Control of diseases and physiological disorder in fresh-cut fruits using phenyllactic acid
bio-compound
Bui Kim Thuy, Nguyen Thi Lan Huong
Vietnam Institute of Agricultural Engineering and Postharvest Technology
126 Trung Kinh str., Trung Hoa, Cau Giay Dist., Hanoi, Vietnam
Tel: +84438687884; Email: [email protected]; [email protected]
Introduction
Vietnam has a great potential for production of fruits and vegetables. Recently, many fruit farms
have been formed in specialized fruit growing zones, giving an even and good quality and larger
quantity supply of fruits. Together with the economic development, consumption demand is
increasing in quality and quantity. The fresh-cut fruit industry need to increase types of
minimally processed fruits to meet the consumer’s requirements for quick and convenient
products that preserve their nutritional value, retain a natural color, flavor and texture, and
contain fewer additives (Jongen, 2002).
However, fresh-cut fruits are highly perishable products due to their intrinsic characteristics and
minimal processing. Microbial growth, sensorial attributes decays, and loss of nutrients are
among the causes that compromise the quality and safety of fresh cut products (Ayala Zavala et
al., 2008a). These drawbacks are caused by the steps in the minimal processing like peeling and
cutting which promote an increment in the metabolic rate, enzymatic reactions, and release juice
(Rapisada et al., 2006). Chemical synthetic additives can reduce decay rate, but consumers are
concerned about chemical residues which could affect their health and environmental pollution,
giving rise to the need to develop alternative methods for controlling fresh cut fruit decay. One
of the emerging technologies for control of postharvest diseases is the application of the natural
bioactive compounds. In recent years, numerous studies on the antimicrobial activity of a wide
range of the natural compounds from different origins have been reported (Ayala Zavala et al.,
2008b). Many microorganism pathogens causing of foodborn diseases and fresh food decay can
be inhibited by the natural compounds (Fisher and Phillips, 2006; 2008). Among these,
phenyllactic acid (PLA) is a promising candidate for its application in fresh-cut fruit and
vegetable products.
PLA is an organic acid produced by some microorganism like lactic acid. PLA showed a wide
antimicrobial spectrum in some positive and negative Gram bacteria, mold and fungi
(Dieuleveus et al., 1998a, 1998b). PLA has been reported to be one of the most abundant
aromatic acids to which antibacterial properties have been attributed and to occur in several
honeys with different geographical origins (Steeg, 1987; Weston, 1999). Effects of PLA on
animals and human health were investigated and showed no toxicity (Oberdoester et al., 2000).
This current study which is the first investigation on PLA properties and applicability in Vietnam
focuses on the evaluation of effects of PLA on contaminated pathogen development and
physiological changes of fresh-cut pineapple during storage in cold temperature (5 oC).
2. Objectives of the action plan
Prolong the shelf-life of fresh-cut pineapple towards higher food quality and hygiene, increase of
economic values and living standard.
3. Methodology
Pineapple fruits were obtained from a local food processors. Fresh-cut pineapple was minimally
processed as in the following diagram.
Input material
↓
Selection and Classification
↓
Cleaning and removing inedible parts
↓
Cut into pieces
↓
NaOCl treatment
↓
Dry in ambient conditions
↓
PLA treatment
↓
Packing
↓
Storage in 5 0C
↓
Sampling for analysis of parameters
Diagram of pineapple minimal processing
3.1. Determination of antimicroorganism activity
3.1.1. Microorganism cultures
The molds Aspergillus niger, Aspergillus flavus, Penicillium digitatum belonging to the micobial
type culture collection of Vietnam Institute of Agricultural Engineering and Postharvest
Technology (Vietnam) were used in this study. Molds were grown on PDA medium individually
at 250C for 5 to 7 days and stored at 40C. Spore inocula were prepared by growing the molds on
PDA medium until sporulation, and the spores were collected by vigorous shaking with sterile
peptone water (0.2%, w/v). The spore concentration was determined using a hemocytometer and
adjusted to 104 spores per milliliter.
