ARTICLE IN PRESS

LWT 41 (2008) 730–738

www.elsevier.com/locate/lwt

Characterization of fermented silver carp sausages inoculated

with mixed starter culture
Yongjin Hua,b, Wenshui Xiaa,, Changrong Geb
a
Key Laboratory of Food Science and Safety, Ministry of Education, Southern Yangtze University, Wuxi 214122, Jiangsu, China
b
School of Food Science and Technology, Yunnan Agricultural University, Kunming 650201, Yunnan, China
Received 16 August 2006; received in revised form 5 April 2007; accepted 5 April 2007

Abstract

To improve the characteristics and functionality, and increase the use of fish muscle, three groups mixed starter cultures (group one:
Lactobacillus plantarum-15, Staphylococcus xylosus-12 and Pediococcus pentosaceus-ATCC33316 [S-PXP]; group two: Lactobacillus
planatrum-15, Staphylococcus xylosus-12 and Lactobacillus casei subs casei-1.001 [S-PXC]; and group three: Staphylococcus xylosus-12,
Lactobacillus casei subsp. casei-1.001 and P. pentosaceus-ATCC33316 [S-XCP]) were inoculated in minced silver carp muscle to produce
a fermented fish product. During the 48 h fermentation at 30 1C, silver carp muscle inoculated with mixed starter cultures resulted in a
rapid pH decrease, suppression in the increase of thiobarturic acid (TBARS) values, total volatile base nitrogen (TVB-N), trimethylamine
(TMA), and the growth of spoilage bacteria and pathogens, and had higher whiteness than the control (without any starter) (Po0.05).
The changes in SDS-PAGE indicated extensive hydrolysis of muscle protein occurred during fermentation. This study showed that the
mixed starter cultures could substantially improve the flavor, digestibility, and nutritional value of the silver carp muscle.
r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Silver carp; Lactic acid bacteria; Staphylococcus xylosus; Fermented sausage; Mixed starter cultures

1. Introduction The fermentation of fish for human consumption has many

Silver carp (Hypophthalmichthys molitrix) may be the
cheapest and most abundant freshwater fish due to its
quick growth and resistance to stress, disease and rough
handling. In China fresh water fish, unlike marine fish,
mainly marketed for fresh condition consumption. The
main processed products are salted fish and fish sauce.
However, these products have high salt concentration
(15–25% w/w) and, therefore, have limited nutrient value
because they cannot be consumed in large quantities
(Aryanta, Fleet, & Buckle, 1991). The development of
new fish products, which would be free of the fishy odor
and taste, and which retained all the nutritional advantages
of fish, would enlarge the range of applications of silver
carp muscle. A promising approach to the creation of such
fish products seems to be through the use of fermentation.
Corresponding author. Tel.: +86 510 85869455;
fax: +86 510 85812812.
E-mail address: xiawshsytu@yahoo.com.cn (W. Xia).
benefits (Cooke, Twiddy, & Alan Reilly, 1987; Gelman,
Drabkin, & Glatman, 2000; Paludan-Muller, Madsen,
Sophanodora, Gram, & Moller, 2002). it could be used
as a low cost, convenient method for the unrefrigerated
preservation of fish muscle and improve the organoleptic
qualities of fish and increase the nutritional value and/or
digestibility of the raw material.
Some fermented meats or fish are very popular in
oriental countries and also in parts of western countries. In
recent years, using pure bacterial cultures to produce a fish-
type product is attracting increasing interest. Various
researchers have stated that using lactic acid bacteria
(LAB) in fish meat has improved the quality of the end
product. According to Kim and Hearnsberger (1994), using
Pediococcus acidilactici to manufacture fish sausage could
accelerate the formation of lactic acid and significantly
inhibit the growth of spoilage bacteria and pathogens,
which consequently extended the shelf life and also
enhanced the safety. Rapid decline of pH not only gives
the products a unique lactic acid flavor, but also increases

