Die Nahrung 39 (1995) 4, 308 -315

Effect of potassium sorbate on development

of biogenic amines during sausage fermentation
and H. A. ABDEL-RAHMAN*
A. R. SHALABY

Food Technology and Dairy Science Department, National Research Centre, Dokki, Cairo, and *Food
Science Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt

Summary
Different concentrations of potassium sorbate were examined for their effect on the formation of
biogenic amines during sausage fermentation using Lactobacillus plantarum and Pediococcus acidilactici
as starter cultures. Microbiological examination revealed that there was a slight decrease in bacterial
counts during aging period of fermented sausages. No differences in bacterial counts could be observed
between treatment up to 0.06% potassium sorbate, although they were lower than that of control.
Tyramine development continued, although potassium sorbate was used in concentration up to 0.06%.
Histamine, tryptamine and phenylethylamine concentrations increased, then decreased in the later
stage of aging to reach undetectable levels. Putrescine, cadaverine and spermidine were not detected
in any sample throughout the study. Statistical analyses proved that there was a positive relation
between total biogenic amine (TBA) content of fermented sausage and aging period. On the contrary,
a negative relation between TBA and potassium sorbate concentration was found. The interrelationship
between the concentration of TBA and the two factors (aging period and potassium sorbate
concentration) with higher determination coefficient (Rz = 0.94) was given.

Zusammenfassung
EinfluD von Kaliumsorbat auf die Entwicklung biogener Amine wahrend der Wurstfermentation
Es werden verschiedene Konzentrationen an Kaliumsorbat auf ihren EinfluB fur die Bildung von
biogenen Aminen wahrend der Fermentation von Wurst unter Venvendung von Lactobacillusplantarum
und Pediococcus acidilatici als Starterkulturen gepriift. Mikrobiologische Untersuchungen zeigen, darj
wahrend der Reifungsperiode der fermentierten Wurst eine leichte Abnahme der Bakterienzahl erfolgt.
Bei Behandlungen mit bis zu 0.06% Kaliumsorbat konnen keine Unterschiede der Bakterienzahlen
beobachtet werden, obwohl sie niedriger sind als bei der Kontrolle. Die Tyramin-Bildung wird
fortgesetzt, obwohl Kaliumsorbat in Konzentrationen bis 0.06% eingesetzt wird. Die Histamin-,
Tyramin- und Phenylethylamin-Konzentrationenerhohen sich zunachst, nehmen aber im spateren
Stadium der Reifung bis zu nicht mehr nachweisbaren Mengen ab. Putrescin, Cadaverin und Spermidin
werden in keiner Probe wahrend der Untersuchungen nachgewiesen. Statistische Analysen zeigen, daB
zwischen dem Gesamtgehalt an biogenen Aminen (TBA) in fermentierter Wurst und dem Reifungspro-
zeD ein positiver Zusammenhang besteht. Im Gegensatz dazu besteht eine negative Relation zwischen
TBA und der Kaliumsorbat-Konzentration. Es wird das Bestehen einer Wechselwirkung zwischen der
Konzentration an TBA und den zwei Faktoren (Reifungsperiode und Kaliumsorbat-Konzentration)
mit hoherem BestimmtheitsmaB (R2= 0.94) gezeigt.

Introduction

Biogenic amines have been associated with several outbreaks of food borne disease in
the world. Histamine toxicity has been reported [27] and its possible potentiators, cadaverine