Bacteria strains of Escherichia coli, Salmonella typhi, Vibrio cholerae used in the study came
from the micobial type culture collection of National Institute of Hygiene and Epidemiology
(Vietnam). Bacteria were grown on NA medium individually at 370C for 3-4 days and stored at
40C. Spores were prepared and collected as described for molds above. The spore concentration
was adjusted to 104 spores per milliliter
3.1.2. Determination of antimicroorganism activity
The inhibition spectrum of tested organisms was determined by using the agar well diffusion
method as described by Magnusson and Schnurer (2001). The agar plates containing 104 spores
of every tested strain per milliliter were prepared. Wells with a diameter of 10 mm (d) were cut
in the agar using a sterile cork borer. 200 µl of PLA was filled in the wells and 200 µl of sterile
water was filled in the control wells. Then, the plates were allowed to diffuse into the agar during
5 hour pre-incubation period at room temperature followed by incubation at optimum
temperature for 48 hours. The diameters of inhibitory halo were determined.
PLA with different concentrations was added in tubes containing PDA media (200µl). 50µl of
organism test culture with spore concentration of 104cfu/ml was rejected in the tubes. Tubes
were incubated at optimum temperature for 24 hours. Then, 10µl of each mixture was spread
onto the surface of the petri dishes containing 10 ml of media followed by incubation at optimum
temperature for 48 hours. After incubation, the samples were collected for the determination of
percentage inhibition calculated by using the formula given below:
Percentage inhibition (%) = (C-T)/ T * 100
Where, - C: Number of spores in control (without PLA)
- T: Number of spores in test (with PLA)
3.2. Determination of physiology changes
3.2.1. Firmness
Flesh firmness of fresh-cut pineaple was evaluated using Fruit Pressure Tester (Bertuzzi,
Italia) with 0.5 cm tip. Firmness (X) was calculated as follow:
F
X 
S
Where, - X: firmness of pineapple flesh (kg/cm2)
- F: Value recorded in the Tester (kg)
- S: Area of the tip (cm2)
3.2.2. Color
Colour of pineapple flesh was measured using a Minolta CR-300 reflectance colorimeter (Japan).
The chroma meter was calibrated against a white tile. The pineapple flesh were measured for L*
(lightness), a*[green (-) to red (+)], b* [blue (-) to yellow (+)].
Color difference (∆E ) is calculated as the following equation:
∆E = [(Li - Lo)2 + (ai - ao)2 + (bi - bo)2 ]1/2
Where,
- Li, ai, bi: Values at the measurement i
- Lo, ao, bo: Standard values (values of samples at the begining of the experiment)
- L (lightness): 0 is black, 100 is white
- a (red-green): Positive values are red; negative values are green and 0 is neutral
- b (yellow-blue): Positive values are yellow; negative values are blue and 0 is neutral
3.2.3. Total acidity
The total acidity of fresh-cut pineapple juice was determined according to Vietnam Standard
3948-84. Adding 10 ml of filtered juice, 20 ml dH2O, and 3 drops of phenolphthalein as indicator
into a flask. Titration was performed by adding NaOH 0.1% until the solution in the flask
appeared light pink color. Record the used volume of NaOH. The results were expressed as citric
acid. This value includes all the substances of an acidic nature in the juice that react with NaOH.
It so happens, however, that in most fruits the chief substances reacting with NaOH are the
organic acids; for this reason, the titratable acidity represents fairly well the organic acid content
of a given juice. Total acidity as citric acid content (%) was canculated as the following formula:
X
M .V2 .N .100
V1 .1000
Where:
-
X: Citric acid contentt (%)
M: Molecular weight of citric acid = 64
N: Concentration of NaOH solution
V1: Volume of juice used for titration, ml
V2: Volume of NaOH 0.1 N solution for titration, ml
4. Result and discussion
4.1. Inhibition activity of PLA against some major harmful microorganisms in vitro
4.1.1. Antifungal activity
Inhibition ability of PLA against three strains of fungi including A.niger, A.flavus, P.digitatum
was investigated using the well diffusion method as described by Magnusson and Schnurer
(2001) (detailed in the methodology). Results were shown in the below table 1:
Table 1: Antifungal activity of PLA against three selected fungal strains
Inhibitory
concentration
(mg/ml)
10
12.5
20
25
40
50
Average inhibitory diameter (D-d, mm)
A.flavus
A.niger
P.digitatum
0.0 ± 0.0
0.0 ± 0.0
2.2 ± 0.01
3.6 ± 0.03
5.4 ± 0.04
9.4 ± 0.02
0.0 ± 0.0
0.0 ± 0.0
3.6 ± 0.02
4.1 ± 0.05
6.7 ± 0.04
10.3 ± 0.02
2.3 ± 0.06
3.7 ± 0.03
5.5 ± 0.05
6.6 ± 0.04
7.7 ± 0.07
12.6 ± 0.02
Data are mean ± SE of three repeated samples
Formation of the inhibitory halo surround the well containing PLA liquid shows the antifungal
activity. The higher the diameter of inhibitory halo is, the stronger the inhibitory activity of PLA
is. PLA concentration of 10mg/ml showed the inhibitory effect on P.digitatum (D-d = 2.3 mm),
meanwhile at this concentration there was non-inhibitory effect on two other strains (inhibitory
halos were formed). Concentration of PLA up 20 mg/ml indicated the obviously inhibitory effect
on three test strains. The highest diameter of inhibitory halo against P.digitatum was 12.6 mm at
PLA concentration of 50mg/ml. This showed that P.digitatum was more sensitive to PLA than
the other strains. A.flavus trended to resist PLA when the lowest diameter of inhibitory halo of
9.4 mm was recorded.
