Food Microbiology, 1984, 1, 177-l 85

Influence of water activity on microbial development in minced, cured pork, in relation to gas headspace composition

Elisabeth Blickstad

Swedish Meat Research Institute. P.O. Box 504, S-244 00 Klivlinge, Sweden

Received 2 March 1984

The water activity (ad of a cured minced meat mixture was reduced with NaCl or glycerol + NaCl and the products stored in air, Ns or COs at 4°C. The lower the a,, the lower was the growth rate of the total microflora except when aW was reduced below 0.98 with glycerol and products were stored in CO,, when the growth rate was independent of a,. At a, 0.94 NaCl was more effective than glycerol + NaCl in reducing the growth rate. In air the final microflora of products stored at a, SO.96 consisted primarily of yeasts while at 0.98 a,, both Lactobacillus (62%) and yeasts (38%) were found. In CO, and N2 Lactobacillus predominated at 0.94-0.98 a, when 2% NaCl or NaCl f glycerol were used. At 0.94 a,, produced using 6% NaCl, a heterogenous final microflora was found, including Micrococcus and Staphylococcus. An increase in the microbial shelf-life of meat products may be obtained by reducing the a,,, particularly for use with storage in Ns- and COs-atmospheres. The type of solute chosen to regulate a, may affect the solubility of COs, which in turn affects the shelf-life.

Introduction Cured meat products are complex systems and the growth of the micro- organisms present in these products is affected by several factors including water activity (a,), NaCl concentration and storage gas atmosphere.

The NaCl concentration is normally 2-4% in cooked cured meat products (Gardner 1983). For raw cured meats (e.g. Wiltshire bacon) dry salting is used in order to improve the shelf-life (Gardner 1983). The addition of NaCl (2-3%) is sometimes also used to improve the storage stability of minced meat raw materials used for sausage production and to facilitate the pumping of these raw materials in a continuous produc- tion line. 074%0020/84/030177 + 09 $02.00

The reduction of a, is used for improv- ing the storage stability of foods. The a, may be reduced by the addition of solutes such as NaCl, sugar and glycerol, by drying or by the addition of dry ingre- dients such as milk powder. The a, of non-dried cured meat products is about 0.98, due to the NaCl concentration, and may be further reduced by slight modifi- cations to the formula, down to O-96 (Leistner et al. 1980). The growth of bacteria is affected if the a, is reduced from 0.98 to O-96, for example the growth rate of Lactobacillus viridescens was reduced by 50% over this a, range (Blick- stad 1984).

In the study by Blickstad and Molin (198313) it was suggested that a, may influence the effect of gas storage on the

0 1984 Academic Press Inc. (London) Limited

178 E. Blickstad

shelf-life of meat products and that a slight reduction in a, in combination with storage in pure CO2 could explain why the total aerobic count of frank- furter sausages did not increase during seven months of storage at 4°C. In the present work the effect of reduced water activity on the microflora of a minced, cured, raw meat product during storage in air, N2 or CO2 is determined.

Methods Experimental design

Four different minced meat mixtures with different water activities (a, = 0.98; O-96; 0.94) were prepared. The water activities were adjusted with glycerol (610%) and/or NaCl (2-6%) (Table 1). All mixtures con- tained 88% minced pork meat, prepared from freshly cut pork loins, and 120 ppm NaN02. Final weight was adjusted with water. The

initial pH of the mixtures was 5.7. Petri dishes (approx. 85 cm) filled with the meat mixtures (about 80 g) were stored in air, Ns or COz at 4°C. For aerobic storage, a gas perme- able film (PVC) was wrapped around the dishes. The packaging material used for Ns- and COs-storage was low permeability Lam- o-Foil 4 ply (polyamid/alufolielpolyamid/ polyethene film; Otto Nielsen Ltd., Lyngby, Denmark). The headspace volume in the storage containers was about 3.2 1.

Samples were analyzed during the storage period and on each used occasion two Petri plates with meat mixture were used.

Gas analysis

The 02, CO2 and N2 concentrations were analyzed as described by Blickstad et al. (1981).

Water activity

The water activity was determined using an electronic hygrometer, Humidat K-11 (Nova Sina AG, Zurich, Switzerland).

Table 1. Microbial counts of cured minced pork, having different a, values, during storage in air at 4°C.