0023-6438/$30.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2007.04.004
 ARTICLE IN PRESS
Y. Hu et al. / LWT 41 (2008) 730–738
the texture firmness and mouthfeel due to the acid
denaturation of muscle proteins (Palumbo et al., 1993).
Gelman et al. (2000) applied LAB fermentation with
Leuconostoc mesenteroides, Pediococcus pentosaceus and
Lactobacillus plantarum to the minced yellowfin tuna.
Under chill conditions the presence of L. mesenteroides
gave changes towards a meaty flavour and juicy texture,
low values for chemical indices of spoilage and extended
shelf-life (over 4 weeks). Yin, Pan, and Jiang (2002) used
LAB to ferment mined mackerel. The fermented products
substantially inhibited the development of volatile basic
nitrogen, suppressed the growth of other microflora, and
hydrolyzed the muscle proteins during fermentation.
Using LAB fermentation in combination with coagulase-
negative Staphylococci (CNS), such as Staphylococcus
xylosus can contribute to the control of microbial safety
and quality (hurdle technology). LAB ensures the safety of
products by reducing the pH through fermentation of
sugars, while CNS influence other technological properties
of fermented meat products. Many authors (Molly et al.,
1997; Talon, Walter, Chartier, Barriere, & Montel, 1999)
suggested that Staphylococcus spp., rather than LAB, could
have a predominant effect on the characteristic flavor of
fermented sausages. In particular S. xylosus with its
catalase activity prevents rancidity (Barriere et al., 2001;
Mauriello, Casaburi, Blaiotta, & Villani, 2004), and
proteolytic and lipolytic activities contributing to the
aroma of fermented sausages by the formation of esters
and other aromatic compounds from amino acids
(Johansson, Berdague, Larsson, Tran, & Borch, 1994;
Montel, Reitz, Talon, Berdague, & Rousset-Akrim, 1996).
That is why mixed cultures are widely used in manufactur-
ing dry fermented sausages. However, little is known of the
microbiological and biochemical changes that occur in
freshwater fish sausage fermentation inoculated with the
combination of LAB plus S. xylosus.
The main aim of this study was to determine the effect of
the three groups mixed starter cultures on the character-
ization of silver carp sausage during the manufacture.
Quality parameters of fermented silver carp, which are
TBARS, pH, water activity, microbial counts, TMA and
TVB-N, were determined. In addition to these, proteolysis
characteristics and whiteness were also analyzed, which
might provide further information on the characterization.
2. Materials and methods
2.1. Preparation of starter culture
Lactobacillus casei subsp. casei-1.001 and P. pentosaceus-
ATCC 33316 were purchased from China General
Microbiological Culture Collection Center, Beijing; L.
plantarum-15, and S. xylosus-12 were obtained from the
Technology Center of the Shuanghui Group (a leading
meat-processing company in China). The LAB was
separately subcultured twice in DeMan Rogosa Sharpe
(MRS) broth (Difco, Detroit, MI, USA) at 30 1C for 2
731
days. Cell pellets were harvested by high-speed refrigerated
centrifuge (Model HSC-20RA, Tuman, Jilin, China) at
10,000g for 15 min at 4 1C and washed with 20 mmol/l KCl-
phosphate buffer, pH 7.0 and then the cell pellets were
resuspended in the same buffer. S. xylosus-12 was
subcultured twice in Nutrient Broth at 30 1C for 3 days
each time. Cells were harvested by centrifugation at 2000g
for 15 min at 4 1C, washed with saline water (0.9% NaCl)
and then resuspended in saline water. Finally, the number
of bacterial cells in each suspension was adjusted to reach
the range of 7–8 log CFU/ml of saline solution by using a
spectrophotometer (Model WFZ-UV-2100, Unico TM,
Shanghai, China) (Fadda, Vignolo, Holgado, & Oliver,
1998).
2.2. Silver carp sausage preparation
Frozen silver carp (2.5–3 kg/fish), purchased from a local
market (Wuxi, Jiangsu, China), was thawed in running tap
water and then gutted, eviscerated, deboned, removed the
scale, skin, pin bones, debris, and filleted. Silver carp fillets
were minced through a deboner (Model 694, New Bedford,
MA, USA) with a drum having 5 mm-diameter perfora-
tions. The processed samples were then mixed with 1
volume of sterile water, 3% NaCl, 3% glucose. Starter
cultures were inoculated to a final level of 6–7 log CFU/g
fish mince and well mixed using a sterile glass rod. Three
separated batches were prepared with different mixed
starter cultures that are S-PXP (L. plantarum-15,
S. xylosus-12, and P. pentosaceus-ATCC 33316 [1:1:1]);
S-PXC (L. plantarum-15, S. xylosus-12 and L. casei
subsp. casei-1.001 [1:1:1]), S-XCP (S. xylosus-12, L. casei
subsp. casei-1.001 and P. pentosaceus-ATCC33316 [1:1:1])
and a batch without any starter (NS) as control were
prepared. Each sausage batters was stuffed into collagen
casings (+38 mm, RL2, Naturin, Weinheim, Germany)
with a filling machine (SZ-200, Guangzhou, China) and
fermented at 30 1C, RH 85% for 48 h. Samples were taken
every 12 h for analysis.
2.3. Microbiological analyses
Silver carp sausage samples (25 g) were aseptically
transferred to a sterile plastic bag and pummeled for
1 min in a Stomacher (IUL Instrument, Barcelona, Spain)
with 225 ml 0.1% peptone water. Appropriate decimal
dilutions of the samples were prepared using the same
diluent and 0.1 ml of each dilution was plated in triplicate
on different growth media (Table 1). Results were
expressed as colony forming units per gram (log CFU/g).
2.4. Proximate chemical analyses
The water activity (Aw) was carried out by using a digital
water activity meter (Rotronic Hygroskop DT, Zurich,
Switzerland) after equilibrium at 25 1C. Protein contents
were determined by the Kjeldahl Method (AOAC, 2002).
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732 Y. Hu et al. / LWT 41 (2008) 730–738