0 VCH Verlagsgesellschaft mbH, D-69451Weinheim, 1995 10$5.OO+.25/0

OO27-769X/9S/O408-03
SHALABY/ABD
EL-RAHMAN:
Biogenic amines in sausage fermentation 309

and putrescine, have been suggested [14, 11. Moreover, there was a relationship between
tyramine content of foods and disease noted after ingestion of such foods [2].
Biogenic amines are especially associated with latic fermented products, particularly wine,
cheese, fish and meat and very low levels occur also in fermented vegetable [13]. The
concentrations of both tyramine and histamine increased dramatically during dry sausage
ripening [5]. Moreover, appreciable levels of both tyramine and histamine in dry sausage
were found [30], while SHALABY [20] found variable concentrations of biogenic amines in
Egyptian sausage.
The factors which influence the formation of biogenic amines in foods include the
availability of free amino acids, the presence of microorganisms that can decarboxylate
amino acids, the favourable conditions for growth of such microorganisms and their
production of decarboxylase enzymes. The increase of free amino acids content during the
ripening of dry sausages has been reported [5]. Microorganisms commonly found in sausage
fermentations include Pediococcus, Lactobacillus, Streptococcus and Micrococcus [4, 71. The
production of lactic acid by Pediococcus cerevisiae seems to contribute to be stability of
some sausage. Pediococcus cerevisiae, Streptococcus sp., and Lactobacillus plantarum were
found to be able to produce biogenic amines [8, 11, 161. Moreover, NOUT1131 stated that
in meat products species of Enterobacteriaceae were associated with cadaverine, and
lactobacilli with tyramine formation. On the other hand, the antimicrobial activity of
sorbate was demonstrated in beef and pork frankfurters [25, 311 and uncooked sausage
[29]. In addition, potassium sorbate was found to be an effective inhibitor of growth and
histamine production by histamine producing bacteria [28].
The aim of this work was to study the effect of potassium sorbate on development of
biogenic amines during sausage fermentation using Lactobacillusplantarum and Pediococcus
acidilactici (Pediococcus cervisiae) as starter cultures, according to the conditions of our
experiments.

Materials and methods

Starter preparation

Lactobaci(lusp1antaruvn DSM 20 174 and Pediococeus acidilactici DSM 20238 cultures were obtained
from the Egyptian Microbial Culture Collection (EMCC), Cairo MIRCEN.
Individual 250 ml ERLENMEYER flasks, containing 100 ml of sterile 1% tryptone - 1 % yeast extract
broth, were inoculated with 1% (V/V) of a 24 h old culture under test. The flasks were incubated
under static conditions at 37 "C for 24 h. At the end of incubation time, the cell crop was harvested,
by centrifugation (Ivan Sorvall, Inc., Norwalk, Cor, USA) at 12000 g for 10 min at 4 "C. Then, the
cells were washed with 10 ml saline solution (0.9%, at 4 "C), and resuspended in sterile saline solution
to their original volume. Twenty ml of each culture suspension were used for inoculation of the sausage
preparation.

Sausage preparation

The composition of the sausage mixture was beef (16.5 kg; containing 10- 12% fat), glucose (500 g),
sucrose (165 g), spices (135 g), garlic (90 g), onion (180 g). and salt (430 9). The beef was obtained
from the local market at Shobra El-Khima, Cairo, Egypt, and was cut into small pieces, which were
tempered overnight at 4- 7 "C prior to formulation. All ingredients including starter, except potassium
sorbate, were added to meat pieces (pH = 5.8), mixed by hand using aseptic gloves to avoid
contamination, passed twice through a home meat mincer (with a 1/8 in plate), and divided into
310 Die Nahrung 39 (1995) 4

6 equal lots. Sorbate was added to the individual lots at concentration of 0% (trt I), 0.02% (trt 11),
0.03% (trt III), 0.04 YO(trt IV), 0.05% (trt V), and 0.06% (trt VI), and dispersed by mixing in a home
mixer.
The sausage mixes were stuffed into natural casings and placed in the air at room temperature
(25 i 2 "C) and 60% relative humidity for fermentation and drying. Samples were taken periodically
for analysis. All data reported are the average of triplicate analyses on each sausage sample.

