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Effective use of nisin to control lactic acid

bacterial spoilage in vacuum-packed
bologna-type sausage

ARTICLE in JOURNAL OF FOOD PROTECTION · OCTOBER 1999

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1004

Journal of Food Protection, Vol. 62, No. 9, 1999, Pages 1004–1010

Copyright Q, International Association of Milk, Food and Environmental Sanitarians

Effective Use of Nisin to Control Lactic Acid Bacterial Spoilage

in Vacuum-Packed Bologna-type Sausage
E. ALISON DAVIES, CATHERINE F. MILNE, HELEN E. BEVIS, RICHARD W. POTTER, JO M. HARRIS,
GRAHAM C. WILLIAMS, LINDA V. THOMAS,* AND JOSS DELVES-BROUGHTON

Aplin & Barrett Ltd. (Cultor Food Science), Technical Department, 15 North Street, Beaminster, Dorset DT8 3DZ, UK

MS 98-338: Received 11 December 1998/Accepted 3 March 1999

ABSTRACT
Lactic acid bacteria (LAB) commonly cause spoilage in minimal heat-treated vacuum-packed cured delicatessen meats.
Predominant species are Lactobacillus sake and L. curvatus. LAB strains isolated from spoiled products of this type (liver
sausage, ham and bologna sausage) were found to be sensitive to low nisin concentrations (maximum of 1.25 mg g21). Addition
of 25 mg g21 nisin (as Nisaplin) inhibited the growth of LAB spoilage organisms inoculated into vacuum-packed pasteurized
bologna-type sausages stored at 88C. Control sausages became spoiled (.108 LAB CFU g21) by day 7, whereas sausages
containing nisin remained unspoiled for .50 days. The effect of three types of phosphates (used as emulsifiers) on nisin
activity in the sausages was compared. LAB growth rate was fastest in samples containing orthophosphate, and slowest in
sausages containing diphosphate. The shelf life was also greatly extended in the latter. Fat content also affected nisin activity.
Nisin activity (as indicated by LAB inhibition) was greatest in samples containing 15% . 25% . 37% (wt/wt) fat. In a
sausage formulation containing 37% fat and incorporating diphosphate as emulsifier, levels of nisin as low as 2.5 mg g21
showed antibacterial effects. A nisin level of 6.25 mg g21 totally inhibited LAB growth for over 4 weeks and 25 mg g21 for
5 weeks. Spoilage control was achieved in the same sausage formulation but with 25% (wt/wt) fat; 12.5 mg g21 nisin prevented
LAB growth for 5 weeks.

