2. Contribution of individual biogenic amines to total levels in raw cabbage and sauerkrauts.

in unstored sauerkrauts in both L. plantarum and L. mesenteroides 4. Discussion

fermentations. After 3 months of storage, the sauerkrauts obtained
with L. plantarum at 0.5% NaCl (Table 3) presented higher amounts
of putrescine and histamine and a lower content of spermidine
than did those obtained at 1.5%, although the differences were
not signicant (P 6 0.05) for cadaverine and tyramine. However,
stored 0.5% NaCl L. mesenteroides sauerkrauts showed lower con-
tents of putrescine and tyramine and higher contents of cadaverine
and histamine than did those fermented with a level of 1.5% NaCl,
whilst no signicant (P 6 0.05) differences were found for sperm-
ine and spermidine (Table 4).
As consequence of fermentation, the contribution of individual
biogenic amines to total content underwent some changes
(Fig. 2). Putrescine and spermidine increased; cadaverine, tyramine
and spermine decreased, whilst histamine did not suffer any
modication compared to their contribution in the unprocessed
cabbage. However, slight changes of the individual amine contri-
butions were found during refrigerated storage (Fig. 2).
The autochthonous microbiota of raw cabbage was dominated
by aerobic mesophilic bacteria and LAB; total coliforms appeared
in lower counts whilst faecal coliforms and moulds/yeasts were
not found. These results are in accordance with those found in
raw cabbage (Peas, Frias, Gmez, & Vidal-Valverde, 2010) and
other Brassica fresh vegetables (Feng-Di, Bao-Ping, Bo, & Bei-Zhong,
2007; Olarte, Sanz, Echvarri, & Ayala, 2009; Vandekinderen et al.,
2009).
During starter-induced sauerkraut production, the increase in
LAB populations was greater than that of aerobic mesophilic bacte-
ria, mainly due to the former being better adapted to the surround-
ing environmental conditions (low pH and anaerobic conditions).
In contrast, total coliforms seemed to be more sensitive than were
these bacterial groups to the inhibition effect of lactic and acetic
acids produced during fermentation. It is known that organic acids
can easily penetrate the bacterial lipid membrane and dissociate
148 E. Peas et al. / Food Chemistry 123 (2010) 143150
into anions and protons once internalised (Cherrington, Hilton, &
Chopra, 1990), producing important problems in some bacterial
populations that must maintain a near neutral pH in their cyto-
plasm to sustain their functionality (Ricke, 2003).
Higher aerobic mesophilic bacteria and LAB populations were
found in sauerkrauts obtained by L. plantarum at 0.5% NaCl than
in those obtained at 1.5% NaCl. These results suggest that growth
of the autochthonous aerobic mesophilic bacteria and LAB of cab-
bage, and also of L. plantarum used as a starter, could be inuenced
by high osmotic pressure. Similar results have been observed pre-
viously when white cabbage was fermented using a mixed starter
culture of L. plantarum and L. mesenteroides (1:1) at 0.5% and 1.5%
NaCl (Peas et al., 2010).
The rises in aerobic mesophilic bacteria and LAB populations
during storage after L. plantarum (Table 1) and L. mesenteroides (Ta-
ble 2) cabbage fermentations, suggest that temperatures of 4 C did
not prevent the proliferation of these populations after
fermentation.
The presence of microorganisms in food is usually accompanied
by the formation of biogenic amines since they are generated by
decarboxylase-positive microorganisms under favourable condi-
tions and as a result of endogenous amino acid decarboxylase
activity in food materials (Shalaby, 1996). Several of the amines
found by us in raw cabbage were previously identied in different
cabbage-like vegetables, and a wide range of their contents has
been reported. In this context, Nishibori, Fujihara, and Akatuki
(2007) found that spermidine (150 mg/kg DM) was the major
amine in cabbage, but its concentration and those of putrescine
(27 mg/kg DM), and spermine (40 mg/kg DM), determined by these
authors, were several-fold higher than those found in the present
work. On the other hand, Simon-Sarkadi and Holzapfel (1994) re-
ported that spermidine was also the major contributor to total
amine content in Chinese cabbage (15.1 mg/kg fresh weight);
putrescine, tyramine and histamine were present at similar con-
centrations (1.8, 1.3 and 1.1 mg/kg fresh weight, respectively)
and spermine was the minor biogenic amine (0.56 mg/kg fresh
weight). High levels of spermidine in raw white cabbage (Brassica
oleracea L. var. capitata cv. Bronco) were expected, since this amine
is widely distributed in vegetables as Valero, Martnez-Romero,
and Serrano (2002) reported, playing important roles in cell divi-
sion and growth (Bardcz, 1995; Flores et al., 1989). The presence
of putrescine was also expected, as this is an intermediate in the
synthesis of spermidine (Walters, 2003). Nevertheless, Moret,
Smela, Populin, and Conte (2005) reported larger concentrations
of putrescine (1.6 mg/100 g fresh weight) in savoy cabbage than
that observed by us in white cabbage, but cadaverine, histamine
and tyramine were present in undetectable concentrations. The
differences observed among results reported by other authors
and those found by us suggest that biogenic amine levels depend
on the Brassica vegetable specie and variety studied. The different
microbiota present in each vegetable also explains the large differ-
ences in biogenic amine content found in the literature, since the
formation of biogenic amines depends on the type and number
of microorganisms involved. Kalac, Spicka, Krizek, Steidlov, and
Peliknov (1999) reported that different strains of the same
microbial species may vary by a magnitude of three orders in their
abilities to produce biogenic amines. Other factors that may affect
the biogenic amine content of vegetables are water availability
(Smith, 1985), temperature and the altitude at which the vegetable
has been grown (Serrano, Martinez-Romero, Guillen, & Valero,
2003).
