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Journal of Food Safety ISSN 1745-4565

IMPROVEMENT IN SHELF LIFE AND SAFETY OF

PASTEURIZED PALM SAP (BORASSUS FLABELLIFER LINN.)
BY THE ADDITION OF NISIN
PHISUT NAKNEAN1
Faculty of Agricultural Product Innovation and Technology, Srinakharinwirot University, Sukhumvit 23, Bangkok, 10110, Thailand

1
Corresponding author. ABSTRACT
TEL: 66-2649-5000 ext. 18304,
FAX: 66-2649-5000 ext. 18304; The objective of this study was to improve the quality and safety of pasteurized
EMAIL: n.phisut@yahoo.com palm sap by the addition of various concentrations of nisin. The pasteurization
condition was 75C for 10 min as considered from no growth of pathogens was
Accepted for Publication September 24, 2013
detected while the concentrations of nisin in the range of 0–40 IU/mL were
doi: 10.1111/jfs.12084
selected for addition in palm sap as considered from sensory evaluation. The
initial pH of pasteurized palm sap was 6.6 and the addition of nisin caused a
decrease in the pH to 6.2, 6.0, 5.8 and 5.6 for the samples added 10, 20, 30 and
40 IU/mL, respectively. The initial polyphenol oxidase activity decreased by
approximately 55 and 76% for the sample without nisin added and the sample
added 40 IU/mL nisin, respectively, from the original value measured in fresh
palm sap while the initial invertase activity was not affected by nisin. During
storage, the loss of sucrose, polyphenol content, DPPH-RSA and the increment in
browning intensity were minimized by nisin treatment, particularly at 30–40 IU/
mL. The combination of pasteurization with the addition of nisin (30 IU/mL) and
low temperature storage could preserve palm sap for 10 weeks whereas the control
sample was spoiled within 2 weeks.

PRACTICAL APPLICATIONS
The sale of pasteurized palm sap is a common practice in Thailand and its con-
sumption has increased over the past years. However, pasteurized palm sap has a
short shelf life (approximately 1–2 weeks). In order to control the growth of spoil-
age microorganisms, the use of natural antimicrobial preservatives has been pre-
ferred in the food industry. For this purpose, nisin was preferred in this study.
Although there are many reports on the use of nisin to prolong the shelf life of
food, research on the utilization of nisin in palm sap is limited. According to the
results, the addition of nisin could reduce the pasteurization temperature and
time. In addition, the use of nisin (30 IU/mL) significantly reduced spoilage
microorganism counts and extended the refrigerated shelf life of pasteurized palm
sap for at least 10 weeks. Therefore, the safety of pasteurized palm sap during
storage could be improved by the addition of nisin. This result may be a practical
application for the producer because of the short shelf life of this product.

Office 2010). In addition, palm sap has also been produced

INTRODUCTION
Palm sap is a local delicacy widely consumed by Asians.
Thailand is one of the largest palm sap producers in South-
east Asia. More than 2,000 palm sap producers are located
in Southern, Thailand (Provincial Agricultural Extension
in many Asian countries such as India, Thailand, Myanmar,
Sri Lanka and Cambodia. In Sri Lanka, the palm sap or
palm sugar cottage industry is a major household income
source among palm tree tappers. It provided more than
101,500 and 188,500 Rs which accounted for 53 and 62% of