Spoilage of minimal heat-treated, cured, vacuum-pack-
aged, chilled-meat products, such as bologna sausages, is
not uncommon. (51). The major spoilage organisms are
gram-positive lactic acid bacteria (LAB) whose growth is
preferentially selected by factors such as the low storage
temperature, presence of nitrite and curing salts, and mi-
croaerophilic conditions. Predominant species are the fac-
ultative heterofermenters: Lactobacillus sake and L. cur-
vatus (1, 6, 7, 17, 18, 23, 25, 29–34, 42, 48–51). L. sake
is reported to constitute 68% and L. curvatus 16.9% of the
total LAB isolated from vacuum-packed processed meats
(25). The growth of these organisms results in the produc-
tion of surface slimes, off color, gas, and odors. Other spoil-
age species include heterofermenting lactobacilli and leu-
conostocs, and Brocothrix thermosphacta (35, 36) (the lat-
ter when the packaging film is permeable). Most gram-pos-
itive bacteria associated with meat (unlike many
gram-negative bacteria) have a relatively high tolerance to
some of the food preservation hurdles used in meat prod-
ucts, such as reduced aw, refrigeration temperatures, and
low pH (25).
If stored at the correct refrigeration temperature, vac-
uum-packed commercial meat products that have been min-
imally heat treated usually have shelf lives of several weeks
(25, 35). In an examination of frankfurter sausages, Blick-
stad and Molin (6) found that counts (mainly LAB) reached
107 organisms g21 after 77 days. In contrast, a sensory eval-
* Author for correspondence. Tel: 144 1308 862018; Fax: 144 1308
863320; E-mail: linda.thomas@cultor.com.
uation of bologna sausages (2) reported a shelf life of 14
days at 38C, 8 days at 68C, and 2 days at 158C. Depending
on the storage temperature and levels of contamination,
LAB spoilage microflora can reach .108 organisms g21
after relatively short times (49). In an investigation of the
distribution and growth of bacteria in delicatessen meats,
Holley (24) reported that significantly greater numbers of
LAB were present on the package surface slices of com-
mercially sliced vacuum-packed bologna sausage compared
to the internal slices. In the same study, heterofermenting
LAB were found to be the predominant organisms in both
commercially packaged sliced cooked ham and bologna
sausage. Pasteurization of increasing severity (the most se-
vere being a core temperature of 638C for 6 min) was re-
ported to alter but not totally remove the (predominant)
LAB population of vacuum-packed Vienna sausages, so
that spoilage still occurred (23). In Europe it is expected
that pasteurization would eradicate the majority of non-
spore-forming bacteria. Postprocessing contamination can
occur as a result of packaging, slicing, and handling, and
this contamination can be difficult to eliminate.
The food preservative nisin is currently recognized in
approximately 50 countries as a safe food preservative and
is produced by Aplin & Barrett Ltd. (Cultor Food Science)
under the tradename Nisaplin. It has a wide spectrum of
activity against gram-positive bacteria, including spore-
formers. Nisin was first used in processed cheese products,
but its use has now been extended to numerous other prod-
ucts, such as dairy desserts, liquid milk, soups, alcoholic
beverages, canned vegetables, and crumpets (14, 15). Nisin
J. Food Prot., Vol. 62, No. 9
can be used to control LAB spoilage in beer, wine, alcohol
production, and low pH foods such as salad dressings.
Bologna sausage, a type of minimally cooked meat