The inhibitory ability of PLA against these mold strains was tested by calculating percentage
inhibition of population of test strains, which was indicated in figure 1.
Figure 1 showed that PLA concentration of 20 mg/ml was inhibitable 50.1% of P.digitatum
population, but only 28.3 and 20.1% of A.niger and A.flavus, respectively. Minimal inhibition
concentration of PLA against P.digitatum found in this experiment was 40 mg/ml, and against
A.niger and A.flavus were 50 mg/ml. These results are corresponding to the findings of
Lavermicocca et al. (2000) reported that 98,6% of population A. niger FTDC3227, 86,5% of
population A. flavus FTD3226 and 100% of polulation P. corylophilum IBT6978 were inhibited
by 50 mg/ml of PLA obtained from the fermentation process of L. plantarum 21B.
Figure 1: The inhibitory ability of PLA against 3 mold strains
Data are mean ± SE of three repeated samples
4.1.2. Antibacteria activity:
Results of the inhibitory activity of PLA against three strains of bacteria were shown in table 2.
Table 2: Antibacteria activity of PLA against three selected bacteria strains
Inhibitory
concentration
(mg/ml)
10
12.5
20
25
40
50
Average inhibitory diameter (D-d, mm)
E.coli
S.typhi
V.cholerea
5.3 ± 0.07
8.6 ± 0.05
12.7 ± 0.02
16.3 ± 0.06
18.4 ± 0.05
24.6 ± 0.04
4.5 ± 0.03
6.3 ± 0.02
10.5 ± 0.04
14.3 ± 0.02
16.7 ± 0.06
20.3 ± 0.05
8.7 ± 0.06
11.3 ± 0.04
21.5 ± 0.02
26.0 ± 0.07
28.1 ± 0.04
31.3 ± 0.05
Data are mean ± SE of three repeated samples
Similarly to inhibition assessment against molds, average inhibitory diameters against 3 test
bacteria strains were obviously different at the different concentration of PLA. Among 3 test
bacteria strains, V.cholerea was the most sensitive to PLA with the highest average inhibitory
diameter of 31.3 mm at PLA concentration of 50 mg/ml. Meanwhile, E.coli was the least
sensitive to PLA (the lowest average inhibitory diameter of 24.6 mm). These findings would be
illustrated more clearly by calculating percentage inhibition of test population that was shown in
figure 2 below.
Percentage inhibition (%)
120
100
80
60
E.coli
40
S.typhi
V.cholerea
20
0
6.25
10
12.5
20
25
30
40
PLA concentration (mg/ml)
Figure 2: The inhibitory ability of PLA against 3 bacteria strains
Data are mean ± SE of three repeated samples
Results showed that PLA concentration of 25 mg/ml absolutely inhibited against V.cholerea and
S.typhi (100% of population) while PLA at this level could inhibit 95.6% of E.coli population.
PLA at concentration of 30 mg/ml could inhibit against E.coli absolutely. These prove that E.coli
relatively resist to PLA in comparison with V.cholerea and S.typhi.
4.1.3. Inhibition activity of PLA against some major harmful microorganisms in fresh-cut
pineaple
From the above research results, we carried out the experiment applying PLA as bio-preservative
for fresh-cut pineapple treated in minimal processing (detailed in the methodology part). In these
trials, PLA was used at concentrations range from 10-25 mg/ml.
Decay caused by microorganism is an important factor lead to the decay of the fresh-cut fruit in
preservation process. Microorganisms use sugar in fruit juice as nutrient to upload the mass and
excrete endoenzymes such as cellulase, pectinase, glucose oxidase,… that decompose peel, cell
membrane and ferment sugar to produce acid. This results in slices of fruit to be soft quickly,
nutritious quality of fruit is thereby reduced, and increased acid content induce the fruit with less
attractive taste and odor. In this study, we checked the growth of aerobic bacteria in fresh-cut
pineapple during storage in each experiment. The results were shown in the below figure 3.
