J. Pac .Sci. Tech. Vol.21 No.3（2012）

Original Paper～～～～～～～～～～～～～～～～～～～～～～～～～～～～

Effect of CO2-flushed Packaging on Quality of Pink Shrimp
Contaminated with Vibrio parahaemolyticus
Yoshio MAKINO*†, Yuki ITO*, Seiichi OSHITA*, Yoshinori KAWAGOE**, Yukie
KAWABUCHI***, Tsuyoshi AYAKI****, Mikio TANAKA****, Kazuhiko HIROSE****,
Tatsuya SUGAWARA*****, and Takashi HIRATA*****
CO2-flushed packaging was effective for maintaining the quality of pink shrimp contaminated with
Vibrio parahaemolyticus. Contaminated shrimp were sealed in Krehalon ML VS-20 film pouches flushedwith air, N2, or CO2 and stored at 20°C. CO2 concentration in the pouch was approximately 87% and was
retained for at least 168 h. The numbers of V. parahaemolyticus in raw pink shrimp after 8 h (initiallyinoculated at 1–9 CFU·g−1) or 12 h (initially inoculated at 100–999 CFU·g−1) of storage and the maximum
number of bacterial cells at steady state as a kinetic parameter of the Gompertz equation were smaller
under the CO2-flushed atmosphere than under the other atmospheres. The period before reaching theenteropathogenic concentration of 106 CFU·g−1 was prolonged by CO2-flushed packaging. These results
indicate that CO2-flushed packaging is effective for suppressing V. parahaemolyticus growth. Shrimp
blackening due to the activity of polyphenol oxidase (EC 1.14.18.1) was inhibited by CO2-flushed
packaging. Although CO2 reduced the pH on the surface of shrimp, sensory scores were not affected. The
results obtained in the present study indicate that CO2-flushed packaging is effective for maintaining theappearance, taste, and hygiene of pink shrimp.
Keywords：Facultative anaerobe, Sea food, Storage, Inorganic gas, Blackening, quality, Gompertz model

1. Introductionof aerobes.3) However, Kimura et al.4) reported

Pink shrimp (Pandalus borealis Krøyer, 1838)that atmospheres containing 20–100% CO2 did

1)

not significantly suppress the growth of V.

Because shrimp can become contaminated withparahaemolyticus purchased from the Institute forpathogens such as Vibrio parahaemolyticus

Fermentation,(Fujino et al., 1951; Sakazaki et al., 1963 emend.;

inoculated on plate count agar and incubated atWest et al., 1986), it is important that shrimp

30°C. Hirose et al.5) reported that 100% CO2

distributed to the market should be safe andatmosphere was not suppressed the growth ofare an important seafood resource worldwide.

Osaka,

Japan,

which

were

2)

anaerobic bacteria though aerobic bacteria were

It is well known that CO2 inhibits the growth

suppressed at 10°C. Conversely, Makino et al.6)

hygienic for consumption.
*

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
College of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, JapanWDB Co. Ltd., WDB Co. Ltd., Marunouchi 2-3-2, Chiyoda-ku, 100-0005, Japan****
Polymer ProcessingTechnology Center, Kureha Corporation, 18-13, Kamitamari, Omitama, Ibaraki 311-3436, Japan*****
Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan†
Corresponding author, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657,
Japan, TEL:03-5841-5361, FAX: 03-5841-8174, Email:amakino@mail.ecc.u-tokyo.ac.jp*****－ 203 －

Effect of CO2-flushed Packaging on Quality of Pink Shrimp Contaminated with Vibrio parahaemolyticus

reportedthatatmosphere

the Gompertz equation, which is a kinetic modelsignificantly suppressed the growth of bacteria

widely used for microbial growth analysis.9,10)

isolated from prawns [Marsupenaeus japonicus;

Simultaneously, the effects of CO2 on shrimp(Bate, 1888)], which were inoculated on pink

blackening, pH, and sensory scores, which areshrimp and incubated at 20°C. This research

considered as important factors determining

demonstrated the novel finding that CO2 isquality, were examined. The experiments in theeffective in suppressing the growth of facultative

present study were conducted at 20°C, because

anaerobes as well as aerobes. However, growth

thiswas measured in a gas-tight acrylic chamber.

investigating the atmospheric influence on V.