Table 1
Media and incubation conditions of testing microorganism

Media Microflora Conditions for incubation Source

PCA Agar Total aerobic bacteria 37 1C for 48 h Plate Count Agar (Oxoid, CM325 )
MRS Agar Lactic acid bacteria 37 1C for 48 h Man Rogosa Sharpe (Oxoid, CM361 )
VRBD Agar Enterobacteriaceae 37 1C for 48 h Violet-Red-Bile-Dextrose-Agar (Oxoid, CM485)
MS Agar Staphylococcus 30 1C for 72 h Mannitol Salt Agar (BD Difco)
GSP Agar Pseudomonas 26 1C for 72 h Pseudomonas-Aeromonas-Selective-Agar-Base (Merck, Nr 10230)
YM Agar Yeasts and molds 25 1C for 96 h Yeast Malt Agar (BD Difco)

the crude fat content was determined by a Soxhlet
extraction apparatus using the solvent diethyl ether
anhydrous (40–50 bp) (AR, Shanghai, China) (AOAC,
2002).
2.5. Determination of pH and titratable acidity (TA)
pH measurements were done according to the procedure
of Wang (2000). Ten gram samples were homogenized with
90 ml deionized water and the pH was measured with a
digital pH meter (Mettler Toledo 320-s, Shanghai, China).
Titratable acidity (TA) expressed as percent lactic acid
were determined according to the method described by
Ikenebomeh (1989).
2.6. Determination of weight loss
Weight loss was determined as described by Nakao et al.
(1991). A sample with casing (100 g) was accurately
weighed before fermentation using an analytical balance
(ESJ120-4, Fuzhou, China). During the manufacture,
sample was taken and then reweighed. Difference in weight
of sausage before and after fermentation was referred to as
weight loss.
2.8. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE)
The water-soluble proteins were isolated by homogeniz-
ing 10 g of mince with 90 ml chilled distilled water and
centrifuging at 5000g for 20 min. The supernatant was used
to determinate the water soluble muscle proteins. The salt-
soluble proteins were isolated by homogenizing 10 g of
mince with 90 ml 0.6 M NaCl-phosphate buffer (pH 7.2).
After 20 min centrifugation at 5000g, the supernatant was
collected and used as the salt soluble muscle proteins.
SDS-PAGE was done using a vertical gel electrophoresis
unit (Mini-Protean-3 Cell. Bio-Rad, Richmond, CA, USA)
according to the methods of Laemmli (1970) and a running
gels containing 10% T (acrylamide plus bisacrylamide),
and a stacking gel containing 4% T (0.125 mol/l Tris, pH
6.8). Aliquots of 15 ml were injected in each well including
standard markers. Electrophoresis was done at 100–120 V.
After electrophoresis was completed, gels were stained at
25 1C, using Coomassie Brilliant Blue R-250 (0.1%) and
destained with a solution of aqueous methanol (methano-
l:acetic acid:water ¼ 1:1:8) until a clear background was
obtained. A high molecular weight (HMW) calibration kits
(Pharmacia, BD, Sigma) (myosin [200 kDa], calmodulin
[160 kDa], galactosidase [116 kDa], phosphorylase-b
[97.4 kDa], serum albumin [66.2 kDa], actin [43 kDa],
arbonicanhydrase [29 kDa], trypsin inhibitor [25 kDa] and
lactalbumin [14.2 kDa]) were used as protein markers.