Total aerobic Yeasts and Lactic acid H202-

Storage time count moulds bacteria producers (days) (logno.g-l) (logno.g-l) (logno.g-l) (logno.g-1)

0.99 a,, no solute 0 2.4 Cl.0 <l-O <l-O 5 4-8 3-7 Cl.2 <l.O 8 7.1 5.1 2.2 <l.O

12 9.0 8.5 4.6 cl.0 O-98 a,, 2% NaCl

7 2.6 <1.2 1.6 <l.O 14 4.4 3.5 3.8 2.7 21 5.3 5-6 4-9 3-9 39 6.3 6.0 5.2 2.6

0.96 a,, 2% NaCl + 6% glycerol 14 2.5 <1.5 <l.O <l.O 18 4.3 1.7 <l.O <l.O 26 5.8 5.4 <1.2 3.4 39 6-8 6-8 4-o <l-O

0.94 a,.,, 2% NaCl + 10% glycerol 11 2.3 <l.O Cl.0 Cl.0 28 4.9 4-3 2.7 <1.8 39 6.3 6.2 5.0 3.8 54 6.8 6.7 4.8 3.9

O-94 a,, 6% NaCl 21 2.0 <l*O <l.O <l.O 49 6.0 6.0 <l.O <l.O 76 6.8 7.0 cl.7 <l.O

Meat product microflora and a,., 179

Green discolourutions

Samples being stored in Nc or COc were, after sampling, stored in air at 4°C for five days in order to detect any green discolouration.

Off odours

Off odours detected at the time of sampling were noted.

Microbiological sampling

A sample (30 g) was homogenized with 270 ml of physiological saline solution [plus 0.1% (w/v) peptonel using a Stomacher 400 (A. J. Seward and Co., Ltd, London) for 3 min. The pH was measured in the homogenate.

Media used for viable counts were tryptone glucose extract agar (TGEA; Difco), Violet Red Bile Dextrose agar WRBD; Oxoid), acet- ate agar (AcA; Enfors et al. 1979), potato dextrose agar (PD; Oxoid), STAA (Gardner 1966) and Marshall agar [as Marshall (1979), except APT, (BBL) was used instead of acetate agarl. Total aerobic count was obtained on TGEA (28”C, 3 days) Enterobac- teriaceae on VRBD (37”C, 1 day), lactic acid bacteria on AcA (28”C, 5 days), yeasts and moulds on PD (22”C, 5 days), Brochothrix thermosphacta on STAA (22”C, 2 days; then flooded with 1% tetramethyl- p-phenylenediamine-dihydrochloride and counting only uncoloured, oxidase negative colonies), hydrogen peroxide producing organisms on Marshall agar (anaerobic incu- bation in Gas Pak Anaerobic Systems, BBL at 25°C for 3 days, followed by aerobic incubation at 22°C for 1 day; dark colonies were counted).

Identification

Isolates were picked from the countable TGEA plates used for the determination of the total aerobic count. From each plate, representing one sample of meat mixture, 15 colonies were picked. The isolates were divided into genus as described by Blickstad and Molin (1983a,b). Yeasts and moulds were not identified.

Micrococcus spp. were tested for acid pro- duction from glucose aerobically and anaer- obically in a medium described by Baird- Parker (1966), and furthermore by Blickstad et al. (1981). Lactobacillus spp. and Leucono- stoc spp. were tested for gas production from glucose in APTG (g 1-i: glucose, 20; peptone, 10; yeast extract, 7.5; NaCl, 5; K2HP04, 2; sodium carbonate, 1.25; Tween 80, 0.5; MgS04.7H20, O-5; MnS04.4H20, 0.05; thia-

mine, 0.0001; agar, 3.0; pH 6.2). The APTG tubes were sealed with agar and incubated at 28°C for a total of three weeks, and were examined weekly.

Results

Effect of a, regulated with glycerol

The water activity (a,) was reduced by adding glycerol to minced meat contain- ing 2% curing salt and with an a, of 0.98. The results are summarized in Fig. 1 and Tables 1, 2 and 3.

(a)

'i

5.0 I /---- lldT

Il.01 , , , , , , -i

m

1.0 -

-I

r (c) .

.

5.0 -

:

,

J I

1 1.0

--I 10 50 1OO

Time (days)

Fig. 1. Change in total aerobic count at different a, levels: 0.99 a,, 0% NaCl, 0% glycerol (0); 0.98a,, 2% NaCl, (0); 0.96a,, 2% NaCl, 6% glycerol (+I; 0.94a,, 2% NaCl, 10% glycerol (W; 0*94a,, 6% NaCl (0). The minced meat samples were stored in (a) air, (b)Nc and (c) COc at 4°C.