Moreover,parahaemolyticus growth as described in our

the

a

>99%

shrimpCO2

weresterilizedbyautoclaving for 15 min at 121°C to remove thetemperature

wassuitable

value

for

previous report.6)

initial microflora. Thus, these experimental
conditions were very different from the practical

2. Materials and Methods

conditions. Therefore, the suppression of V.

2.1

parahaemolyticus

growthby

CO2Materials

Pink shrimp (17.5 ± 1.0 g per shrimp)

required

harvested from the Tajima fishery harbor (Japan)

examination under practical conditions.
The appearance of shrimp degrades very

were transported to the laboratory within a day of

rapidly after harvesting because of the enzymatic

harvest. They were not treated with any chemical

activity

to suppress blackening.

ofpolyphenol

oxidase(PPO,

EC

V. parahaemolyticus isolated by the authors6)

1.14.18.1), which results in the appearance of
black spots on the shrimp surface.7) Chen et al.8)
reported that high CO2 pressure inhibited the
7)

was

used

in

the

present

study.

V.

11)

were

parahaemolyticus, stored by liquid drying,activity of PPO, and Gonçalves et al. suppressedsuspended in a small amount of sterilized Salt

the blackening of pink shrimp using a modified

Polymyxin

atmosphere with a high CO2 concentration. These

incubated in the same medium for 18 h at 30°C.

findings suggest that CO2 is effective in

The incubated broth containing 109 CFU·mL−1 ofmaintaining both the appearance and hygiene of

microbial cells was used for this study.

6)

shrimp. However, Makino et al.

Broth“Nissui”,

andstatically

could notLaminated film pouches (Krehalon ML VS-20,

examine the effect of CO2 on the suppression ofKureha Corporation, Tokyo, Japan, polyethylene

shrimp blackening because PPO was inactivatedterephthalate / nylon / ethylene vinyl alcohol /bypolyethylene) were used to store the shrimp. The

autoclaving,

which

was

used

as

the

properties were as follows: surface area, 0.2 m2;

sterilization method.
In the present study, the suppressive effect offilm thickness, 40 µm; O2 transmission rate, 25

CO2-flushed packaging, as a practical method for

mL·m−2·d−1·atm−1 [at 23°C, 80% relative humidity

the safe distribution of pink shrimp, on V.

(R.H.)]; water vapor transmission rate, 20

parahaemolyticus growth was investigated usingg·m−2·d−1 (at 40°C, 90% R.H.); standard plate- 204 -

J. Pac .Sci. Tech. Vol.21 No.3（2012）

count (SPC), <4 CFU·m−2.

with air were simply sealed. N2 and CO2 gases
were sealed in the pouches as described in the

2.2

previous section. Atmospheres of air or high CO2

Measurement of atmospheric changes

concentration were used as the control and test,in the laminated pouches
The ability of the laminated pouches to retain

respectively.

Theatmosphere

of

high

N2

atmosphere was examined before the storage test

concentration (equivalent to air without O2) wasfor contaminated shrimp. N2 (99.9995 vol%) or

prepared to confirm the effect of O2 on V.

CO2 (99.995 vol%) gas was flushed into a pouch

parahaemolyticus growth because the absence ofthrough a sterilization filter (pore size, 0.1 µm)

O2 in the high CO2 atmosphere might affect V.

and immediately sealed using an impulse sealer.

parahaemolyticus growth, making it difficult to

The preparations were stored for 168 h at 20°C.

judge whether CO2 suppressed the growth by

Changes in O2, CO2, and N2 concentrations were

simply comparing the growth at air and CO2.

measured using a GC-14A gas chromatograph

The preparations were stored for 48 h at 20°C.

(Shimadzu Co., Kyoto, Japan) equipped with a

CFU of V. parahaemolyticus was measured every

thermal conductivity detector, according to the

4 h using thiosulfate citrate bile sucrose (TCBS)

method of Makino et al.

12)This experiment wasagar plates as the selection media for thebacterium, according to the method of Makino etrepeated twice.

al.6) The contaminated shrimp before storage were
2.3

used for the colony count at 0 h.