product originating in Italy, is made with finely ground,
cured beef and pork, with seasonings similar to frankfurters.
Nisin has recently been approved in similar meat systems
in Brazil (Mercosur). The purpose of the present study was
to evaluate the effectiveness of nisin (used as the commer-
cial concentrate Nisaplin) for the control of LAB spoilage
in refrigerated, vacuum-packed bologna sausages. In order
to investigate the feasibility of this preservation system, an
initial study was undertaken to examine the nisin sensitivity
of LAB spoilage isolates from this type of meat product.
This was conducted in order to establish that nisin was a
suitable preservative to use in the sausage samples to con-
trol spoilage by LAB. A preliminary study was also con-
ducted to investigate whether nisin recovery from meat sys-
tems was affected by fat content, as reported by Bell and
de Lacey (4). Finally, we investigated the influence of phos-
phate type and fat content on the inhibition of LAB growth
by nisin in bologna-type sausage samples stored at 88C.
MATERIALS AND METHODS
Cultures. L. curvatus strain 860; L. sake strains 511, 513,
and 550; Leuconostoc mesenteroides subsp. mesenteroides strain
824; Leuconostoc mesenteroides subsp. dextranicum 882 and
3A8; Leuconostoc carnosum strains 174, 558, 719, and 973 were
obtained from TNO Nutrition and Food Research Institute, Zeist,
The Netherlands. The strains had been isolated from spoiled,
cooked cured meat products (liver sausage, ham, and bologna sau-
sage) and were maintained on glass beads at 2808C.
Determination of nisin sensitivity of LAB spoilage iso-
lates. One hundred-microliter aliquots of appropriate concentra-
tions of nisin were added to 9.8 ml de Man, Rogosa, Sharpe
(MRS; Oxoid, Basingstoke, Hants., UK) broths to give final con-
centrations of 0 to 25 mg g21. Nisin is quoted throughout this
paper in units of pure nisin (mg g21). Pure nisin has an activity
of approximately 40 3 106 IU g21, and the commercial product
Nisaplin (manufactured by Aplin & Barrett Ltd., Cultor Food Sci-
ence) has a standard activity of 1 3 106 IU g21. For control tests,
sterile deionized water was used as a replacement. Overnight MRS
broth cultures grown at 378C, (ca. 109 CFU ml21) were appropri-
ately diluted in maximum recovery diluent (MRD; Oxoid) and
100 ml inoculated into the test broth to give an initial concentra-
tion of approximately 103 CFU ml21. The cultures were incubated
at 308C, apart from L. curvatus that was incubated at 378C.
Growth was assessed by the visual appearance of turbidity.
Nisin bioassay. This plate diffusion procedure using Micro-
coccus luteus NCIB 8166 as the test organism was conducted
according to the methodology described by Fowler et al. (22).
Preliminary investigation of the influence of fat/lean meat
ratio on nisin extraction. The fat and lean contents of pork chops
were separated and each part was homogenized in a blender to
achieve an emulsion-like consistency. Samples were prepared con-
taining different lean/fat proportions. To each sample 2.5% NaCl
(wt/wt) was added to aid emulsification, and the samples were
mixed using a stomacher. Then, 2.5 mg g21 nisin was added per
10-g sample. The samples were mixed again, heat-sealed, and
heated to a core temperature of 758C for 2 to 3 min. The samples
BOLOGNA SAUSAGE PRESERVATION USING NISIN 1005
were incubated at 88C and examined at day 0, day 28, and day
35 to determine level of Nisaplin retention.