The density of the aerobic bacteria in 6 experiments increased with storage time in different
levels. In the control, the total aerobic bacteria of fresh-cut pineapple fold about 3 log units after
5 day storage, and this count over the allowed limit (> 104 cfu/g) after 6 day storage. Meanwhile,
the growth of the aerobic bacteria of fresh-cut pineapple treated by PLA and ascorbic acid
increased slowly, among them the total aerobic bacteria of pineapple treated by PLA
concentration of 20 mg/ml and 25 mg/ml were the lowest and lasted shelf-life of pineapple till 12
day storage.
Figure 3: Total aerobic bacteria of fresh-cut pineapple
Data are mean ± SE of three repeated samples
4.2. Physiological changes of fresh-cut pineapple
Food commerce in markets was assessed basing on objectives such as nutritious, hygiene, and
sensory quality. In which, the sensory quality always plays an important role deciding in
commerce. In our study, the nutritious and sensory qualities of fresh-cut pineapple in
experiments were tested basing on 3 indexes including firmness, color, and total acid content.
4.2.1. Color
Color is a key factor in the sensory quality of fresh-cut pineapple. Change in color of fresh-cut
pineapple during storage was expressed in the figure 4.
Difference between L, a, b values in experiments leaded to the difference of ∆E values. ∆E value
was the highest in the control (∆E increased from 0.0 up to 11.27 after 6 day storage), and the
lowest in the test treated by PLA of 20 mg/ml (∆E was only 6.48 after 12 day storage). This
proved that fresh-cut pineapple treated with PLA could have the shelf-life longer than nontreated one.
Figure 4: Color change of fresh-cut pineapple
Data are mean ± SE of three repeated samples
4.2.2. Firmness
Firmness is also one of the important quality indexes for fresh-cut pineapple. Firmness reduced
during preservation is a serious problem inducing decreased sensory value, and short shelf-life as
well. Firmness of fresh-cut pineapple was determined as described in the method part. Results
were shown in figure 5.
Figure 5: Firmness of fresh-cut pineapple
Data are mean ± SE of three repeated samples
We found that the firmness of fresh-cut pineapple trended to decrease following preservation
time. In the control, the firmness of fresh-cut pineapple reduced rapidly (reduced 29% after 6
days). Meanwhile, the firmness of fresh-cut pineapple treated by PLA and ascorbic acid reduced
slowly, especially sample of pineapple treated by 20mg/ml of PLA had the least reduce level
(only 16% after 12 days). This could be explained that PLA at certain concentrations limited the
physiological changes and inhibited the activity of microorganisms as well as enzymes inducing
flesh of fruit become soft.
4.2.3. Total acidity content
Organic acid occupies a relative quantity in total organic compounds of fresh-cut pineapple.
Organic acid of pineapple is major citric acid that affect on quality of fresh-cut pineapple during
preservation, contributes in producing specific smell of product.
Figure 6 showed that total organic acid content of fresh-cut pineapple in all experiments
gradually decreased. However, this decrease level in the control was faster than that in the tests.
In detail, after 6 day preservation the total organic acid content of fresh-cut pineapple in the
control reduced about 27% compared to that in the test about 26% after 12 day preservation
(treated by 20mg/ml PLA). This demonstrated that fresh-cut pineapple treated by PLA slowed
the decrease process of organic acid content. However, the acid content of pineapple samples
increased at different times. Cause of this could be explained by the fermentation and decay of
microorganism in fresh-cut pineapple occurred at certain period of preservation.
Figure 6: Total organic acid content of fresh-cut pineapple
Data are mean ± SE of three repeated samples
5. Conclusion
Results of the study in vitro showed that PLA had a board antimicrobial spectrum that not only
controlled some harmful fungi, but also inhibited pathogenic bacteria. PLA at concentration of
40-50 mg/ml can absolutely inhibit the fungi such as A.niger, A.flavus, and P.digitatum.
However, the inhibitory activity of PLA against some pathogen bacteria is lower than. 100% of
population of E.coli, S.typhi, and V.cholerea was inhibited by PLA at concentration of 25-30
mg/ml. In vivo study, PLA acts as a bio-preservative in fresh-cut pineapple in minimal
processing when it can ensure the quality of fresh-cut pineapple for 12 day preservation.
Findings of the study demonstrated PLA acts as a prospective bio-preservative compound
applicable in food industry.
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