Microbiological analysis

Twenty grams of sample were taken aseptically from different places and homogenized with 180 ml
of sterile 0.1 % peptone solution by sterile blender. Dilutions were prepared in 0.1 YOpeptone. One ml
from appropriate dilutions were placed in petridishes and poured with melted medium and cooled.
Plate count agar (PCA) medium (Difco labs), was used to enumerate the total bacterial counts, and
MRS agar (Difco labs), was used to enumerate the lactic acid bacterial counts (LAB). Duplicate plates
were done for each dilution. MRS plates were overlayed with the same media and incubated at 37 "C;
PCA plates were incubated aerobically at 30 "C. The plates were enumerated for colony-forming units
(cfu) after 48 h.

Amine analysis

Amines were extracted from sausage samples by 5% trichloroacetic acid (TCA) as recommended
by ZEEet al. [32], and isolated by solvent partitioning procedure as described by MIETZet al. 1121.
Derivatization was done according to SPINELLI et al. [26], and determination was performed using
HPLC (Waters Associates with 3 Model 6000 A solvent delivery systems, Model 720 system controller,
Model 730 data Module, U6k injector and differential UV detector) using the conditions described
by METZ et al. [12]. The accuracy of the method in terms of percentages of recovery [20], the precision
[21] and the limits of detection [19, 221 were estimated by SHALABY [19-221. The sensitivity of the
method is directly related to the apparatus used.

Statistical analysis

Analysis of variance was performed using PROC ANOVA of SAS [17]; Geometic means were
separated by DUNCAN'S multiple range test according to SNEDECOR
et al. [23]. PROC REG of SAS
[ 171 was used to calculate the regression coefficients.

Results and discussion

The changes in populations of total counts and lactic acid bacteria during 45 days
ripening of fermented sausage are given in Table 1. At zero time, the log population count
of total bacteria was 7.34, while log lactic acid count was 6.98. It seems that lactic acid
bacteria required for the fermentation were the predominate microflora where they have
a significant inhibitory action inherent gram-negative bacteria [IS]. The statistical analysis
of the obtained data (Tables 2 and 3) indicates that no significant differences (P < 0.05)
in counts could be observed within treatments (at 45 days storage), on the contrary,
significant differences (P < 0.05) were obtained between treatments and the control. It
reflects that bacterial counts have been inhibited by the potassium sorbate, and the effect
of potassium sorbate was significantly clear (at P < 0.05) by extending the storage period
(Tables 2 and 3). However, shifts in microbial population during ripening period could be
observed, e.g. treatment I11 at day 30 (Table 2), and treatments V and VI at day 30 (Table 3),
 SHALABY/ABD
EL-RAHMAN:
Biogenic amines in sausage fermentation 311

Table 1
Changes in bacterial counts during aging of fermented sausages as affected by potassium sorbate
concentration
Batch No. and Aging period Log bacterial counts [cfu/g]
(sorbate [%I) [days]
APC* LAB**
0 7.34 6.98
15 7.15 7.08
30 6.98 6.00
45 6.34 5.70
I1 15 7.08 6.90
(0.02) 30 6.42 6.40
45 5.40 5.18
I11 15 7.20 7.04
(0.03) 30 6.18 6.04
45 5.60 5.23
IV 15 6.36 6.11
(0.04) 30 5.89 5.51
45 5.63 5.23
V 15 6.04 5.92
(0.05) 30 5.81 5.61
45 5.42 5.28
VI 15 5.96 5.40
(0.06) 30 5.85 5.17
45 5.40 5.08
* Aerobic plate counts
** Lactic acid bacteria

where no significant differences could be obtained (at P < 0.05) between these treatments
and control. The shifts observed proved that the inhibitory action of sorbate against bacteria
is not as comprehensive and appears to be selective. These results are in agreement with
SOFOSet al. [24], who stated that various species and strains of microorganisms exhibit
different sensitivities to inhibition by sorbate leading to shifts in microbial population