In accordance with our ndings, fermentation with L. plantarum
or with L. mesenteroides enhances the formation of the individual
biogenic amines, increasing the total bioactive amine concentra-
tion two- or threefold in comparison with that of unprocessed cab-
bage. During fermentation, aerobic mesophilic bacteria and LAB
underwent an important proliferation as they found optimal eco-
logical conditions within the fermenting substrate, especially
LAB. Many of these bacteria have decarboxylase enzymes, which
enhance the formation of biogenic amines from available free ami-
no acids of cabbage. Some strains of LAB, which are frequently in-
volved in cabbage fermentation, such as Lactobacillus spp.,
Lactobacillus curvatus, Leuconostoc mesenteroides and Pediococcus
damnosus, have proved to have important amino acid decarboxyl-
ase activity (Halsz, Brath, & Holzapfel, 1999; Moreno-Arribas,
Polo, Jorganes, & Muoz, 2003; ten Brink et al., 1990; Voigt & Ein-
tenmiller, 1977). Furthermore, Halsz et al. (1994) and Halsz et al.
(1999), observed that the applied starter cultures may affect the
production of biogenic amines during fermentation. Hence, the
biogenic amine formation observed in sauerkrauts obtained in
the present work may be mainly attributed to the metabolic activ-
ity of L. plantarum and L. mesenteroides used as starter cultures. In
addition, some aerobic mesophilic decarboxylase-positive micro-
organisms that proliferate during cabbage fermentation may
contribute to the biogenic amine production, since the contaminat-
ing vegetable microbiota are responsible for the high biogenic
amine production in fermented products (Voigt & Eintenmeller,
1977).
In general, the concentration of total biogenic amines in
sauerkrauts was lower when fermentation was carried out with
L. mesenteroides than that with L. plantarum (Fig. 1). These results
are in accordance with the lower aerobic mesophilic bacteria and
LAB counts in the former. Similarly, the contents of individual bio-
genic amines were lower in L. mesenteroides sauerkraut, with the
exception of cadaverine, which was slightly higher than that in
sauerkrauts obtained with L. plantarum at both salt levels. These
data suggest a higher activity of the enzymes responsible for the
formation of bioactive amines in L. plantarum CECT 748 than in L.
mesenteroides CECT 219, used in the present study.
There are only a few studies in the literature on the effect of
starter culture addition during sauerkraut production in the for-
mation of biogenic amines (Halsz et al., 1999; Kalac, Spicka, Kri-
zek, & Peliknov, 2000; Spicka, Kalac, Bover-Cid, & Krizek, 2002),
but there are no published data about the effect of L. mesentero-
ides addition, during cabbage fermentation, on the individual and
total amine concentrations in sauerkraut. Halsz et al. (1999) re-
ported values for putrescine, cadaverine, spermidine and sperm-
ine in the range of 6070 mg/kg fresh cabbage, 110130 mg/kg
fresh cabbage, 140150 mg/kg fresh cabbage and 4050 mg/kg
fresh cabbage, respectively, in sauerkraut obtained after fermen-
tation for 71 days, using L. plantarum as starter culture. These
authors also studied the biogenic amine content in spontaneously
fermented sauerkraut and concluded that total amine content
was higher than when a starter culture was used. Similar results
were reported by Spicka et al. (2002). The data reported by
Halsz et al. (1999) are expressed in fresh weight, whilst, in
the present study, the results are expressed in dry matter.
Because of fermented cabbage products present high water con-
tents (90%), the values found by Halsz et al. (1999) are much
higher than those found by us. These differences can be ex-
plained by the different fermentation conditions used in the
two studies (strain, time and temperature).
Several authors have examined the biogenic amine content in
commercial sauerkrauts and results vary widely, depending on
the fermentation conditions, the manufacturing country and the
post-fermentation treatment (pasteurisation and sterilisation).