2.7. Determination of TVB-N, TMA contents and TBARS 2.9. Color evaluation

TVB-N and TMA contents were determined by the
micro-diffusion method of Conway (Cobb & Thompson,
1973).
TBARS was determined according to the method of
Buege and Aust (1978). A sample (5 g) was homogenized
with 25 ml of TBARS solution (0.375% TBA, 15% TCA,
and 0.25 N HCl). The mixture was heated for 10 min in
boiling water (95–100 1C) to develop a pink color. Then the
mixture was cooled with running water and centrifuged at
5500g for 25 min. The absorbance of the supernatant was
measured at 532 nm using a spectrophotometer (Model 135
WFZ-UV-2100, UNICOTM, Shanghai, China). The
TBARS value was calculated from the standard curve of
malonaldehyde and expressed as mg malonaldehyde/kg
sample.
The color of the samples was measured as the L ; a ; b
values of CIE using a color meter (Model TC-Pa G, Beijing
optical Instruments Factory, Beijing, China). L , a and b
indicate lightness, redness/greenness and yellowness/blueness,
respectively. Whiteness was calculated using the following
equation (Lanier, Hart, & Martin, 1991):
Whiteness ¼ 100  ½ð100  L Þ2 þ ða Þ2 þ ðb Þ2 1=2 .
2.10. Statistical analyses
Duncan’s multiple range test was employed to determine
the significance of difference within treatments for each
treatment. Three replicates were performed and the mean
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Y. Hu et al. / LWT 41 (2008) 730–738 733

values were calculated. Values were considered significantly
different when Po0.05. Date were analyzed for degree of
variation and significance of difference using an analysis of
variance (ANOVA) (Ramsey & Schafer, 1997). All
statistical analyses were performed using SPSS statistic
program (Version 11.0 for windows, SPSS Inc., Chicago,
USA, 2001).
3. Results and discussion
3.1. Microbiological analyses
The initial total aerobic plate count (APC) in the sausage
batter was in the range of 3.7–4.7 log CFU/g. However,
during 48 h fermentation at 30 1C, the batches with added
cultures significantly increased (Po0.05) to the level of
high log CFU/g after 36 h fermentation and then started to
stabilize or decline during later stages (Table 2). It is
seemed likely that the higher acidity and bacteriocin
produced by starters could have suppressive action against
the growth of APC in the end product (Yin et al., 2002).
LAB counts increased during fermentation for both the
control and starters added samples (Table 2). Initial LAB
counts in the starter cultures added samples were
significantly higher than in the control samples (Po0.05)
due to the inoculation of starter strains. During 36 h
fermentation, LAB numbers increased and reached levels
up to 9.2 log CFU/g of sausage in all batches of sausage
inoculated with mixed starters, which indicated that silver
carp is suitable for the growth of LAB. In contrast, the
control samples LAB increased more slowly, LAB counts
increased to 7.32 log CFU/g during 36 h. Likewise, the
initial counts for Micrococcaceae among samples ranged
from 4 to 7 log CFU/g, increasing with starters addition.
The counts of Micrococcaceae were significantly increased
in the batches inoculated with mixed starters to a level of
7–8 log CFU/g at 12 h, thereafter, the counts decreased
significantly in all batches except the control (Table 2).
Several authors have reported that the acidification and
anaerobic conditions inhibited the growth of Micrococca-
ceae during ripening of fermented sausages (Hugas &
Monfort, 1997; Aksu & Kaya, 2004).
Enterobacteriaceae and Pseudomonas counts of sausages
with or without starters were between 2.1 and 3.6 log CFU/g
at the beginning of fermentation; no differences between
batches were observed. After 48 h of fermentation, the
sausages inoculated with starter cultures significantly
inhibited the growth of Enterobacteriaceae and Pseudomonas

Table 2
Microbiological changes in silver carp sausage during fermentation

Microorganisms Sample Ferment time (h)

0 12 24 36 48
a a a
Aerobic bacteria NS 3.7870.22 6.7170.18 7.5870.22 8.7970.20 8.9270.16a
S-PXC 4.6870.15 7.6270.16b 9.1270.23b 9.7670.14b 9.4270.13b
S-PXP 4.4270.20 7.3270.18b 9.2470.17b 9.5870.15b 9.2870.19b
S-XCP 4.6270.12 7.6270.14b 9.3470.16b 9.8870.14b 9.6870.20b