Preparation of bologna-type sausage. The sausages were
prepared according to the following recipe (g per kg): lean beef,
320; lean pork, 202; lard, 370; water, 40; starch, 40; sodium glu-
tamate, 0.5; sodium ascorbate, 0.5; phosphate, 3; NaCl, 20; so-
dium nitrite, 0.12. Trisodium orthophosphate was used unless oth-
erwise stated. This formulation had a fat content of 37% (wt/wt).
The sausage mix was divided into stomacher bags (2 3 199
g), and 0.5 ml of an appropriate Nisaplin solution was added to
give the desired final nisin concentration. The mixtures were
mixed using the stomacher for 2 min, pasteurized (a core tem-
perature of 708C for 2 min), and stored overnight at 48C, prior to
inoculation with the test strains.
Variation of type of phosphate compound in the bologna-
type sausage samples. Three different phosphate types were com-
pared: trisodium orthophosphate, diphosphate, and triphosphate.
The latter two phosphate types were commercial mixtures (Fibri-
sol Service Ltd., London). For this investigation 2 3 1 kg of
sausage was prepared, mixed, and divided into 5 3 199 g bags.
Phosphate was added at a level of 3 g kg21 (as in the original
formulation). Nisaplin solution (0.5 ml) was added to each bag to
give a nisin concentration of 0, 12.5, or 25 mg g21.
Variation in the fat content of the bologna-type sausage
samples. The fat content of the sausages was varied by using
different levels of lean and fat meat to give fat contents of 15%,
25%, and 37% (wt/wt). For each investigation fat was replaced
with starch as necessary. (There was no other change in the sam-
ple formulation.) Nisaplin solution (0.5 ml) was added to each
bag to give a nisin concentration of 0 or 6.25 mg g21.
Inoculation of bologna-type sausage. The samples were in-
oculated with a cocktail mixture of LAB (L. curvatus 860; L. sake
550; Leuconostoc mesenteroides subsp. mesenteroides 824, and
Leuconostoc carnosum 558).
The cocktail inoculum was prepared by removing the strains
from storage at 2808C and incubating on MRS agar anaerobically
at 258C for 3 days. They were then subcultured twice in MRS
broth anaerobically at 308C for 24 h before use. Each culture (ca.
5 3 108 CFU ml21) was diluted 1022 in MRD and 1 ml of each
added together to make the cocktail. Then, 0.5-ml aliquots of the
cocktail were inoculated into 199-g bags of sausage mix to give
ca. 104 CFU g21, and the contents of the bags were mixed thor-
oughly for 2 min using a stomacher. The sausage samples were
then aseptically divided into 10-g portions and vacuum packed
(15 mbar) before incubation at 88C.
Analysis of samples. LAB counts of inoculated packs, con-
ducted at regular intervals were conducted on MRS agar incubated
at 258C anaerobically. Measurements of pH and nisin bioassays
were performed on samples on the day of inoculation and at the
end of the incubation period. Chemical analysis of representative
samples from each experiment was undertaken at Campden &
Chorleywood Food Research Association. This included analysis
of: moisture content (TES-SC-097, air oven at 1038C), protein
content (TES-AC-414, Dumas methodology), sodium content
(TES-AC-099, atomic absorption spectroscopy), fat content (TES-
AC-378, Werner Schmid extraction), starch content (TES-AC-272,
alkaline hydrolysis, enzymic digestion, glucose by Boehringer en-
zyme kit), phosphorous content (TES-AC-095, vanadate-molyb-
date colorimetry), nitrite content (TES-AC-382 colorimetric meth-
od), and water activity (TES-MB-042).
1006 DAVIES ET AL. J. Food Prot., Vol. 62, No. 9