Measurement of V. parahaemolyticus

This experiment was repeated three times.

growth in stored shrimp
SPC of the purchased shrimp was measured
using Petrifilms
Tokyo,

TM

Japan)

parahaemolyticus.
containing

10

2

Experimental data for the plate counts were(AC plates; Sumitomo 3M Ltd.,statistically evaluated by analysis of variance

before(ANOVA)Aorinoculationwithsuspension

(0.1

4

−1

10

CFU·mLofV.

honestly

significant difference (HSD) tests (P < 0.05) using

V.

JMP™ ver. 8.0.2 software (SAS Institute Inc.,
Cary, NC, USA).
Changes in plate counts over time were plottedshrimp. This was equivalent to an initial
2

Tukey–Kramer

mL)

parahaemolyticus was inoculated into the raw
0

with

−1

on scatter graphs. Growth curves were generatedof shrimp. Shrimp were also sterilized byfrom experimental data obtained in this study

autoclaving (121°C, 15 min) to confirm the effectusing the Gompertz equation (Eq. 1), which hasof the initial SPC on V. parahaemolyticusgrowth.

been most often employed9,10) in conjunction with

A suspension (0.1 mL) containing 102 CFU·mL−1

SigmaPlot® ver. 9.01 (SPSS Inc., Chicago, IL,

of V. parahaemolyticus was inoculated into theUSA), a nonlinear least-squares fitting program

sterilized shrimp.

that

inoculation at 10 (1–9) or 10 (100–999) CFU·g

Shrimp were sealed in the laminated film

employsalgorithm:

pouches flushed with air, N2, or CO2. Pouches－ 205 －

theMarquardt–Levenberg

Effect of CO2-flushed Packaging on Quality of Pink Shrimp Contaminated with Vibrio parahaemolyticus

N t = N 0 + Ce −e− B (t − M )

(1)

L=M −

where N is the plate count (CFU·g−1), C is the

1
B

(2)

difference between the initial and maximum plate

2.4

counts (CFU·g−1), e is the Naperian base, t is the

shrimp stored under various atmospheres

storage time (h), M is the time at which theabsolute growth rate is maximum (h), B is the
maximum relative growth rate (relative growthrate at M) (h−1), and subscript 0 represents thestart of storage.
The lag phase duration L (h) was estimated as

Observation of blackening of pink

Blackening
contaminated

of

pink

shrimp

0

−1

with

10

CFU·g

initiallyofV.

parahaemolyticus was recorded by taking images
using a Coolpix L16 digital camera (Nikon
Corporation,

Tokyo,

Japan)

duringtheexperiments mentioned in section 2.3.

shown in Eq. 2 from the Gompertz equation:13)
2.5

Measurement of pH and sensory scoresfor pink shrimp stored under various
atmospheres
Pink shrimp without inoculation were sealed in
the laminated film pouches by the method
described in section 2.3. The preparations werestored for 4 h at 20°C. After storage, the pH on
the shrimp surface was measured using a YK-21
pH meter (Sato Shoji Corporation, Tokyo, Japan)
equipped with a flat sensor for measuring pH on
the surface of a sample.
Taste, flavor, and texture of the pink shrimp
were evaluated using a four-star scale (1, bad; 2,
poor; 3, fair; 4, good) by three trained panelists.
These data were statistically evaluated by
ANOVA with the least significant difference(LSD) test (P < 0.05) using JMP™ ver. 8.0.2.

3. Results and Discussion
3.1 Gas barrier properties of the laminatedpouches
Changes in the atmospheres in the laminatedpouches over time are shown in Fig. 1.
Fig. 1 Changes in O2 ({), N2 ( ), and CO2 (U) in
the laminated film pouches (Krehalon ML VS-20)
over time. (A) and (B): Initially flushed with CO2
and N2, respectively.

