PREDICTIVE MODELING AND GROWTH MODELS OF AEROBIC

MESOPHILIC BACTERIA ON FRESH-CUT LETTUCE BY

HYPOCHLORITE-WASHING

ZHAO-XIN LU1, FENG-XIA LU, LI-KUI ZHANG, XIAO-MEI BIE and

XIAO-KUI ZOU

College of Food Science and Technology

Nanjing Agriculture University
1 weigang Nanjing
210095, P.R. China

Accepted for Publication June 23, 2006

ABSTRACT

Response surface methodology was used to evaluate the effect of three

selected factors (chlorine concentration, washing time and water-to-lettuce
ratio) on reducing aerobic mesophilic bacteria on fresh-cut lettuce. According
to statistical analysis, the model established was effective in predicting the
reduction of aerobic mesophilic bacteria on fresh-cut lettuce by washing with
chlorine. In addition, best-fit Gompertz-modified models were described to
evaluate aerobic mesophilic bacteria growth on fresh-cut lettuce during
storage at 0, 4 and 25C, respectively. The final load of aerobic mesophilic
bacteria and shelf life of fresh-cut lettuce could be predicted in various storing
temperatures with the growth models.

PRACTICAL APPLICATIONS

Response surface methodology was effectively able to optimize the

washing conditions with chlorine to reduce aerobic mesophilic bacteria on
fresh-cut lettuce, and the models established were validated in effectively
predicting the reduction of aerobic mesophilic bacteria on fresh-cut lettuce by
washing with chlorine.
Gompertz models were effective in describing the effect of the storage
temperature in inhibiting the growth of aerobic mesophilic bacteria, and in
predicting maximal growth value of aerobic mesophilic bacteria on fresh-cut
lettuce and shelf life of fresh-cut lettuce during storage at 0, 4 and 25C.

1
Corresponding author: TEL: 0086-25-4396583; FAX: 0086-25-4396431; EMAIL: fmb@njau.edu.cn

Journal of Food Safety 27 (2007) 157–168. All Rights Reserved.

© 2007, The Author(s) 157
Journal compilation © 2007, Blackwell Publishing
158 Z-X. LU ET AL.

Therefore, both the predictive model and Gompertz growth models estab-
lished will be potentially applied in fresh-cut lettuce production for maintain-
ing the quality.

INTRODUCTION

Predictive food microbiology is a rapidly developing science and has

made great advances in recent years. Predictive model of microorganisms can
be used to predict microbial safety and shelf life of produce, to find critical
points in the process and to optimize production and distribution chains
(Zwietering et al. 1991; Nauta 2002). Sumner and Krist (2002) reported the
application of predictive microbiology in the Australian meat industry. Pre-
dictive modeling was employed to examine the effect of temperature fluctua-
tions on microbiological and sensory qualities of equilibrium-modified
atmosphere (EMA)-packaged minimally processed vegetables in a simulated
cold distribution (Jacxsens et al. 2002). Koseki and Itoh (2001) showed that
predictive modeling was used to determine the effects of storage temperature
on growth of microbial populations on acidic electrolysed water-treated fresh-
cut lettuce and cabbage.
There are several sigmoid functions describing a bacterial growth curve,
such as Gompertz, Stannard, Richard, Schnute and Logistic model and others.
However, increasing reports have shown that Gompertz function is used
widely in the field of predictive food microbiology. Growth models of some
pathogens have been established in laboratory media for Staphylococcus
aureus (Sutherland et al. 1994), Bacillus cereus (Sutherland et al. 1996),
Yersinia enterocolitica (Sutherland and Bayliss 1994), Clostridium botulinum
(Graham et al. 1996) and Escherichia coli 0157:H7 (Sutherland et al. 1997;
Pood et al. 2001). However, no report was found about growth models of total
aerobic mesophilic bacteria on fresh-cut vegetables. Actually, the microbio-
logical safety and shelf life of fresh-cut vegetables was mostly affected with
various microorganisms, not only pathogen, but also spoilage bacteria. There-
fore, it is more important and more feasible for food safety and predictive shelf
life to study the growth model of total bacteria on fresh-cut vegetables than one
or two kinds of bacteria.
Producing fresh-cut vegetables usually involves cleaning, trimming,
coring, slicing, shredding, washing, centrifugal drying and packaging (Yildiz
1994). However, fresh-cut vegetables may undergo substantial mechanical
injury because of cutting, which may result in rapid growth of microorganisms
upon exposure to nutrient juices. So, fresh-cut lettuce may have high levels of
microorganisms without disinfection treatment, which may shorten the shelf
life of these products greatly. Therefore, it is necessary to take measures to
 AEROBIC MESOPHILIC BACTERIA ON FRESH-CUT LETTUCE 159