180 E. Blickstad

In air and Nz the total aerobic count final concentration being l-3 log. no g- 1 increased most rapidly at the highest a, lower than the total aerobic count. levels (0.98 a,; Fig. l(a), (b)). In COa a, Organisms producing hydrogen peroxide had no effect when a, was reduced by a were found at a, levels d 0.98. At the combination of NaCl and glycerol (Fig. end of storage the CO2 and the O2 l(c)). Irrespective of the a, and the concentrations (sampled between the storage gas atmosphere the total aerobic surface of the minced meat and the PVC count increased to about 7 log. no. g+i. film) were 0.4 and 21.2%, respectively.

The initial pH was 56-57 and did not vary during storage. No off odours were detected on any occasion. Brochothrix thermosphacta or Enterobacteriaceae were not found during storage.

The determined a, value varied slightly and irregularly during storage (air, a, < 0.011; Na, a, 9 0.006; COz, a, d 0.012).

Table 1 shows the counts for samples stored in air. The number of yeasts and moulds increased to a final level corre- sponding to the level of the total aerobic count (Table 1). At the end of storage all samples were visibly mouldy. The num- ber of lactic acid bacteria increased, the

Table 2 gives the counts of samples stored in Nz. No significant increase in the level of yeasts and moulds was found. Lactic acid bacteria grew at 0*94- 0.98 a, with the highest counts being found at 0.94 a, (2% NaCl) where the count of lactic acid bacteria was about equal to the total aerobic count. The highest number of HsOs-producers (4.6 log. no g-i) were found at 0.96 a, but such organisms could be found at all a, levels. No CO2 was produced during storage (CO2 < 0.1%) and small concen- trations of O2 (~0.7%) were detected in almost all samples.

The counts of samples stored in COz

Table 2. Microbial counts of cured minced pork, having different a, values during storage in Nz at 4°C.

Storage time (days)

Total aerobic Yeasts and Lactic acid HzOz- count moulds bacteria producers

(logno. g-l) (logno. g-l) (logno. g-l) (log no. g-l)

0.98 a,.,, 2% NaCl 7

19 39

0.96 a,,,, 2% NaCl + 6% glycerol 7

28 39 54

0.94 a,, 2% NaCl + 10% glycerol 19 28 54 70

0.94 a,, 6% NaCl 49 70 92

105

2.5 <l.O Cl.0 4.2 -cl.0 2.6 6.8 <1.2 5.2

2.2 <l.O 4.1 <l.O 5.8 <2+3 6.9 <l.O

2.3 <l,O cl.3 cl.0 2.2 <l.O <l.O cl.0 5.8 <l.O 5.1 <1.9 6.5 <2.8 6.2 ~2.4

1.8 2.0 4.3 3.5

<1.3 <l.O <l.O <l.O

Cl.0 Cl.0 c3.1 2.8

5.4 3.7 4.8 4.6

<l.O Cl.0 <3.3 (2.8

Cl.0 2Y5 3.5

cl.0 <lG3

2.6

a Not analyzed.

Meat product microflora and aw 181

Table 3. Microbial counts of cured minced pork, having different a, values, during storage in CO2 at 4°C.

Total aerobic Yeasts and Lactic acid Hz%?-

Storage time count moulds bacteria producers (days) (logno. g-l) (logno. g-l) (logno. g-l) (log no. g-1)

0.98 aw, 2% NaCl 14 2.3 K1.0 Cl.0 -cl.0 39 4.8 Cl.0 135 cl.0 54 6.6 <l.O 4,o 2.6 70 6.4 4.4 5.5 <l.O

0.96 a,, 2% NaCl + 6% glycerol 14 2.3 cl.0 cl.0 <l.O 32 4.2 K1.0 <2,7 cl.0 54 5-9 cl.7 cl.0 <2.2 70 6.7 3.6 6.1 cl.0

0.94 a,, 2% NaCl + 10% glycerol 21 2.6 Kl.0 c2.2 cl.0 32 4.6 Cl.0 3-9 <l-O 54 6.2 cl.0 5.9 4.5 70 6.6 cl.0 5.2 cl.0

0.94 a,, 6% NaCl 21 1.1 cl.0 cl-0 <I.0 70 1.8 il.0 cl.0 <l.O 97 1.5 K1.0 Cl.0 cl.0

are given in Table 3. A significant amount of yeasts and moulds (about 4 log. no g-1) were found at the end of storage at 0.98 a, and 0.96 a, but not at 0.94 a,. The number of lactic acid bac- teria increased at all a, levels, but the number detected was lower than the total aerobic count, while higher than the number of yeasts and moulds. HsOs- producers were found only after 54 days of storage, but not any earlier or any later, and the highest amount was found at 0.94 a,. The O2 and COz concentra- tions in the bags were s 0.3% and 2 99.0%, respectively.