TABLE 1. The influence of the fat/lean meat ratio on nisin ex-

traction
Pork sample % nisin recovery (25 mg g21 addition)
(% fat/
% lean) Day 0 Day 28 Day 35

0/100 104 43 46
20/80 98 74 52
40/60 106 87 43
60/40 117 50 46
70/30 110 60 Not tested

Statistical analysis. Data were analyzed using general linear

modeling (PROC GLM on SAS Institute, Cary, N.C.), and means
were discriminated using Duncan’s multiple range test. All micro-
bial data were transformed to logarithm before analysis.
RESULTS
Determination of nisin sensitivity of LAB spoilage
isolates. For all the LAB isolates tested, a concentration of
approximately 102 to 103 cells ml21 was inhibited by a nisin
concentration of 1.25 mg g21 for 14 days at 308C (except
for L. curvatus that was tested at 378C).
Preliminary investigation into the influence of the
fat/lean meat ratio on nisin extraction. Initial nisin re-
covery was high in all the samples tested (Table 1). At day
28, nisin recovery was lowest in the 100% lean/0% fat sam-
ple. At day 35, residual nisin was similar in all samples;
50% activity loss was observed.
Preliminary investigation into the effect of nisin on
the growth of LAB in vacuum-packed bologna-type sau-
sage samples stored at 88C. These sausages had a fat con-
tent of 37% (wt/wt) and contained trisodium orthophos-
phate. Initial counts for control samples were ca. 104 CFU
g21. A slight bactericidal effect was observed with the ad-
dition of nisin (25 mg g21), reducing the initial level to 5.2 FIGURE 1. The effect of phosphate type on the efficacy of nisin
3 103 CFU g21 in samples thus treated. Control samples to control LAB spoilage in vacuum-packed bologna-type sausages
stored at 88C. The sausages had a fat content of 37%. Counts
were spoiled (LAB count $108 CFU g21) by day 7, where-
incubated on MRS agar at 258C anaerobically. (a) Orthophos-
as samples containing nisin remained unspoiled for .50
phate, (b) diphosphate, (c) triphosphate. Symbols: V, control (no
days. No marked change in LAB numbers from the inoc- addition of nisin); v, addition of 12.5 mg g21 nisin; m, addition
ulation level was observed in the nisin-treated samples. of 25 mg g21 nisin.
The effect of phosphate type on the efficacy of nisin
in controlling LAB growth in vacuum-packed bologna- life was considerably extended. There was a significant dif-
type sausage samples stored at 88C. The growth rate of ference (P , 0.01) between growth at both nisin levels and
the inoculated LAB was much greater in the orthophos- no nisin. By completion of the trial (71 days, data not plot-
phate samples compared to all the other phosphate emul- ted), nisin-containing samples had not reached spoilage lev-
sifier samples, achieving spoilage levels .108 CFU g21 by els.
day 6 in samples not containing nisin (Fig. 1a). The shelf Sausages containing triphosphate but not nisin became
life of these sausages was extended by 9 days with a nisin spoiled by day 28 (Fig. 1c). The shelf life was extended by
addition of 12.5 mg g21, and .55 days with a nisin addition 35 days by the addition of 12.5 mg g21 nisin. No spoilage
of 25 mg g21. There was a significant difference (P , 0.01) had occurred in sausages containing 25 mg g21 by the end of
between growth in the presence of 25 mg g21 nisin and in the trial. There was a significant difference (P , 0.01) be-
the absence of nisin, but there was no significant difference tween growth in the presence of both nisin levels and no nisin.
between growth in the presence of 12.5 mg g21 nisin and Residual nisin levels indicated an average loss of 29%
with no nisin. following processing and approximately 50% loss by the
LAB growth was slowest in nisin-treated sausages that end of the incubation period (Table 2).
contained diphosphate as the phosphate type (Fig. 1b). At The effect of fat content on the efficacy of nisin in
both levels of nisin (12.5 mg g21 and 25 mg g21), the shelf controlling LAB growth in vacuum-packed bologna-
J. Food Prot., Vol. 62, No. 9 BOLOGNA SAUSAGE PRESERVATION USING NISIN 1007