control microbial population on fresh-cut products during process operations.

Washing after cutting reduces microbial contamination levels and enzymatic
oxidation during further storage (Sinigaglia et al. 1999). Many antimicrobial
solutions have been reported but probably the most widely used for vegetables
is a hypochlorite solution (Mermelstein et al. 1998; Izumi et al. 1999).
In this work, the objectives were as follows: (1) to establish the model of
evaluating the effect of three factors (chlorine concentration, washing time and
water-to-lettuce ratio) on the reduction of aerobic mesophilic bacteria in order
to reduce initial load of aerobic mesophilic bacteria on fresh-cut lettuce; (2) to
establish growth model of aerobic mesophilic bacteria during storage at 0, 4
and 25C in order to predict shelf life of fresh-cut lettuce.

MATERIALS AND METHODS

Lettuce Processing
The processes of preparing fresh-cut lettuce were shown in Fig. 1. Fresh
lettuce was purchased from local markets. The damaged leaves were removed

Fresh Lettuce

Pre-washing

Cutting

Washing with chlorine solution

Drying

Packaging

Weighing

Storage at 0°C 4°C and 25°C

FIG. 1. DIAGRAM FOR THE PREPARATION OF FRESH-CUT LETTUCE

160 Z-X. LU ET AL.

TABLE 1.
OPERATION FACTORS AND THEIR LEVELS

Experimental factors Symbol Levels

Coded value Actual value

Chlorine concentration (mg/L) x1 1 150

0 75
-1 0
Washing time (min) x2 1 8
0 5
-1 2
Water-to-lettuce ratio (L/kg) x3 1 35
0 30
-1 25

and the rest were cut aseptically by hand to 2 cm in width with a sharp stainless
steel knife, and prewashed in tap water. Ten grams of prewashed cut lettuce was
put in a 500-mL glass beaker and submerged in water, and then stirred by a
magnetic stirrer (85–2 constant temperature magnetic stirrer, Guohua Electric
Machine Limited Company, Changzhou City, China) at the rate of 180 rpm at
a constant temperature of 17C. Fresh-cut lettuce was blown dry using a super
clean bench (SW-CJ, Suzhou AnTai Air Tech Co. Ltd., Suzhou City, China).
Washing solution was prepared by adding appropriate amount of sodium
hypochlorite solution with 50 mg/L available chlorine (Chemical Purity,
Lingfeng Chemical Reagent Limited Company, Shanghai City, China), and the
fresh-cut lettuce was washed according to the experimental design (Table 1).
Then they were dried in a clean bench at room temperature for 1 h and
packaged into aseptic polyethylene with thickness of 0.02 mm. Finally,
samples were weighed for 200 g and refrigerated at 0, 4 and 25C.

Microbiological Analysis
Bacterial load on fresh-cut lettuce was regularly analyzed during storage.
Each sample (10 g) was mixed with 90-mL sterile physiological saline (0.85%
NaCl) with sterile glass rods, and the mixture was shaken in a 250 mL triangle
bottle for 3 min and serially diluted (1:10). The serially diluted samples
(100 mL) were surface-plated in triplicate onto plate count agar, incubated 24 h
at 37C for determining aerobic mesophilic bacteria as colony-forming unit per
gram (cfu/g).