After 168 h of storage, the atmosphere in thepouches initially flushed with CO2 contained- 206 -

J. Pac .Sci. Tech. Vol.21 No.3（2012）

approximately 87% CO2, 2.8% O2, and 10% N2.
The atmosphere in the pouches initially flushedwith N2 contained approximately 0.38% CO2,
1.3% O2, and 98% N2. These barrier properties
are suitable for gas-flushed packaging of pink
shrimp contaminated with V. parahaemolyticus
because this bacterium grows rapidly within 48h.6) Although 99.995% CO2 and 99.9995% N2
were flushed into the pouches, a certain amount of
air may enter the pouch before sealing. Because
air contains approximately 78% N2, the N2
concentration in the atmosphere shown in Fig.
1(B) may be higher than that shown in Fig. 1(A).
The CO2 concentration in Fig. 1(B) was expected
to be approximately 0.03%, the usual CO2 level inair. Because the CO2 and N2 concentrations in Fig.
1 were lower than those in the acrylic chamber inour previous report,6) the influence of these gases
on microbial growth in the present study may besmaller than that in the previous study.
3.2

Effects of various atmospheres on V.

parahaemolyticus growth in stored shrimp
Changes

in

the

plate

countsofV.

parahaemolyticus over time are shown in Fig. 2.
Differences

betweenthenumbersofV.

parahaemolyticus after the same storage period
were compared by the Tukey–Kramer HSD test.
SPC of raw pink shrimp used in this study was
log2.3 CFU·g−1. V. parahaemolyticus grow on
PetrifilmsTM (AC plates); however, the initial
number of V. parahaemolyticus in the raw shrimp
was below the limit of detection using TCBS agar
plates. Therefore, microorganisms other than V.
parahaemolyticus

accounted

for

the

Fig. 2 Changes over time in plate counts of V.
parahaemolyticus in pink shrimp stored inpouches flushed with air ({), N2 ( ), and CO2(U)at 20°C. (A): raw shrimp, 100 (1–9) CFU·g−1
initial plate count. (B): raw shrimp, 102 (100–999)
CFU·g−1 initial plate count. (C): autoclaved
shrimp, 100 (1–9) CFU·g−1 initial plate count.
Means ± standard error of three observations areplotted.

initialmicroflora in the raw shrimp. Although the initial－ 207 －

Effect of CO2-flushed Packaging on Quality of Pink Shrimp Contaminated with Vibrio parahaemolyticus

microflora was unclear, effects of SPC on V.

the initial number of bacteria by chemicals, etc., isparahaemolyticus growth can be determined byeffective in delaying growth. The number ofcomparing the results in Fig. 2(A) and 2(C). No

bacteria under the CO2-flushed atmosphere in Fig.

significant differences were observed between

2(A) was lower than that in Fig. 2(B) until 28 h.

these results. This indicates that the initial SPC in

This suggests that the difference in the initial

the shrimp did not affect the plate count data for V.

number of bacteria affected the number before

14)

reaching a steady state. O2 in the air did not affectreported that the number of V. parahaemolyticus

the growth of the bacteria because there were noinparahaemolyticus

thisstudy.

Kimura5cells increased from 10 to 10 cells during 5 h atsignificant differences between the numbers of

30°C and that the growth rate was considerably

bacteria in pouches flushed with air and N2 inhigher than that of the bacteria growing on a

Fig. 2.

standard plate. Therefore, rapid growth of

The growth kinetic parameters in the Gomperz

inoculated V. parahaemolyticus may suppress theequation are presented in Table 1.

growth of other bacteria.

maximum number calculated as N0 + C under the

The

The number of bacteria after 8 h of storage

CO2-flushed atmosphere was significantly lower

under the CO2-flushed atmosphere in Fig. 2(A)than that under the other atmospheres, as

was lower than that under the other atmospheres.

indicated in Fig. 2. However, the maximumThis suggests that the CO2-flushed atmosphere isnumber was higher than that under the >99% CO2

effective in suppressing V. parahaemolyticus

atmosphere reported by Makino et al.6) This may

growth. Although the CO2 concentration in thebe due to the difference in the CO2 concentration.

present study was lower than that in the previous

The lag phase duration, as the period just before

6)

according to the result in Fig. 1(A),the start of rapid growth, is important forbacterial growth was still suppressed. The number

evaluating storage methods and conditions. Theof bacteria after 12 h of storage under theduration for shrimp contaminated with 100

CO2-flushed atmosphere in Fig. 2(B) was also

CFU·g−1 and stored under the CO2-flushedlower than that under the other atmospheres. The

atmosphere was slightly longer than that forlater suppressive effect of CO2 in Fig. 2(B)shrimp under the other atmospheres. However,compared with that in Fig. 2(A) may be due to thethis tendency was unclear. In our previous study,6)difference in the initial inoculation concentrations.

the lag period under the >99% CO2 atmosphere

However, the result at 12 h of storage in Fig. 2(B)was shorter than that under air. These results

is in agreement with the result reported bysuggest that evaluation of the period using thestudy,

6)

Makino et al.
contaminatedin which shrimp were initiallywithparahaemolyticus.