Table 2
Statistical analysis of aerobic plate counts (APC)
Batch No. and APC (Geometric mean X lo6)
(sorbate [YO])
Day 0 Day 15 Day 30 Day 45
1 (0) 20.1" 11.7OaS 8.43"*' 2.1 3bs
I1 (0.02) 20.1" 1 1.20"31 2.28b*2 0.22c.2
111 (0.03) 20.1" 1.5.7Oa3' '
14.60". 0.38b,2
IV (0.04) 20.1= 2.26b,2 0.76'*3 0.43"~~
V (0.05) 20.1" 1.13b.
'3 ' '
0.64b1 0.25"~~
VI (0.06) 20.1a 0.94b*3 0.7 1b. 0.24c.z
8' b. ': Means having different letter exponents within rows are significantly different (P < 0.05)
2, 3: Means having different numeric exponents within columns are significantly different (P < 0.05)
312 Die Nahrung 39 (1995) 4

Table 3
Statistical analysis of lactic acid bacterial counts (LAB)
Batch No. and LAB (Geometric mean X lo6)
(Sorbate [%I)
Day 0 Day 15 Day 30 Day 45
1 (0) 9.39a.b 11.90"3 ' 2.13b*'*2 1.94b3'
I1 (0.02) 9.39" 7.83". 2.1 2b' 1 * 2 0.14'p2
I11 (0.03) 9.39" 10.70". ' 1.03b.1 . 2 , 3 0.16'.2
IV (0.04) 9.39" 1.29'. 0.3 1'. 0,l 7d'
V (0.05) 9.39" I .69b*2 3.88"-b, ' 0.17's2
VI (0.06) 9.39" 0.25b.3 0.51'323 0.12d.2
a,b,c.d.
. Means having different letter exponents within rows are significantly different (P < 0.05)
3: Means having different numeric exponents within columns are significantly different ( P < 0.05)

during storage of foods. It was also reported that the addition of sorbic acid and its salts
promoted growth of desirable bacteria and inhibited the spoilage bacteria [3].
Table 4 shows the effect of potassium sorbate concentration on the development of
biogenic amines during aging of fermented sausages. Cadaverine, putrescine, spermine
and spermidine were not detected in all samples analysed throughout the study. Tyramine

Table 4
Changes in biogenic amine content during aging of fermented sausages as affected by potassium sorbate
concentration
Batch No. and Aging Biogenic amines* [mg/kg]
(Sorbat [XI) period
[days1 TYR HIS TRY PHE TBA**
0 25.2 4.2 1.3 8.8 39.5
15 36.7 6.0 2.5 60.0 132.2
30 159.0 7.5 ND 90.0 256.5
45 318.0 5.1 ND 50.0 383.1
I1 15 63.7 6.0 2.5 45.0 117.2
(0.02) 30 127.0 6.0 ND 60.0 193.0
45 317.5 ND ND 20.0 337.5
111 15 63.7 6.0 1.3 25.0 96.0
(0.03) 30 95.3 6.0 ND 40.0 141.3
45 285.8 ND ND 9.5 295.3
IV 15 47.7 6.0 1.3 10.2 65.2
(0.04) 30 93.5 ND ND 18.5 112.0
45 254.0 ND ND ND 254.0
V 15 31.8 5.1 ND 9.5 46.8
(0.05) 30 79.4 ND ND ND 79.4
45 222.3 ND ND ND 222.3
VI 15 31.8 4.9 ND ND 36.7
(0.06) 30 63.5 ND ND ND 63.5
45 222.3 ND ND ND 222.3
* Cardaverine, putrescine, spermine and spermidine not detected in any sample analysed
** TBA: Total biogenic amines
ND: Not detected
SHALABY/ABD
EL-RAHMAN:
Biogenic amines in sausage fermentation 313