Experimental Design
In this experiment, a central composite design was adopted (Box et al.
1978). The response expressed as Log N0/Nt, (N0, Nt is the number of aerobic
 AEROBIC MESOPHILIC BACTERIA ON FRESH-CUT LETTUCE 161

mesophilic bacteria before and after washing, respectively) is the log reduction
of aerobic mesophilic bacteria. For each factor, three levels are given as a
second-order polynomial model. The second-order polynomial model used
was as follows:

y = b0 + b1 x1 + b2 x2 + b3 x3 + b11 x12 + b22 x2 2 + b33 x32 + b12 x1 x2 + b13 x1 x3 + b23 x2 x3

where y is each response in terms of three factors, x1 (chlorine concentration),

x2 (washing time) and x3 (water-to-lettuce ratio); b0, b1, b2, b3, b11, b22, b33, b12,
b13 and b23 are regression coefficients. Experimental factors and their levels are
shown in Table 1.
Growth curve is defined as the logarithm of the relative population size
(y = Log Nt/N0) as a function of time (t). Gompertz equation modified by
Zwietering et al. (1991) can describe this growth curve with three parameters
(A, mm, l):

{ μ e
y = A exp − exp ⎡⎢ m ( λ − t ) + 1⎤⎥
⎣ A ⎦ }
where y is the relative growth size as Log Nt/N0 at the time t; the asymptote A,
which is the maximum value reached; the maximum specific growth rate mm,
which is defined as the tangent in the inflection point; the lag time (l), which
is defined as the x-axis intercept of this tangent.

Statistical Analysis
Design Expert software (Version 6.0.5, 2001; Stat-Ease, Inc., Minneapo-
lis, MN) and MATLAB software (The Language of Technical Computing,
Version 6.5.0.180913a, Release 13, Jun 18, 2002, The Mathworks, Inc.) were
used to perform analysis of variance (ANOVA) and to establish the second-
order polynomial equation and Gompertz equations, respectively. The signifi-
cance of models established was determined by R2 (multiple determination
coefficients), F value and P value.

RESULTS AND DISCUSSION

Predictive Model of Aerobic Mesophilic Bacteria Reduction by Washing

The number of aerobic mesophilic bacteria on fresh-cut lettuce before
washing was 8 ¥ 105 cfu/g. The reduction of aerobic mesophilic bacteria on
fresh-cut lettuce after hypochlorite-washing is shown in Fig. 2. The model
obtained was:
162 Z-X. LU ET AL.

3.43 2.93
LogNt/N0

2.68
6.50
2.43
1.92

1.17 1.42 1.92

0.92

B: B
5.00
0.42

3.50

8.00
150.00
6.50
112.50 2.00
5.00 75.00 0.00 37.50 75.00 112.50 150.00
B: B 3.50 37.50
A: A
2.00 0.00
A: A
a
2.68

2.01 32.50
LogNt/N0

1.34
2.43

0.67
C: C

30.00
0.92
1.42 1.92
0.00

27.50

35.00
150.00 25.00
32.50
112.50 0.00 37.50 75.00 112.50 150.00
30.00 75.00
C: C 27.50 37.50
A: A
25.00 0.00
b A: A

2.42

2.09
32.50
LogNt/N0

1.77

1.45
C: C

30.00
1.12 1.72 1.92
1.62 1.82

1.52

27.50
1.42

35.00
8.00
32.50 25.00
6.50
30.00 5.00 2.00 3.50 5.00 6.50 8.00
C: C 27.50 3.50
B: B
25.00 2.00
c B: B

FIG. 2. RESPONSE SURFACES AND CONTOUR PLOTS IN TERMS OF VARIOUS FACTORS

(a) Response surface and contour plot of the effect of chlorine concentration and washing time on
aerobic mesophilic bacteria reduction in water-to-lettuce ratio = 30L/kg. (b) Response surface and
contour plot of the effect of chlorine concentration and water-to-lettuce ratio on aerobic mesophilic
bacteria reduction in washing time = 5 min. (c) Response surface and contour plot of the effect
of washing time and water-to-lettuce ratio on aerobic mesophilic bacteria reduction in chlorine
concentration = 75 mg/L (N0, Nt is the number of total bacteria on fresh-cut lettuce before and after
washing; A, chlorine chlorine concentration, B, washing time, C, water-to lettuce ratio).
 AEROBIC MESOPHILIC BACTERIA ON FRESH-CUT LETTUCE 163