TABLE 2. Initial and final pH values and nisin levels (determined

by bioassay)
Nisin % nisin
added pH recovery
(mg
Sample g21) Initial Final Initial Final

Figure 1a 0 6.06 5.14 — —

12.5 6.06 5.48 64 51
25 6.06 6.07 59 51
Figure 1b 0 5.76 5.44 — —
12.5 5.76 5.75 81 48
25 5.76 5.80 73 48
Figure 1c 0 5.86 5.21 — —
12.5 5.86 5.79 77 62
25 5.86 5.87 70 56
Figure 2a 0 5.8 5.6 — —
6.25 5.8 5.8 85 52
Figure 2b 0 5.8 5.7 — —
6.25 5.8 5.7 86 55
Figure 2c 0 5.8 5.5 — —
6.25 5.8 5.7 83 54
Figure 3 0 5.8 5.7 — —
6.25 5.8 5.7 88 92
12.5 5.8 5.9 85 64
Figure 4 0 5.6 Not done — —
2.5 5.6 Not done 98 86
6.25 5.6 Not done 63 37
25 5.6 Not done 69 62

type sausage samples stored at 88C. The viable count data

for the growth of the LAB are shown in Figure 2 (other
analyses in Tables 2 and 3). The inhibitory effect of nisin
was most pronounced in sausages containing the least
amount of fat (15%) (Fig. 2a). Effective control was ap-
parent over the 80-day incubation period of this trial, at the
end of which the LAB counts were ,103 CFU g21, com-
pared to an approximate count of 106 CFU g21 in the sau-
sages without nisin.
Nisin reduced the LAB for a period of up to 40 days
in sausages containing 25% fat (wt/wt) (Fig. 2b). At a fat
concentration of 37% (wt/wt) (Fig. 2c), the effect of nisin
was still apparent but less than in the sausages with lower
fat formulations.
For all fat levels there was a significant difference (P
, 0.01) between the growth data in the absence or presence
of nisin (6.25 mg g21).
Residual nisin levels indicated a 16% loss following
processing, and approximately 50% loss by the end of the
80-day period. Fat had no effect on the retention or loss of
nisin (Table 2), and the fat levels achieved were close to
target levels (Table 3).
Evaluation of the minimum inhibitory concentra-
tion of nisin for the control of LAB spoilage at 88C in
vacuum-packed bologna-type sausage samples with a fat
content of 25% (wt/wt) and with incorporation of di-
phosphate as emulsifier. Concentrations of 6.25 mg g21
and 12.5 mg g21 of nisin were effective in the control of
LAB growth for a minimum period of 4 weeks (Fig. 3). As
expected, the higher level of nisin achieved greater inhibi-
FIGURE 2. The effect of fat content on the efficacy of nisin to
control LAB spoilage in vacuum-packed bologna-type sausages
stored at 88C. The sausages contained orthophosphate. Counts
incubated on MRS agar at 258C anaerobically. (a) 15% fat con-
tent; (b) 25% fat content; (c) 37% fat content. Symbols: V, con-
trol (no addition of nisin); v, addition of 6.25 mg g21 nisin.
tion of the LAB, but a noticeable inhibition occurred even
at the lower level of nisin tested. There was significant dif-
ferences (P , 0.01) between the growth data at all three
nisin concentrations (0, 6.25, and 12.5 mg g21).
This particular sausage formula (with 25% fat [wt/wt]
and diphosphate) showed good residual levels of nisin: an
average of 13% loss following processing, and only 22%
loss over 75 days storage.
Evaluation of the minimum inhibitory concentra-
tion of nisin for the control of LAB spoilage at 88C in
vacuum-packed bologna-type sausage samples with a fat
content of 37% (wt/wt) and with incorporation of di-
phosphate as emulsifier. Nisin at all levels tested had an
antibacterial effect (Fig. 4). Then, 6.25 mg g21 nisin addi-
tion achieved a marked reduction in the LAB growth for a
period of over 4 weeks in the sausages containing 37% fat
(wt/wt), and 12.5 mg g21 nisin inhibited LAB growth for 6
1008 DAVIES ET AL. J. Food Prot., Vol. 62, No. 9

TABLE 3. Results of chemical analysis of sausage samples

Chemical analysis

Moisture Protein Sodium Fat Starch Phosphorus Nitrite Water

Sample (g/100 g) (g/100 g) (mg/100 g) (g/100 g) (g/100 g) (mg/100 g) (mg/kg) activity

Figure 1a 43.0 13.0 2.1 35.5 3.2 153 15.0 0.97

Figure 1b 42.3 12.7 2.3 38.1 4.3 189 12.9 0.97
Figure 1c 41.1 13.4 1.9 41.1 4.4 211 8.9 0.97
Figure 2a 44.7 11.9 Not done 14.3 20.4 234 9.7 0.96
Figure 2b 43.8 11.7 Not done 26.9 12.3 207 30.6 0.96
Figure 2c 43.1 12.5 Not done 36.4 3.4 199 31.7 0.97
Figure 3 45.3 12.9 Not done 23.3 13.6 192 Not done 0.97
Figure 4 42.8 11.7 2.3 33.6 4.0 187 6.7 0.97