Effect of a, regulated with NaCl

The effect of a,, when regulated with NaCl (2%, 6%; a, 0.98, 0.94) is shown in Fig. 1 and Tables 1,2 and 3. Samples with 0% NaCl were only stored in air. The growth rate of bacteria was reduced by the addition of NaCl to the samples and by increase in the NaCl concentration

from 2-6% when stored in air, Ns and COz (Fig. 11. The difference between growth rates at 2 and 6% NaCl is more pronounced in Ns and COs than in air. When no NaCl was added and the sam- ples were stored in air the total aerobic count increased to 9 log. no g-l, while with NaCl added the count increased only up to 7 log. no g-r. For samples with 6% NaCl stored in COz no increase in the total aerobic count was found during the 97 days of storage.

The only occasion when the pH value diverged from the initial value of 56- 5.7, was at the end of storage of samples with 6% NaCl stored in Ns, when the pH was 54. This was also the only occasion where any off odour was detected (sour). No B. thermosphacta were found in any of the mixtures to which NaCl had been added. In the mixture without NaCl (i.e. uncured minced meat) being stored in air, the number of B. thermosphacta increased up to 5.4 log. no g-l during 12

Tabl

e 4.

The

m

icro

bial

flo

ra

of

cure

d m

ince

d po

rk

havi

ng

diffe

rent

a,

va

lues

, af

ter

stor

age

in

air,

N2

and

CO

2 at

4°

C.

Org

anis

ms

Air

Dis

tribu

tion

(%)

NZ

0.98

a,

0.96

a,

0.94

a,

0.94

a,

0.98

a,

0.

96

a,

0.94

a,

0.

94

a,

(2/O

’) 0.

98a,

0.

96a,

0.

94a,

0.

94a,

(2

16)

(2/1

0)

(6/O

) (2

/O)

(216

) (2

110)

(6

/O)

(2/1

0)

(216

) (2

110)

(6

10)

Acin

etob

acte

r sp

p.

Cor

ynef

orm

ba

cter

ia

Gra

m-n

egat

ive

cocc

i Ku

rthia

sp

p.

Lact

obac

illus

spp.

ho

mof

erm

enta

tive

hete

rofe

rmen

tativ

e Le

ucon

osto

c sp

p.

Mic

roba

cter

ium

sp

p.

Mic

roco

ccus

sp

p.

Mic

roco

ccus

va

rians

Ps

eudo

mon

as

spp.

S

taph

yloc

occu

s sp

p.

Yea

st

Uni

dent

ified

b

spp.

- -

- -

- 23

-

- -

- -

- 3

- -

- 3

- -

- -

- -

- -

3

21

- 41

-

- z

93

73

83

- 3

27

- -

- -

- -

- 20

-

- -

- -

- -

- -

- -

3 -

- -

- -

- -

10

- -

- -

- -

3

G---

II-

27

100

100

100

3 -

- -

- -

- 17

6

- -

7

- 3

1 -

- -

10

70

93

80

3 -

- -

- - 7

- -

- 20

-

17

- -

3 -

- 10

27

-

- 3

20

24 3

Tota

l ae

robi

c co

unt

(log

no

g-l)

Stor

age

time

6.3

6.8

6.8

6.8

6.8

6.9

6.5

3.5

6.4

6.7

6.6

1.5

39

39

54

76

39

54

70

105

70

70

70

97

(day

s)

a %

NaC

l s

glyc

erol

1,

b

Dead

.

Meat product microflora and a,,, 183

days of storage. No Enterobacteriaceae were found.

In air the number of yeasts and moulds increased to a final level corresponding to the level of the total aerobic count at all NaCl concentrations tested (0, 2 and 6%; Table 1). At the end of storage all of these samples were mouldy. The number of lactic acid bacteria increased in sam- ples with 0 and 2% NaCl but not in samples with 6% NaCl. The final concen- tration of lactic acid bacteria was lower than the total aerobic count. H202- producers were only detected in samples containing 2% NaCl. The final CO2 concentration was only measured for samples with 2% NaCl when 0.3% CO2 was found.

In Na no increase in yeasts and moulds was found in the presence of 2 or 6% NaCl (Table 2). The number of lactic acid bacteria increased during storage and was about 1 log. no g-l lower than the total aerobic count. HzOs-producers were detected both at the 2 and the 6% NaCl level. No CO2 was produced and the O2 concentration was d 0.6%.

In COz no increase in the bacterial counts was found in samples with 6% NaCl (Table 3). At the 2% NaCl level high concentrations of lactic acid bac- teria together with yeasts and moulds were found. HzOz-producers were not frequently detected. The O2 concentra- tion was d 0.2% and the CO:! concentra- tion 3 99.0%.