This

10

3

CFU·g

suggests−1

that

Gompertz equation is difficult or that the effect ofV.

CO2 on extending the lag period is small orthenon-existent.
Sakazaki et al.15) reported that administration of

suppression by CO2 is delayed by an increase in
the initial number of bacteria. Thus, reduction of

approximately- 208 -

106

viable

cells

of

V.

J. Pac .Sci. Tech. Vol.21 No.3（2012）

parahaemolyticus caused gastroenteritis in a male

3.3 Effects of atmospheres on the appearance

subject. Therefore, the storage periods for theof

6

number of bacteria to reach 10 CFU·g

−1

are

pink

shrimpcontaminated

with

V.

parahaemolyticus

presented in Table 1 as an index for evaluating

Blackening of stored raw shrimp initiallyenteropathogenicity per gram of shrimp. The

contaminated with 100 CFU·g−1 was observed, as

storage periods under the CO2-flushed atmosphereshown in Fig. 3.

0

2

The appearance of the cephala of shrimp stored

CFU·g bacterial cells were longer by 10 h and 5

under air and N2-flushed atmospheres wash, respectively, than those under the otherblackened after 4 h and 16 h, respectively. This

6)

atmospheres. According to our previous study,

suggests O2 effects blackening. Gonçalves et al.7)

the maximum number at steady state under thereported that the enzymatic activity of PPO,

for shrimp initially contaminated with 10 and 10
−1

6

−1

>99% CO2 atmosphere did not reach 10 CFU·g .

which requires O2, results in the appearance ofThus, CO2-flushed packaging is suggested to be

black spots on pink shrimp. Therefore, reduction

effective in maintaining better shrimp hygiene,of O2 in the storage atmosphere may be effective

and the effect is higher with an increased CO2

in suppressing shrimp blackening.

concentration.
Table 1. Kinetic parameters in the Gompertz equation (Eq. 1), and the period of time for the number of bacteria toreach 106 CFU·g−1 under various atmospheres at 20°C.
Container

Autoclaving

Initial plate
count, CFU·g−1

100
No
Flexible
pouch

10

2

N0

C

B

M

L

Perioduntil 106
CFU·g−1, h

Air

0

9.4

0.16

10

3.9

15

N2

0

9.3

0.16

10

4.2

15

CO2

0

8.3

0.10

14

4.6

25

Air

2.2

7.2

0.15

7.7

1.2

11

N2

2.7

6.5

0.20

8.7

3.7

11

CO

2.2

6.1

0.14

11

3.2

16

N2

0

9.4

0.15

10

3.4

15

CO2

0

7.9

0.14

15

7.4

24

Air

3.4

6.2

0.17

13

7.6

14

N2

3.1

6.1

0.14

12

5.0

14

CO2

3.2

2.5

0.22

10

5.9

∞

Atmos-p
here

Kinetic parameter

Reference

Present
Study

2
Fig. 3 Appearance of pink shrimp initiallycontaminated with 100 CFU·g−1 V. parahaemolyticus
stored in pouches flushed with air (A),(C) at 20°C.
The
numbers
Air N2 (N),0 and
9.4CO2 0.15
10
3.3 hyphenated15
denote the storage period0 (h).

YesAcrylic
gas-tight
chamber

Yes

10

10

3

Makinoet al.6)

N is the plate count (CFU·g−1), C is the difference between the initial and maximum plate counts (CFU·g−1), e is the Naperian base, t is thestorage time (h), M is the time at which the absolute growth rate is maximal (h), B is the maximum relative growth rate (relative growth rateat M) (h−1), and subscript 0 represents the start of storage, and L is the lag phase duration (h): L = M − 1/B.