TABLE 2.
ANALYSIS OF VARIANCE OF REGRESSION MODEL OF HYPOCHLORITE-WASHING

Source df Sum of square Mean square F value Prob > F

Model 9 13.75 1.53 17.12 0.0013**

x1 1 10.95 10.95 122.81 <0.0001**
x2 1 2.00 2.00 22.37 0.0032**
x3 1 0.36 0.36 4.03 0.0916
x12 1 0.052 0.052 0.58 0.4734
x22 1 0.12 0.12 1.34 0.2912
x32 1 0.059 0.059 0.67 0.4455
x1x2 1 0.22 0.22 2.41 0.1714
x1x3 1 0.037 0.037 0.41 0.5448
x2x3 1 0.0002 0.0002 0.0025 0.9620
Residual 6 0.54 0.089
Lack of fit 5 0.53 0.11 237.65 0.0492*
Pure error 1 0.00045 0.00045

* P ⱕ 0.05; ** P ⱕ 0.01.

y = 1.70 + 1.05 x1 + 0.45 x2 + 0.19 x3 − 0.14 x12 + 0.21x2 2 − 0.15 x32 + 0.16 x1 x2 +
0.068 x1 x3 − 0.005 x2 x3

(R2 = 0.9625, P = 0.0013, Xi standing for coded value as related to Table 1)

According to Table 2, it was seen that the interaction (x1x2, x1x3 and x2x3)
and quadratic terms (x12, x22 and x32) were not significant for the model. The
nonsignificant variables were removed from the model. The final model was
refitted to the data resulting in:

y = 1.65 + 1.05 x1 + 0.45 x2 + 0.19 x3

(R2 = 0.9319, P < 0.0001, xi standing for coded value as related to Table 1).

The values of regression coefficient of the three factors (x1, x2, x3) were
greatly different: x1 was maximal, x2 middle and x3 minimal, which indicated
that the efficacy in reducing aerobic mesophilic bacteria was largely influenced
by x1 (hypochlorite concentration), moderately by x2 (washing time) and only
less by x3 (water-to-lettuce ratio).
According to Fig. 2a, it can be seen that the response was increased with
increasing of x1 and x2 in x3 = 30 L/kg. Fig. 2b showed that the reduction of
aerobic mesophilic bacteria was affected by only x1 when x2 was 5 min,
164 Z-X. LU ET AL.

indicating the hypochlorite effect on killing bacteria. From Fig. 2c, washing
with 75 mg/L chlorine concentration for 6.5 min made aerobic mesophilic
bacteria on fresh-cut lettuce reduce by about 2 logs. Therefore, washing with
sodium hypochlorite was effective in reducing aerobic mesophilic bacteria
on fresh-cut lettuce. In addition, the same reduction of aerobic mesophilic
bacteria could be obtained through adjusting the washing time and water-to-
lettuce ratio, considering the problem of chlorine residue in the practical
process of fresh-cut lettuce.

Models Validation
The established model equation was tested by using a chlorine concen-
tration of 75 mg/L, a washing time of 6.5 min and a water-to-lettuce ratio of
30 L/kg. According to this model equation, the predicted value for the reduc-
tion of aerobic mesophilic bacteria was 1.90, but the mean experimental value
was 2.08 ⫾ 0.1273, indicating that the predicted value was in good agreement
with the actual value.