concentration at zero time was 25.2 mg/kg and its formation continued throughout the
aging period reaching a maximum of 318 mg/kg at 45 days, for the control. The maximum
values recorded for histamine, tryptamine and phenylethylamine were 7.5 (at 30 days, trt I),
2.5 (at 15 days, trt I and 11) and 90 mg/kg (at 30 days, trt I), respectively, but these values
decreased by extended ripening. The decreases noted in the later stage of ripening were
probably due to decomposition of the amines formed [16] since amines could be utilized
as energy source by other organisms [9, 10, 181.
From the data given in Table 4 it could be noted that potassium sorbate retarded the
formation of biogenic amines. The effect of potassium sorbate increased as its concentration
in the sausages increased (at the same aging period) as well as its effect was more distrinct
by extending the aging period (at the same concentration). The decrease noted in biogenic
amines as a result of using potassium sorbate could be due to that this agent decrease the
possibility of development of a microflora that would posses arnine decarboxylase activity.
TAYLOR et al. [28] stated that potassium sorbate showed promise as an effective inhibitor
of growth and histamine production by histamine-producing bacteria. Moreover, according
to statistical analysis of the microbiological data (Tables 2 and 3), where no significant
changes in bacterial counts were observed within treatments (at day 4 3 , and findings of
EITENMILLER et al. [6], where no significant lower tyramine level was reported between the
use of P . cervisiae and L. plantarum and when no starter culture was used for sausage

Table 5
of TBA as a result of interrelation
Comparison between experimental values (y) and predicted values (j)
between sorbate concentration (A',)and aging period (A',)

Sorbate [%] Aging period Total biogenic amines [mg/kg]

(XI) Pays1 (X,)
Experimental Predicted Difference
value (y) value G) 0, - 3
0 40 50 - 10
0 15 132 154 -22
30 256 257 -1
45 383 36 1 22
0.02 15 117 101 16
30 193 204 -11
45 338 308 30
0.03 15 96 74 22
30 141 178 - 37
45 295 28 1 14
0.04 15 65 48 17
30 112 151 - 39
45 254 255 -1
0.05 15 46 21 24
30 79 125 -46
45 222 228 -6
0.06 15 37 -5 42
30 64 99 - 35
45 222 202 20
314 Die Nahrung 39 (1995) 4

fermentation, the effect of potassium sorbate on the development of biogenic amines may
be due to its inhibitory effect on the decarboxylase enzymes rather than the bacterial growth .
The formation of total biogenic amines (TBA) as a function of the concentration of
potassium sorbate used and aging period with higher determination coefficient (R2 = 0.94)
was given by the following formula:
Y = 50.169 - 2643.718Xl + 6.901X2
where :
Y = Total biogenic amines [mg/kg]
X, = Potassium sorbate used [%]
X , = Aging period [days].
The formula obtained revealed that there is a positive relation between TBA content of
fermented sausage and aging period (during 45 days) as well as there is a negative relation
between TBA and potassium sorbate concentration used (0 -0.06%). The experimental
values and the predicted values obtained from the given formula along with the differences
were given in Table 5.
Finally, it could be concluded that potassium sorbate was advocated in fermented sausage
manufacture for its effect on the inhibition of spoilage bacteria, as well as biogenic amine
content of the final product will be decreased by using this agent. It is of importance to
mention here that the amounts of sorbate used in foods are in the range of 0.02-0.3%,
and these concentrations have no impact on food quality [24].

References

[l] BJELDANES, L. F., D. E. SCHUTZand M. M. MORRIS,Food Cosmet. Toxicol. 16 (1978) 57.