LAB NS 4.1170.14a 6.4370.16a 6.8870.20a 7.3270.18a 7.3870.21a

S-PXC 6.0670.13b 7.2270.12b 9.2270.16b 9.6470.14b 9.4270.24b
S-PXP 6.1270.10b 7.0470.22b 9.4670.16b 9.7870.13b 9.6270.19b
S-XCP 5.8670.13b 7.1270.16b 9.0270.20b 9.3470.18b 9.2270.18b
Enterobacteriaceae NS 2.5670.11 5.8270.18a 8.1270.23a 8.6670.20a 8.1370.24a
S-PXC 2.3670.12 2.7670.21b 2.8870.16b 3.2870.14b 2.5670.20b
S-PXP 2.1870.11 2.3170.17b 2.5270.12b 2.7870.12b 2.1270.14b
S-XCP 2.3670.13 2.7670.12b 2.8870.11b 3.2870.18c 2.5670.20c
Micrococaceae NS 4.2170.18a 5.5270.11a 5.7870.21a 6.8870.25a 7.2170.22a
S-PXC 6.2170.21b 7.6870.25b 7.4370.17b 7.0270.22b 6.8870.13b
S-PXP 6.3170.21b 7.4270.19b 7.2670.21b 7.1670.14b 6.6570.20b
S-XCP 6.3570.24b 6.8970.17b 7.1670.14b 7.1270.20b 6.4270.17b

Pseudomonas NS 3.5670.22 5.6670.20a 7.6570.25a 8.1370.31a 8.5870.22a

S-PXC 3.2170.28 3.4470.15b 3.6270.22b 2.6670.20b 1.6870.15b
S-PXP 3.2870.20 3.1670.19b 3.4370.17b 2.4470.24b 1.9870.14b
S-XCP 3.3670.22 3.6870.24b 3.8870.20b 2.9670.22b 2.2270.17b
Yeasts and molds NS 1.5670.12 4.2570.20a 6.6870.17a 6.7270.24a 6.9070.19a
S-PXC 1.3670.12 2.5670.18b 4.2670.20b 2.3370.16b 1.3270.14b
S-PXP 1.4470.11 2.7870.15b 4.0370.13b 2.1370.18b 1.0170.18b
S-XCP 1.3270.14 2.6670.21b 4.3270.16b 2.6270.11b 1.6870.14b
 NS: no starter added; S-PXP: L. plantarum-15, S. xylosus-12 and P. pentosaceus-ATCC33316 mixed culture; S-PXC: L. plantarum-15, S. xylosus-12
and L. casei subsp. casei-1.001 mixed culture; S-XCP: S. xylosus-12, L. casei subsp. casei-1.001 and P. pentosaceus-ATCC33316 mixed culture.
a–c
Values with unlike superscript letters in the same column are significantly different (Po0.05).
 ARTICLE IN PRESS
734 Y. Hu et al. / LWT 41 (2008) 730–738

Table 3
Proximate chemical composition of fermented silver carp sausages

Parameter Sample

BF NS S-PXP S-PXC S-XCP

a c b b
Moisture (g/100 g) 78.6870.69 77.4270.09 78.1670.10 78.1270.12 78.3270.10ab
Protein (g/100 g DM) 31.201470.20a 24.2470.20b 23.5670.20c 22.6870.32c 22.7270.42c
Fat (g/100 g DM) 5.6470.31 5.2670.20 5.2870.10 5.3470.15 5.2670.10
Ash (g/100 g DM) 1.2170.02 1.2170.01 1.2070.01 1.1970.02 1.2070.01
NaCl (g/100 g DM) 1.5570.01 1.6270.01 1.4670.02 1.5270.01 1.5070.01

DM ¼ dry matter.
 BF: before fermentation; NS: no starter added; S-PXP: L. plantrum-15, S. xylosus-12 and P. pentosaceus-ATCC33316 mixed culture; S-PXC: L.
plantrum-15, S. xylosus-12 and L. casei subsp. casei-1.001 mixed culture; S-XCP: S. xylosus-12, L. casei subsp. casei-1.001 and P. pentosaceus-ATCC33316
mixed culture.
a–c
Values with different superscript letters in the same row are significantly different (Po0.05).