Modeling of Growth Curves of Aerobic Mesophilic Bacteria

in Storage at 0, 4 and 25C
The growth model of total bacteria was investigated in order to find the
growth parameters of mesophilic bacteria and to predict the shelf life of
fresh-cut vegetable in different storage temperature. The number of aerobic
mesophilic bacteria on fresh-cut lettuce was 3.30 ¥ 105 cfu/g before washing
and was 2.72 ¥ 103 cfu/g after washing with 75 mg/L chlorine concentration,
30 L/kg water-to-lettuce ratio and 6.5 min washing time, respectively. The
growth curve of aerobic mesophilic bacteria is shown in Fig. 3 during storage
at 0, 4 and 25C. Through the analysis of experimental data, growth models
of aerobic mesophilic bacteria on fresh-cut lettuce during storage were estab-
lished with MATLAB software. The parameters of Gompertz models are
shown in Table 3.
The significance of regression equations was evaluated by R2 value and F
value. From analysis of variance of Table 4, Gompertz models established
were highly significant (a = 0.01, a is confidence level). The fitting curves
(Fig. 3) showed that the growth of aerobic mesophilic bacteria was greatly
affected by storage temperature. On the eighth day at 0C storage, the popula-
tion of aerobic mesophilic bacteria on fresh-cut lettuce was 105 cfu/g, which
was lower than that at 4C storage. However, the population of aerobic meso-
philic bacteria reached 5.90 ¥ 109 cfu/g on fourth day at 25C storage. After
5.5 days of storage at 25C, the general appearance of fresh-cut lettuce was too
poor.
 AEROBIC MESOPHILIC BACTERIA ON FRESH-CUT LETTUCE 165

FIG. 3. GROWTH CURVES OF AEROBIC MESOPHILIC BACTERIA FITTED WITH THE

GOMPERTZ EQUATION

TABLE 3.
PARAMETER VALUES OF GOMPERTZ MODELS
ESTABLISHED

Storage temperature A mm l

0C 1.5879 0.3346 0.0750

4C 2.5710 0.7070 0.9995
25C 5.7906 1.8950 0.2683

A, the maximum value reached; mm, the maximum specific growth

rate; l, lag time.

From Table 3, it was showed that mm and A at 0C storage were 0.3346 and
1.5879, respectively. They were lower than those of 4 and 25C, indicating that
the storage at low temperature greatly suppressed the growth of aerobic meso-
philic bacteria on fresh-cut lettuce. From Table 3, if the initial load of aerobic
mesophilic bacteria was 103 cfu/g, the final bacteria load on the eighth day
could reach 104.6 cfu/g at 0C and 105.6 cfu/g at 4C on the eighth day and
108.8 cfu/g at 25C on the fifth day, by the growth models. Therefore, storing at
0C and 4C was effective in extending the shelf life of fresh-cut lettuce.
166 Z-X. LU ET AL.

TABLE 4.
ANALYSIS OF VARIANCE FOR GOMPERTZ MODELS ESTABLISHED AT 0, 4 AND 25C

Storage temperature Source df Sum of squares Mean square F value Significance

0C Model 2 3.0951 1.5476 103.2052 *

Error 12 0.1799 0.0150
Total 14 3.2750
R = 0.9721 R2 = 0.9451
4C Model 2 10.1421 5.0710 90.5291 *
Error 12 0.6722 0.0560
Total 14 10.8143
R = 0.9684 R2 = 0.9378
25C Model 2 45.3339 22.6669 67.6982 *
Error 9 3.0134 0.3348
Total 11 48.3473
R = 0.9683 R2 = 0.9377

* Stands for high significance (F value >F0.01[2,9] = 8.02; F value >F0.01[2,12] = 6.93).
Total means total sum of squares.

CONCLUSIONS

According to the model of hypochlorite-washing, the same desired reduc-

tion of aerobic mesophilic bacteria on fresh-cut lettuce could be achieved by
extending washing time properly rather than increasing chlorine concentra-
tion. In this work, washing with 75 mg/L chlorine concentration for 6.5 min,
which reduced aerobic mesophilic bacteria by 2 logs was regarded as an
optimal condition for treating fresh-cut lettuce.
Gompertz models were effective in describing the effect of the storage
temperature on inhibiting the growth of aerobic mesophilic bacteria, and in
predicting maximal growth value of aerobic mesophilic bacteria on fresh-
cut lettuce during storage at 0, 4 and 25C. The observations made in this work
recommend that fresh-cut lettuce be stored at 0C in order to extend effectively
the shelf life, assure the safety and keep the freshness of this product. There-
fore, washing with chlorine and storing at 0C may offer an alternative to
reducing microbiological deterioration and shortening tissue decay of fresh-
cut lettuce.

ACKNOWLEDGEMENTS

The authors greatly appreciated the financial support provided by the

Ministry of Science and Technology of China (2001BA501A10).
 AEROBIC MESOPHILIC BACTERIA ON FRESH-CUT LETTUCE 167

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