[2] BLACKWELL, B., and L. A. MABBITT, Lancet 1 (1965) 938.
131 Corn Products Company (Delaware), Improvements in or relation to processing milk-based foods.
British Pat. 946, 927 (1964).
[4] DEIBLE, R. H., C. F. NIVENJR. and G. D. WILSON,Appl. Microbiol. 9 (1961) 156.
[5] DIERICK, N., P. VANDEKERCKHOVE and D. DEMEYER, J. Food Sci. 39 (1974) 301.
[6] EITENMILLER, R. R., P. E. KOEHLER and J. 0. REAGAN, J. Food Sci. 43 (1978) 689.
[7] FRAZIER, W. C., and D. C. WESTHOFF,in: Food microbiology. Ed. by W. C. FRAZIERand
D. C. WESTHOFF, p. 218. McCraw-Hill, New York, USA, 1988.
[8] GREIF,G., M. DRDAKand M. GREIFOVA, Possibilities of reduction of biogenic amines in vegetable
substrate by controlled fermentation. No. BS-I 1/16. 7th International Congr. of Bacteriology and
Applied Microbiology, Prague, Czech Rep., 3 - 8 July 1994.
[9] JANZ,I., Arch. Exper. Vet. Med. (Leipzig) 39 (1985) 37.
[lo] JANZ,I., Arch. Exper. Vet. Med. (Leipzig) 39 (1985) 121.
[ l l ] MAYER, K., G. PAUSEand U . VETSCH,Obst Gemuse Verwert. Ind. 58 (1973) 307.
1121 MIETZ,J. L., and E. KARMAS, J. Assoc. Off. Anal. Chem. 61 (1978) 139.
[I31 NOUT,M. J. R., Food Res. Int. 27 (1994) 291.
[14] PARROT, J. L., and G. NICOT,in: Handbuch der Experimentellen Pharmakologie. Vol. XVIII,
p. 148. Ed. by M. ROCHAe SILVA.Springer-Verlag, New York 1966.
[I51 RAO,L. O., F. A. DRAUGHON and C. C. MELTON,J. Food Sci. 49 (1984) 334.
[I61 RICE,S. L., and P. E. KOEHLER, J. Milk Food Technol. 39 (1976) 166.
[17] SAS Institute Inc., SASjSTAT User’s Guide Release 6.04 Edition, SAS Institute, Cray, North
Carolina, USA, 1988.
[I81 SCHEIBNER,G., Mh. Vet. Med. 42 (1987) 120.
1191 SHALABY, A. R., Correlation between freshness indices and degree of fish decomposition. Ph. D.
Thesis, Fac. of Agric., Ain Shams University, Cairo, Egypt, 1990.
SHALABYIABD EL-RAHMAN:
Biogenic amines in sausage fermentation 315

[20] SHALABY, A. R., J. Sci. Food Agric. 62 (1993) 219.

[21] SHALABY, A. R., Food Chem. 49 (1994) 305.
[22] SHALABY, A. R., Food Chem. 52, in press.
[23] SNEDOCOR, G. W., and W. G. COCHRAN,Statistical Methods, 7th Edition. The Iowa State
University Press, Ames, USA, 1980.
[24] SOFOS,J. N., and F. F. BUSTA,in: Antimicrobials in Foods, Ed. by M. P. DAVIDSORR and
L. A. BRANEN, p. 49. Marcel Dekker, Inc., New York, Basel, Hong Kong 1993.
[25] SOFOS,J. N., F. F. BUSTA,K. BHOTHIPAKSA and C. E. ALLEN,J. Food Sci. 44 (1979) 668.
[26] SPINELLI, A. M., L. LAKRITZand A. F. WASSERMAN, J. Agr. Food Chem. 22 (1974) 1026.
[27] TAYLOR, S. L., J. Y. HUI and D. E. LYOUS,in: Seafood Toxins. Ed. by E. P. RAGILS.p. 417. ACS
Symposium Series 262. Washington, D.C., 1984.
[28] TAYLOR, S. L., and M. W. SPECKHARD, J. Food Prot. 47 (1984) 508.
[29] TOMPKIN, R. B., L. N. CHRISTANSEN, A. B. SHAPARIS and H. BOLIN,Appl. Microbiol. 28 (I 974) 262.
[30] VANDEKERCKHOVE, P., J. Food Sci. 42 (1977) 283.
[31] WAGNER, M. K., and H. H. BUSTA,J. Food Sci. 48 (1983) 990.
[32] ZEE,J. A., R. E. SIMARD and L. L’HEUREUX, J. Food Prot. 46 (1983) 1044.

Dr. ALI R. SHALABY,

Food Technology & Dairy Science Dept., National Research Centre, Dokki,
Cairo, Egypt

Received 15 November 1994

Revised manuscript received 18 April 1995