(Table 2). Our result was agreement with the result of 0.950
8
Aryanta et al. (1991) who reported that Enterobacteriaceae
counts were significantly decreased with starter cultures 7
added to Turkish Soudjoucks (a fermented meat product) 0.925
after fermentation. This result was also similar to that 6

Weight losses (g/100g)

obtained by with LAB fermented mackerel (Yin et al. 0.900 5
2002). The yeast and mold counts increased in the batches
Aw

inoculated with mixed starters to a level of 4.0–4.5 log 4

CFU/g at 24 h, thereafter, the counts decreased signifi- 0.875
cantly (Po0.05) with processing time to 1–2 log CFU/g. In 3
contrast, the control showed higher (Po0.05) counts of
yeasts and molds in final products (Table 2). 0.850 2

3.2. Proximate chemical composition, Aw, weight losses,
and WHC
No significant differences (P40.05) were seen for fat,
ash and salt content among all batches of sausages (Table
3). However, moisture content was significantly (Po0.05)
lower in the control resulting in a higher protein content.
Initially, the Aw for sausages was 0.91, which decreased
significantly (Po0.05) with fermentation to a final range of
0.82–0.89. Mixed starter culture batches showed a slightly
higher Aw than the control (Fig. 1). Weight losses
significantly increased with processing time and reached
to maximum of 7.78% in the control. In contrast, silver
carp sausages inoculated with mixed starter cultures
showed a lower weight losses than the control (Fig. 1).
3.3. pH value and TA
As fermentation time increased, silver carp sausages
inoculated with mixed starter showed a lower pH than the
control (Fig. 2). The pH gradually decreased to 4.24, 4.31,
and 4.40 within 48 h for S-PXP, S-PXC and S-XCP,
respectively. The pH values for all batches of silver carp
sausages inoculated with mixed starters were not signifi-
cantly different (P40.05). Since fermented sausage is
normally eaten uncooked to eat, it is recommended that
silver carp sausage with pH lower than 4.4 is safe for
consumption (Paukatong & Kunawasen, 2001), i.e., free of
1
0.825
0
0 12 24 36 48
Fermentation time (h)
Fig. 1. Changes in Aw and weight losses in silver carp sausage with/
without starters during fermentation (NS O; S-PXC K; S-PXP &; S-XCP
’). NS: no starter added; S-PXP: L. plantarum-15, S. xylosus-12 and P.
pentosaceus-ATCC33316 mixed culture; S-PXC: L. plantarum-15, S.
xylosus-12 and L. casei subsp. casei-1.001 mixed culture; S-XCP: S.
xylosus-12, L. casei subsp. casei-1.001 and P. pentosaceus-ATCC33316
mixed culture.
pathogenic microorganisms that natural contaminants in
silver carp. The samples without a starter decreased slightly
the first during the 12 h of fermentation and then increased
gradually to 7.2. The pH rose in the control was probably
due to the formation of TVB-N (Table 4). An appreciable
increase in TA was noted in the silver carp sausages
inoculated mixed starter cultures after 24 h of fermenta-
tion. At the end of 48 h fermentation, TA in the samples
inoculated starters increased from 0.28% to 5.98%, while
TA declined from 0.3% to 0.24% in the control.
3.4. Lipid oxidation
The highly unsaturated lipids in fat-rich fish are easily
susceptible to oxidation that results in a rancid smell and
taste as well as alterations in texture, color and nutritional
 ARTICLE IN PRESS
Y. Hu et al. / LWT 41 (2008) 730–738 735