－ 209 －

Effect of CO2-flushed Packaging on Quality of Pink Shrimp Contaminated with Vibrio parahaemolyticus

Fig. 3 Appearance of pink shrimp initially contaminated with 100 CFU·g−1 V. parahaemolyticus storedin pouches flushed with air (A), N2 (N), and CO2 (C) at 20°C. The hyphenated numbers denote thestorage period (h).
Table 2. pH and sensory scores for shrimp stored under various atmospheres.
AtmospherepH
Taste
FlavorTextureAir

7.3 ± 0.031a

3.7 ± 0.35a

4.0 ± 0.00a

3.7 ± 0.35aN2

7.3 ± 0.0067a

4.0 ± 0.00a

4.0 ± 0.00a

4.0 ± 0.00aCO2

6.9 ± 0.031b

3.7 ± 0.35a

4.0 ± 0.00a

3.7 ± 0.35a

Means ± standard error of three observations are presented. Within the same measurement,
values followed by the same letter are not significantly different (P > 0.05; One-way ANOVA
with the LSD test).
- 210 -

J. Pac .Sci. Tech. Vol.21 No.3（2012）

Conversely, the appearance of the shrimp
storedunder

theCO2-flushedatmosphereremained unchanged through all storage periods,

in pH caused by an increase in CO2 dissolved in
the culture medium may not affect the growth of
microorganisms.

Therefore,

although the images are shown only until 28 h.

metabolicThis result is in agreement with the research

suppressed bacterial growth.

7)reported by Gonçalves et al.

pathwaysinhibition
3)

by

CO2

may

of
have

that a modified

The results of sensory tests are also presented

atmosphere with high CO2 concentration wasin Table 2. In consideration of the safety of theeffective in suppressing the blackening of pink

panelists, the storage period of the samples for6)these tests was 4 h. The samples were tested afterV.

the pH measurements. No significant differences

parahaemolyticus in high CO2 concentration was

were observed between the storage atmospheres.

reported because the stored shrimps were initially

Therefore, CO2-flushed storage may not affect thesterilized

eating quality of the shrimp.

shrimp. In the previous study by Makino et al.,
only

the

growthbysuppression

autoclaving.

This

of

operation

inactivated all enzymes, including PPO, and madeThe storage period for the measurement of pH

it impossible to observe blackening of the samples.

and evaluation of sensory scores may have been

Therefore, the suppressive effects of CO2 on V.

much shorter than the average storage period of

and

are

shrimp contaminated with bacteria. In future, we

simultaneously confirmed in the present study for

expect to measure pH after long-term storage.

the first time.

However, evaluation of sensory scores after

parahaemolyticus

blackening

long-term storage may be difficult.
3.4
Effects of atmospheres on pH andsensoryscoresforpinkshrimpcontaminated with V. parahaemolyticus
The pH values for shrimp stored under theCO2-flushed packaging for maintaining the safety

three types of atmospheres for 4 h are presented in

These

Table 2. pH under the CO2-flushed atmosphere

improvements in distribution and storage methods

was significantly lower by 0.4 than that under thefor shrimp.

In the present study, the possibility ofand quality of pink shrimp has been demonstrated.
novel

resultsmaycontributetoother atmospheres. This indicated that CO2
affected the pH on the surface of the shrimp.

5. Acknowledgment

However, V. parahaemolyticus can grow at a pH

We thank Mr. T. Mori (Hyogo Prefecturalrange of 5.6–9.6 (optimum pH, 7.6–8.0).16)

Technology Center for Agriculture, Forestry, andTherefore, the values in Table 2 alone did not

Fisheries) for providing us with pink shrimp.

have a suppressive effect on the growth, although

Financial support was provided to our research

pH under the CO2-flushed atmosphere was further

group by the Japan Society for the Promotion of

from the optimum pH than that under the other

Science as Grant-in-Aid for Scientific Researchatmospheres, and this may have affected bacterial(A) (Project no. 19208020).

growth. Dixon and Kell3) reported that a decrease－ 211 －

Effect of CO2-flushed Packaging on Quality of Pink Shrimp Contaminated with Vibrio parahaemolyticus

Science and Technology, Japan, p. 23

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