Thus, values reported for commercial sauerkrauts in the literature
(expressed in fresh weight) ranged from 1 to 550 mg/kg for putres-
cine, 2 to 300 mg/kg for cadaverine, 2 to 200 mg/kg for histamine, 2
to 900 mg/kg for tyramine and 6 to 50 mg/kg for spermidine (Hal-
sz et al., 1999; Kalac et al., 1999; Kalac et al., 2000; Mayer & Pause,
1972; Spicka et al., 2002). The recommended maximum levels of
 E. Peas et al. / Food Chemistry 123 (2010) 143150
histamine and tyramine in fermented foods are 50100 mg/kg and
100800 mg/kg, respectively (Halsz et al., 1994; Taylor, 1986; ten
Brink et al., 1990). On the other hand, for good quality sauerkrauts,
Knsch, Schrer, and Temperli (1989) recommended maximum
values of 10, 20, 50 and 25 mg/kg for histamine, tyramine, putres-
cine and cadaverine, respectively. These concentrations are higher
than those found in the present work for sauerkrauts obtained by L.
plantarum and L. mesenteroides fermentations, indicating that they
are safe from the point of view of their biogenic amine contents.
Starter-induced fermentation of white cabbage (Brassica olera-
cea L. var. capitata cv. Bronco) was performed at two salt concen-
trations and it was observed that 1.5% NaCl brought about larger
total biogenic amine content than did 0.5% NaCl. These results
may be related to stimulation of the activity of microbial decarbox-
ylase enzymes at higher osmotic pressure, rather than the prolifer-
ation of bacterial populations, since, in general, only slight
differences in microbial counts between the two salt levels were
observed. Our results are in accordance with those reported by
Halsz et al. (1999), who found higher total amounts of biogenic
amines in sauerkrauts obtained at 5% NaCl than at 2% NaCl when
the process was carried out by spontaneous fermentation or by L.
plantarum addition.
The highest bioactive levels observed after three months of
storage (Tables 3 and 4) corresponded to the highest microbial
counts in all sauerkrauts (Tables 1 and 2), indicating a good corre-
lation between the microbial proliferation and biogenic amine for-
mation. Kalac et al. (2000) reported an increase in tyramine
concentrations during storage at 56 C for 6 months, whilst no
differences in putrescine, cadaverine and spermidine levels were
found from 4 to 12 months of storage. The optimal activity of ami-
no acid decarboxylases and that of the enzymes responsible for the
bioactive amine oxidation of different bacteria are affected by the
temperature and pH, which may explain the different biogenic
amine changes during storage obtained in our study compared to
those found by Kalac et al. (1999).
The individual and total biogenic amine concentrations found in
all the sauerkrauts obtained in the present study were lower than
those reported by Spicka et al. (2002) for sauerkrauts obtained
with 1.5% of NaCl by natural fermentations and by L. plantarum
inoculated at different doses (5  104, 1  105, 5  105 CFU/g of
shredded cabbage) and stored at 56 C for 6 months. They found
values in the range of 7495 mg/kg of fresh cabbage for putrescine,
246 mg/kg of fresh cabbage for cadaverine, 3 mg/kg of fresh cab-
bage for histamine, 24209 mg/kg of fresh cabbage for tyramine,
728 mg/kg of fresh cabbage for spermidine, and about 2 mg/kg
of fresh cabbage for spermine. Moreover, they were below the
maximum recommended values reported in the literature for fer-
mented products (Halsz et al., 1994; ten Brink et al., 1990), and
also below the concentrations recommended for good quality sau-
erkrauts (Knsch et al., 1989). It is noteworthy that histamine is in-
volved in most frequent foodborne intoxication caused by biogenic
amines, and the fermentation conditions used in the present work
led to very low histamine levels in sauerkraut products (2.1
11.3 mg/kg DM) which can be considered an important goal for
sauerkraut manufacture.
5. Conclusions
The results of the present study revealed that fermentation and
subsequent storage decreased the total coliform loads and in-
creased those of aerobic mesophilic bacteria and LAB. The rise in
these populations parallelled increases in the individual and total
biogenic amine contents in sauerkrauts. 0.5% NaCl led to the
highest microbial counts in L. plantarum sauerkrauts, whilst no
statistical differences were observed between the two salt levels
149
in L. mesenteroides sauerkrauts. Biogenic amine content in sauer-
kraut was lower at 0.5% than 1.5% NaCl in both starter-induced
fermentations. The addition of L. mesenteroides as inoculant
resulted in smaller amount of total bioactive amines than did
addition of L. plantarum. Spermidine was the major contributor
to the total amine content, followed by putrescine, whilst hista-
mine, that has one of the highest toxic biological activities of
all the biogenic amines, was present in the lowest concentration.
The fermentation and storage conditions used in the present
work led to lower histamine and tyramine levels in sauerkrauts
than the upper limits recommended for fermented food products,
results that can be considered an important goal for sauerkraut
manufacture. White cabbage (Brassica oleracea L. var. capitata cv.
Bronco) fermented with L. mesenteroides as starter and 0.5% NaCl
was optimal for obtaining sauerkrauts with excellent quality from
a safety perspective.
Acknowledgements
This work was supported by the Spanish Commission of Science
and technology (CICYT), Project Number AGL2007-62044. E.P. is in-
debted to a JAE-doc grant funded by CSIC.
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