value (Ólafsdóttir et al., 1997). TBARS value is a widely
used indicator for the assessment of degree of lipid
oxidation. It has been suggested that a maximum TBARS
value, indicating the good quality of the fish, is 5 mg
malonaldehyde/kg (Sikorski, Kolakowska, & Burt, 1990).
In the present study, the initial TBARS values ranged in
0.16–0.18 mg of MDA/kg in samples. After 48 h fermenta-
tion, the control sample had the highest TBARS value
(1.46 mg MDA/kg), whereas starter S-PXP inoculated
sausages had the lowest (1.05 mg MDA/kg) (Fig. 3). Since
the LAB, particularly P. pentosaceus-ATCC33316 and S.
xylosus-12 showed their antioxidant effects on unsaturated
fatty acids, the TBARS values for products added with
such organisms could be definitely lower than the TBARS
values found in the control. This result also confirmed the
result of Talon, Walter, and Montel (2000), who have been
reported that the antioxidant action of starter P. pentosa-
ceus in lipid oxidation.
3.5. TMA, TVB-N, and proteolysis
TVB-N and TMA are directly related to the microbial
spoilage in various species of fish during their processing
and storage (Dalgaard, 2000). The changes in TMA
content in the fermented silver carp sausages and the
control are shown in Table 4. The initial TMA value
ranged from 0.85 to 0.86 mg/100 g muscle, which then
increased very slowly during the first 24 h of fermentation,
reaching low values of 1.38, 0.99, 1.06, and 1.10 mg/100 g
for each of the control, S-PXP, S-PXC, and S-XCP
samples, respectively. By the 36 h of fermentation and
thereafter, the TMA value of control samples increased
rapidly, attaining 10.16 mg/100 g by the end of the
fermentation (48 h), whereas significantly (Po0.05) lower
values of 2.62, 2.78, and 2.86 mg/100 g sausages were
detected for the samples fermented with S-PXP, S-PXC, S-
XCP, respectively. Formation of TMA in the fish muscle is

2.5
12 4.0
11
TBARS (mg of malondialdehyde/kg)

10 4.5
2.0
9
5.0
Titratable acidity (%)

8
1.5
7 5.5
pH

6
5 6.0 1.0
4
6.5
3
0.5
2
7.0
1
0 7.5 0.0
0 12 24 36 48 0 12 24 36 48
Fermentation time (h) Fermentation time (h)

Fig. 2. Changes in TA and pH in silver carp sausage with/without starters
during fermentation (NS O; S-PXC K; S-PXP &; S-XCP ’). NS: no
starter added; S-PXP: L. plantarum-15, S. xylosus-12 and P. pentosaceus-
ATCC33316 mixed culture; S-PXC: L. plantarum-15, S. xylosus-12 and L.
casei subsp. casei-1.001 mixed culture; S-XCP: S. xylosus-12, L. casei
subsp. casei-1.001 and P. pentosaceus-ATCC33316 mixed culture.
Fig. 3. Changes in TBARS in silver carp sausage with/without starters
during fermentation (NS O; S-PXC K; S-PXP &; S-XCP ’). NS: no
starter added; S-PXP: L. plantarum-15, S. xylosus-12 and P. pentosaceus-
ATCC33316 mixed culture; S-PXC: L. plantarum-15, S. xylosus-12 and L.
casei subsp. casei-1.001 mixed culture; S-XCP: S. xylosus-12, L. casei
subsp. casei-1.001 and P. pentosaceus-ATCC33316 mixed culture.

Table 4
Changes in TMA and TVB-N in silver carp sausages during fermentation

FT (h) TMA (mg/100 g) TVB-N(mg/100 g)

NS S-PXP S-PXC S-XCP NS S-PXP S-PXC S-XCP

0 0.8670.02 0.8670.01 0.8570.02 0.8670.02 1.170.22 1.1470.11 1.1170.12 1.1270.11

12 0.9870.02 0.9370.04 0.9170.02 0.9370.02 9.5470.06a 1.2970.22b 1.2470.31b 1.2970.16b
24 1.3870.03a 0.9970.05b 1.0670.03b 1.1070.03b 16.3570.32a 1.8870.18b 1.8970.13b 1.9370.42b
36 4.2270.04a 1.7670.06b 1.8270.03b 1.8870.02b 38.6670.26a 3.2470.55b 3.6470.32b 3.7570.43b
48 10.1670.06a 2.6270.04b 2.7870.02b 2.8670.04b 56.7170.19a 5.7870.32b 5.0170.48b 5.9270.71b
 FT: fermentation time.
Refer to the footnote of Table 1.
a–c
Values with unlike superscript letters in the same row are significantly different (Po0.05).
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736 Y. Hu et al. / LWT 41 (2008) 730–738

Fig. 4. Changes in SDS-PAGE profile of salt-soluble protein (A) and water-soluble protein (B) in fermented silver carp sausages. (S: protein markers, BF:
before fermentation, NS: no starter added; S-PXP: L. plantarum-15, S. xylosus-12 and P. pentosaceus-ATCC33316 mixed cultures; S-PXC: L. plantarum-
15, S. xylosus-12 and L. casei subsp. casei-1.001 mixed cultures; S-XCP: S. xylosus-12, L. casei subsp. casei-1.001 and P. pentosaceus-ATCC33316 mixed
cultures).