Identification of the final microfZora

In air, yeasts totally dominated the final microflora of samples with 0.96 and 0.94 a, (Table 4). At 0.98 a,, 62% of the final microflora consisted of Lactobacillus, while the rest was yeasts.

In N2, Lactobacillus dominated the microflora at all a, levels tested except at 094 a,, in the presence of 6% NaCl, where a heterogenous flora including, e.g., Staphylococcus, Acinetobacter, Leu-

conostoc and Micrococcus was found (Table 4).

In COs, the situation was similar to that of N2. Lactobacillus dominated, except at 0.94 a,, 6% NaCl. In the presence of 6% NaCl, for example, Micro- coccus, Staphylococcus and Kurthia could be found (Table 4).

Discussion

The expected effect of reduced water activity values in a meat product is a reduction in the growth rate of the bacteria present. This was also found in the present study, when the products were stored in air or in nitrogen. However, when stored in COs and when glycerol was used to reduce the a, from 0.98 to O-94 the rate of increase in the total aerobic count was unaffected by the a, level (Fig. l(c)). However growth rate was reduced when NaCl was used to reduce a, to O-94. Carbon dioxide is known to be inhibitory to the growth of many bacteria, and the inhibitory effect increases with increasing COa concen- tration (Enfors and Molin 1980, Molin 1983, Blickstad and Molin 1984). The solubility of COz is affected by, e.g. the temperature (Enfors and Molin 1981) and the concentration of solute, e.g. ethanol (Jones and Greenfield 1982). Thus, in the present study decreasing the a, with glycerol may have affected the solubility of COs and the growth inhibitory effect expected from the reduced a, may have been concealed by the reduction in the solubility of COa.

The type of solute chosen to regulate the water activity is known to affect the response of bacteria to reduced a, levels. NaCl is generally more inhibitory than, e.g., glycerol (Sperber 1983). However, pure culture studies with bacteria have demonstrated that some, especially those being isolated from an environ- ment containing NaCl, are more tolerant towards NaCl than to glycerol (Marshall

184 E. Blickstad

et al. 1971, Blickstad 1984). In the present study it was demonstrated that the spontaneously developing microflora in a meat product was distinctly more restricted by NaCl than by glycerol.

Lactobacillus, i.e., the normal spoilage flora of vacuum-packaged meat pro- ducts, tolerate a, values down to 0.94 in pure culture studies (Blickstad 19841. Lactobacillus spp. were competitive at a, 3 0.94 in the present study, except in the presence of a high concentration of NaCl (6%), where instead e.g. Micrococcus and Staphylococcus were found. Staphylococ- cus may tolerate 0.86 a, when regulated with NaCl (Marshall et al. 1971). The growth of Micrococcus is favoured in vacuum-packaged bacon by a high NaCl concentration (Garner 1983). Thus, meat products with a high NaCl concentration are spoiled by organisms other than Lactobacillus. On the other hand a reduced water activity may lead to a domination of Lactobacillus SPP.9 depending on how the a, is reduced and the composition of the gas atmosphere used in storage.

Aerobically stored meat products con- tain high numbers of yeasts (Gardner 1971; Blickstad and Molin 1983a). In the present study high final levels of yeasts were found after storage in air irrespec- tive of a, (0.94 a,-0.98 a,) and NaCl concentration.

For fresh meat stored in Ns the flora is dominated by Pseudomonas spp. and the shelf-life is not prolonged, as compared to aerobic storage (Enfors et al. 1979). However, the situation for cured meat products is different. Lactobacillus totally dominated in the minced meat with 2% NaCl stored in Nz (Table 4). The

References

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No increase in the total aerobic count of frankfurter sausages stored in pure COs was found during a 210 day period (Blickstad and Molin 198313). It was speculated whether a combination of reduced a, and COs totally inhibited the growth. The present study demonstrates that a, 3 0.94 (controlled with glycerol) together with a CO2 atmosphere do not totally inhibit growth. It should be poin- ted out that the presence of glycerol may have decreased the solubility of COz. Thus, if the a, is reduced in such a way that the solubility of COz is unaffected a greater reduction in growth rate than the one obtained in the present study, may be expected.

An increase in the shelf-life of minced cured pork is obtained, at least with regard to microbial spoilage, by reducing the a, of the product and particularly for storage in N2- and COs-atmospheres. When combining reduced a, with C02- storage the type of solute chosen to regulate a, is important, since the solute may affect the solubility of C02. However, COB gives a longer shelf-life than N2 at similar a, levels.

Acknowledgements The author thanks Mr Jan-Ake Sand- berg for his skilful technical assistance.

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