weeks. A quantity of 2.5 mg g21 nisin reduced the LAB
count for 2 weeks. There was a significant difference (P ,
0.01) between the growth data from samples with and with-
out nisin. There was also a significant difference (P , 0.01)
between the growth data in the presence of 2.5 mg g21 nisin
compared to that at higher nisin levels (6.25 and 25 mg g21).
There was an average loss of 23% residual nisin fol-
lowing processing and approximately 40% loss by the end
of the incubation period (Table 2). There appeared to be a
greater loss of nisin from the sausages containing 6.25 mg
g21 nisin, and yet the nisin worked very effectively in these
samples.
DISCUSSION
The nisin sensitivity of the LAB strains tested in the
present study confirms previous reports. Collins-Thompson
et al. (11) tested 30 LAB strains from different meat prod-
ucts (including bologna sausage). Most strains were sensi-
tive to #0.625 mg g21. Some strains were sensitive to
,0.125 mg g21 nisin and did not increase resistance to nisin
even after passaging in increasing concentrations of nisin.
Furthermore it was observed that nisinase activity was rare.
These results supported the premise that nisin would be an
effective preservative to use to control LAB spoilage in
food products.
Reports of nisin activity in meat products have described
variable effectiveness often requiring high nisin concentra-
tions. Nisin efficacy, however, is dependent on both the na-
ture of the meat system and the target organisms (3, 8, 16,
21, 26, 39, 43, 45, 46). Poor efficacy can be a result of nisin
binding to meat proteins, uneven mixing, and poor absorp-
tion into the meat matrix, interference of meat phospholipid
with the nisin activity, and nisin heat lability in neutral pH
conditions (16). These problems can often be overcome in
particular meat products. For example, the stability of nisin
on raw meat surfaces can be improved by immobilizing the
preservative in a calcium alginate gel (12, 19).
In contrast, there have also been studies describing the
effective use of nisin in meat products. Caserio et al. (10)
reported that outgrowth of Clostridium perfringens spores
was completely inhibited by a combination of 5 mg g21 of
nisin and 75 ppm of nitrite. Similarly, 1.875 to 2.5 mg g21
nisin in combination with 40 ppm nitrite effectively inhib-
ited outgrowth of C. sporogenes spores for 5 days in meat
slurries incubated at 378C (38). This combination proved
superior to 150 ppm nitrite alone. Incorporation of nisin
into frankfurters prevented discoloration when the sausages
were inoculated with LAB before pasteurization (44). A
dose of 12.5 mg g21 nisin was successfully used in com-
bination with sorbic acid (0.125%) and monlaurin (0.25 to
0.5%) to control spoilage by Bacillus licheniformis as well