mainly by the bacterial action on the TMAO content and
the presence of specific spoilage organisms in the fish
(Huss, 1988). Mixed starters cultures treated samples
showed relatively lower amount of TMA values than in
the control. The decrease in pH inhibits the growth of
contaminant microorganisms present in the raw materials,
which suppress the accumulation of TMA.
The initial TVB-N values were in the range of
1.11–1.14 mg/100 g, which significantly increased with
processing time and reached to maximum of 56.71 mg/
100 g in the control (Table 4). In contrast, only slight
increase in the TVB-N values was observed in the silver
carp sausages inoculated with mixed starter cultures. This
result agreed to the result of Yin et al. (2002), who reported
that the use of LAB in meat fermentation could inhibit the
accumulation of TVB-N by producing lactic acid and
bacteriocins, which could neutralize the TVB-N or inhibit
the growth of spoilage bacteria and pathogens.
Salt and water soluble proteins were noticeably degraded
during fermentation of silver carp muscle and the intensity
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Y. Hu et al. / LWT 41 (2008) 730–738
Table 5
Hunter color analysis for silver carp sausages
Parameter Sample
BF NS
a b
L* 38.7870.56 44.6870.45
a* 2.1170.11a 1.1270.12b
b* 7.8670.48a 9.770.78b
Whiteness 38.2871.66 43.8370.96a
Refer to the footnote of Table 3.
a–c
Values with different superscript letters in the same row are significantly different (Po0.05).
of proteolysis was more intense in the mixed culture added
samples (Fig. 4). Compared with the samples before the
fermentation and the control, the bands at 200, 160, 116,
and 105 kDa molecular weight for the salt soluble proteins
disappeared as a result of fermentation and the intensity of
the band at 70 kDa for the water soluble proteins markedly
decreased. The bands 66, 26, 22, and 14 kDa appeared to
be more intense during fermentation. These results for the
degradation were very similar to the results of Hughes
et al.(2002) for semi-dry fermented sausages, where starter
culture had a more pronounced effect on protein degrada-
tion than the control without starters. These phenomena
suggested that the proteolysis occurred rapidly during
fermentation because of the action of proteinases from S.
xylosus or LAB as well as the hydrolysis of muscle proteins
caused by acid (Astiasaran, Villanueva, & Bello, 1990).
3.6. Color
Table 5 shows the effects of mixed starter cultures on the
Hunter L , a , b values and the texture profile for four
different batches of fermented sausage. The Hunter L* and
whiteness of samples with starter cultures were significantly
higher than those without starter and those before
fermentation (L : increased from 38.78 to 63.28 to 65.42,
whiteness: from 38.28% to 60.02% to 62.20%, Po0.05).
However, the Hunter a value of those samples with
starters was significantly lower than that without starter
and that before fermentation (Po0.05). Hashimoto and
Watabe (1988), and Ochiai, Chow, Watabe, and Hashi-
moto (1988) ascribed the increase in Hunter L of frozen
tuna meat to the decrease in water holding capacity, which
might be due to the hydrolysis of muscle proteins or
aggregation of myofibrillar proteins during frozen storage.
In this study, the decrease in Hunter a and the increase in
Hunter L and whiteness might be due to the hydrolysis of
water-soluble and salt-soluble proteins (Yin et al., 2002)
and pigment proteins, which consequently make the
fermented products more transparent and much whiter.
The whiter color of Som-fug, an indigenous fermented fish
mince of Thailand, was reported to contribute to consumer
acceptability (Riebroy, Benjakul, Visessanguan, Kijron-
grojana, & Tanaka, 2004).
737
S-PXP S-PXC S-XCP
c c
63.2871.02 63.6471.12 65.4271.26c
0.5770.22c 0.5070.12c 1.3970.18c
15.8070.36c 15.6270.40c 15.2270.18c
60.0271.16b 60.4271.04b 62.2070.68b
4. Conclusions
Using the combinations of LAB along with S. xylosus
could substantially inhibit the accumulation of TVB-N and
TMA, suppress the growth of the main microflora present
in the raw materials, and hydrolyze the muscle proteins and
decreased lipid oxidation during fermentation. In addition,
sausages with mixed starter cultures added had lower
TBARS values, which indicating less lipid oxidation. These
results suggest the high potential in using mixed starter
cultures in the fermented fish products and developing
novel fish food products.
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