3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
sample was weighted into the plastic test tube. A volume
of 0.5 ml of an internal standard, 1,7-diaminoheptane, with
concentration 1 mg ml –1 was added to the sample and it was
extracted with 20 ml of 5% trichloroacetic acid for two minutes
using a disintegrator Heidolph Diax 900 (Heidolph Instruments,
Germany). Suspension was then centrifuged for
10 minutes (3,000 rpm, 4 °C). The supernatant was filtered
through paper filter (Filtrak no. 390) and the solid residue
was extracted for the second time (using method described
above). Both extracts were collected into one tube, filled in
50 ml with 5% trichloroacetic acid and filtered through the
nylon membrane filter (13 mm, 0.45 μm, Chromatography
research Supplies, Edison, USA). An aliquot 1 ml of an extract
was mixed with 0.5 ml of saturated sodium carbonate
solution (Sigma-Aldrich, s.r.o, Prague, Czech Republic) and
derivatized with 1 ml of acetonic solution of dansyl chloride
(5 mg ml –1 ) for 1 hour at 40 °C. The excess of unreacted
derivatisation agent was removed by reaction with 250 μl
10mM ammonia solution. Derivatized biogenic amines were
then extracted into diethyl ether (3 × 1 ml); etheric extract
was evaporated to dryness and dissolved in 500 μl of acetonitrile.
The same procedure was applied on standard solution
of biogenic amines (Sigma-Aldrich). Biogenic amines
were determined as described in Burdychova and Komprda 11
and Burdychova and Dohnal 12 by high performance liquid
chromatography with UV detection at 254 nm. The formation
of biogenic amines by starter and probiotic cultures was
screened as described by Burdychova and Dohnal 12 .
Results
M i c r o b i a l C o u n t s
A number of health benefits have been claimed for probiotic
bacteria such as Lactobacillus casei. Because of the
potential health benefits, this organism is increasingly incorporated
into fermented foods. However, studies have shown
low viability of this probiotic in market preparations 13 .
In both bathes with probiotic bacteria, L. casei was detected
at concentration of 10 4 CFU g –1 during ripening period
and stayed at this level during the whole storage period. The
changes in probiotic flora during ripening and storage of sausages
are shown in Fig. 1. Initial LAB counts in probiotic
batches were significantly higher than in the control samples
without probiotics (p < 0.05), due to the inoculation of starter
strains. During first 14 days of ripening LAB numbers
reached levels up to 10 7 CFU g –1 in all batches; thereafter,
during next 14 days of ripening, the counts of LAB significantly
decreased in all batches to the level of 10 6 CFU g –1 .
The total microbial count reached levels up to 10 8 CFU g –1
in all batches during first 14 days of ripening and their counts
gradually decreased to the level of 10 7 CFU g –1 during next
14 days of ripening and 21 days of storage (data not shown).
To provide health benefits, the suggested concentration
for probiotic bacteria is 10 6 CFU g –1 of a product 13 and daily
consummation of 100 g of such product is recommended.
However, the minimal dose is depended on several factors
such as individual person, probiotic strain and type of food
s602
product 14 . On the other hand, dry sausages are products,
which may be suitable carriers for probiotics into the human
gastrointestinal tract. The question still is, if those types of
meat products can be called functional foods and consumed
daily.
Fig. 1. Changes of probiotic l. casei during ripening (0–28)
and storage (28–49) of sausages „Herkules“, ▲ starter + probiotic
l. casei, ♦ starter 2 + probiotic l. casei
B i o g e n i c A m i n e s F o r m a t i o n
Biogenic amines are formed by the microbial decarboxylation
of free amino acids in food 15 and they are generally
present in dry sausages as reviewed by Maijala 16 and eerola
et al. 17 As described by Buckenhüskes 18 , the absence of biogenic
amines formation should be a selection criterion for
strains used as meat starter cultures. In our study was confirmed
that neither starter nor probiotic cultures formed biogenic
amines (results not shown). All sausages were analyzed
for biogenic amines by HPLC. The concentrations of BA in
the raw material were low but during ripening, fermentation
and storage period a sharp rise occurred in both control and
probiotic batches. The only BA which were at low levels
during the whole ripening and storage periods was histamine,
which concentrations varied from 0.5 to 2.5 mg kg –1 in all
batches (data not shown). nout 19 pointed out that histamine
contents should be in the range of 50–100 mg kg –1 in sausages
processed according to „Good Manufacturing Practice“, the
amount of histamine measured in this study is in agreement
with this rule.
Putrescine was the main amine formed in both control
batches, followed by cadaverine and tyramine. The concentration
of putrescine increased from 1.9 to 101.2 mg kg –1
(control batch 1) and from 4.6 to 350.5 mg kg –1 (control
batch 2). The amount of cadaverine increased from 2.0 to
39.9 mg kg –1 (control batch 1) and from 11.7 to 81.6 mg kg –1
(control batch 2). The level of tyramine increased from 6.3 to
204.4 mg kg –1 (control batch 1) and from 10.5 to 300.4 mg kg –1
(control batch 2), respectively. In this study, putrescine, cadaverine
and tyramine accumulation was significantly inhibited
in probiotic sausages when compared with controls without
probiotic L. casei culture. Putrescine concentration increased
gradually during ripening and storage period, reaching levels
53.7 mg kg –1 (probiotic batch 1) and 163.5 mg kg –1 (probio-