FIGURE 3. The effect of nisin concentration on the growth of
LAB at 88C in vacuum-packed bologna-type sausages with a fat
content of 25% and incorporation of diphosphate as emulsifier.
Counts incubated on MRS agar at 258C anaerobically. Symbols:
V, control (no addition of nisin); v, addition of 6.25 mg g21
nisin; m, addition of 12.5 mg g21 nisin.
FIGURE 4. The effect of nisin concentration on the growth of
LAB at 88C in vacuum-packed bologna-type sausages with a fat
content of 37% and incorporation of diphosphate as emulsifier.
Counts incubated on MRS agar at 258C, anaerobically. Symbols:
V, control (no addition of nisin); v, addition of 2.5 mg g21 nisin;
m, addition of 6.25 mg g21 nisin; 3, addition of 25 mg g21 nisin.
J. Food Prot., Vol. 62, No. 9
as the natural spoilage microflora in a pasteurized cured
meat product (5). The fat/lean ratio in this product was 1:3.
Nisin protects against gram-positive organisms, but
combinations of nisin with other preservatives can extend
this protection to achieve a wider spectrum antimicrobial
effect. In an investigation into the feasibility of replacing
sulfur dioxide with organic acids and nisin in raw pork
sausage, Scannell et al. (41) found that a combination of
sodium lactate and nisin was effective in reducing the total
bacterial count of the product. The combination of lactate
and nisin protected against Staphylococcus aureus and Sal-
monella species (common pathogenic contaminants). In an-
other study, 10 mg g21 nisin used alone or in combination
with 1,000 ppm sorbic acid and 2.5% (wt/vol) polyphos-
phate delayed the spoilage of raw British fresh sausages
(27). In a trial into the antibacterial activity of various bio-
preservatives in refrigerated vacuum-packaged raw beef,
samples treated with 12.5 mg g21 nisin showed the greatest
reduction of aerobic plate counts and LAB, and the bacte-
rial population was kept below 106 CFU g21 for 8 weeks
at 38C (40). A nisin spray treatment was reported to reduce
the incidence of Listeria innocua and B. thermosphacta on
vacuum-packed beef (13).
There are certain problems that can be encountered us-
ing nisin as a preservative in meat products. First, these
products are often heterogeneous in composition and it is
important to ensure that the nisin is distributed evenly
throughout the product. Inefficient mixing would permit
bacterial growth in discrete niches containing insufficient
preservative. In the present study, the sausage samples were
thoroughly mixed with the added nisin, and the observed
LAB inhibition was an indication that nisin was present and
active throughout the sausage.
A certain amount of nisin can be lost during the pro-
cessing of the meat product. In the present study, there was
a small and acceptable loss occurring as a result of the
pasteurization treatment. The nisin remaining, however, was
of sufficient quantity to inhibit LAB spoilage. Loss in nisin
activity depends on the heat treatment and pH of the prod-
uct; heat stability is improved in conditions of lower pH.
Cooked cured meat products vary considerably in for-
mulation. There can be wide variation in fat content, par-
ticle size, salt content, phosphate type, nitrite content, emul-
sifier, etc. The present study found that nisin is more effec-
tive in lean meat, and the type of phosphate used as emul-
sifier can also be influential. The optimum sausage
formulation devised in this study incorporated 25% fat and
diphosphate as an emulsifier. In this product formulation,
nisin significantly increased protection from LAB spoilage
(although even in sausages with higher fat content there
was increased protection). In the current climate of consum-
er awareness of health-related issues, there is greater effort
being targeted into producing lower fat content products.
Fat influences the binding properties and texture of meat
products, and various procedures have been investigated
with the aim of reducing fat levels without impairing prod-
uct quality. According to U.S. Department of Agriculture
Food Safety and Inspection Service regulations, the fat con-
tent of an emulsion-type sausage (such as bologna) can be
BOLOGNA SAUSAGE PRESERVATION USING NISIN 1009
a maximum of 30% fat with 10% added water, or as low
as 5% with 35% added water. For the latter, however, a fat
replacement must be selected that will provide the neces-
sary water-holding capacity and textural properties of a
higher fat product. In the present study, starch replaced fat
in certain samples. Starch has been previously suggested as
a possible fat replacement in meat products (28, 37), and
the effect of starch replacement on the binding and texture
of meat emulsions as related to fat content has been studied
by Carballo et al. (9).
Nisin is poorly recovered from meats and meat prod-
ucts, resulting in misleading and variable bioassay results.
The poor recovery is believed to be due to the strong ad-
sorption onto meat proteins (20, 47). Bell and de Lacey (4)
showed that rate of recovery was not greatly affected by
the curing salts, particle size, or meat/extractant ratio but
was significantly affected by the fat content of the meat.
They reported that when 2.5 mg g21 was added to minced
beef emulsions, recovery of nisin ranged from 26% from
samples with 3% fat content, to 76% nisin recovery from
samples with 83% fat. The present study is in contrast to
this; the recovery of nisin from the simple pork meat emul-
sion (Table 1) and the bologna-type sausage samples (Table
2) was not affected by the fat content. In the final product
formulation examined in the present study, there was good
recovery of nisin at both the beginning and end of the stor-
age period (Table 2). Care was taken to mix the sausage
samples thoroughly, to ensure that nisin was homogeneous-
ly distributed.
In conclusion, nisin levels of 6.25 to 25 mg g21 were
able to inhibit effectively the growth of a mixed LAB in-
oculum and thus extended the shelf life of vacuum-packed
bologna-type sausages stored at 88C. Although fat content
and phosphate type were found to influence the nisin effi-
cacy, nisin still gave significantly increased protection
against LAB spoilage.
ACKNOWLEDGMENTS
We thank Marie-Louise Kant-Muermans of TNO Nutrition & Food
Research Institute, the Netherlands for advice on sausage formulations and
the method of manufacture. We thank Peter Putnam of Cultor Food Sci-
ence Ltd. (New York) for advice and help with the statistical analysis. We
also thank Christine Stenning for technical support.
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