Journal of Food Safety 33 (2013) 515–525 © 2013 Wiley Periodicals, Inc. 515
IMPROVEMENT THE SHELF LIFE BY NISIN
annual total household income of individual tappers and
group members (Kumarasiri 2010).
Fresh palm sap is a natural product obtained from the
Palmyra palm tree (Borassus flabellifer Linn.). Traditionally,
palm sap is collected manually from each inflorescence of
the Palmyra palm tree. The fresh palm sap is oyster white in
color, has a nearly neutral pH, and is already inherently
sweet-tasting by nature (Ho et al. 2008; Phaichamnan and
Meenume 2009). Kiam wood (Cotylelobium lanceotatum
Carih.) and Payorm wood (Shorea talura Roxb) are gener-
ally added to the receptacle used for collection because it
can delay spoilage in palm sap by reducing microbial popu-
lations as well as keeping the quality of a product (Naknean
et al. 2010; Phaichamnan et al. 2010). However, palm sap
is highly susceptible to spontaneous fermentation, initially
alcoholic, followed by acidic fermentation due to the
collection of palm sap is carried out under nonaseptic
conditions. The dominant microorganisms are yeasts, par-
ticularly Saccharomyces cerevisiae and lactic acid bacteria
(Obahiagbon and Oviasogie 2007). The maintenance,
cleaning and storage of equipment and containers have not
been sanitary. Clean equipment is stored together with
equipment that has not been cleaned. In addition, the palm
sap producers show unsanitary behaviors. These improper
hygienic practices cause the contamination of palm sap,
resulting in promotion of fermentation. Thus, high pro-
cessing temperature and long time were required to pas-
teurize palm sap. In Thailand, the pasteurization of palm
sap is generally performed by heating at boiling tempera-
ture. However, thermal deterioration can take place during
thermal processing and this has effects on the quality of the
product, especially the color, flavor and nutritional values.
In order to reduce the thermal degradation during produc-
tion, it is necessary to minimize the heating temperature
and time. However, the reduction in pasteurization tem-
perature and time generally affects the shelf life of the
product. In an alternative way, natural antimicrobial agents
could be applied and combined with the utilization of
lower heating temperatures and times to reduce the thermal
degradation of the food properties and extend the shelf
life of food. The food industry perceives that consumers
demand convenient, fresh, healthy products free of syn-
thetic preservatives. As an alternative, the antimicrobial
properties of natural compounds, such as nisin, have been
recognized as being safe for use in food preservation (Li
et al. 2012).
Nisin is a heat-stable bacteriocin peptide produced by
certain strains of Lactococcus lactis subsp. lactis, and it is the
only bacteriocin recognized as safe for the food industry
by World Health Organization (De Arauz et al. 2009; Li
et al. 2012). There is no documentation on the use of nisin
as a preservative agent in pasteurized palm sap. The main
objective of this research was to evaluate the possible utili-
516
P. NAKNEAN
zation of nisin as a preservative agent to improve the quality
and safety of pasteurized palm sap during storage under low
temperature (4C).
MATERIALS AND METHODS
Sample Collection and Preparation
Fresh palm sap was obtained from a farm contacted in
Phetchaburi province, Thailand. The palm sap was kept in
an icebox during transportation to the Faculty of Agricul-
tural Product Innovation and Technology, Srinakharinwirot
University. After that, the palm sap was stored immediately
under 4C and then subjected to pasteurization within 1 h
of arrival.
Preparation of Nisin Solution
Nisin solution was prepared according to the method of
Pathanibul et al. (2009). Powdered nisin (106 IU/g; 2.5%
actual nisin) was purchased from Sigma-Aldrich (St. Louis,
MO). The nisin (0.1 g) was mixed with 10 mL of 20 mM
hydrochloric acid (10,000 IU/mL). The stock solution was
immersed in boiling water (100C) for 4.5 min. The stock
solution was then refrigerated until ready for use within a
day of preparation.
Experiment 1 Determination of
Pasteurization Time
The aim of pasteurization is to eliminate pathogenic
microbes. A suitable processing time was assessed to obtain
the minimum pasteurization time. The palm sap samples
with nisin added (10 IU/mL) and no nisin added were
pasteurized (75C) at various times including 3, 5, 7, 10 and
12 min. After pasteurization, each sample was taken out and
immediately cooled in an ice bath for further analysis. The
coliforms/Escherichia coli and Staphylococcus aureus were
evaluated by Petrifilm TM E. coli/Coliform Count Plate
and Petrifilm 3 M Rapid St. aureus count plates. This was
done according to the manufacturer’s recommendations
and expressed in term of positive (+) detection or negative
(−) detection. The minimum pasteurization time was iden-
tified as the state where no pathogen was detected.
Experiment 2 Determination of
Suitable Nisin Concentration Range
by Sensory Evaluation
Initially, the stock nisin solution was added to palm sap with
to a final concentration of 10, 20, 30, 40 and 50 IU/mL sap.
After that, each sample was pasteurized at 75C, then imme-
diately cooled in an ice bath for further sensory evaluation.
Journal of Food Safety 33 (2013) 515–525 © 2013 Wiley Periodicals, Inc.
P. NAKNEAN
The pasteurization time was obtained from the experiment
1. The pasteurized palm sap without nisin added was used
as a control sample. After production, sensory assessment
was done for each sample. The consumers scored the
acceptability of the samples using a 9-point hedonic
category scale (where 1 = dislike extremely and 9 = like
extremely), rating flavor, taste and overall acceptability.
Thirty untrained panelists were asked to rate each sensory
attribute. The mean score of the overall acceptability (≥5)
was used as the criterion to select a sample for further study.
Experiment 3 Study of the Quality Changes
of Pasteurized Palm Sap with Added Nisin
during Storage
Stock nisin solution was added to the palm sap at various
concentrations as obtained from the experiment 2 that
considered by the mean score of overall acceptability (≥5).
After that, each sample was pasteurized according to the
procedure described in experiment 2 and then immediately
packed in opaque polyethylene containers and subsequently
cooled in ice water for 5 min. The pasteurized palm sap
without nisin added was used as a control sample. After-
ward, each selected sample was monitored for the quality
changes during storage under low temperature (approxi-
mately 10C) at 2 weeks interval.
Determination of Microbial Load
Total viable count (TVC) and lactic acid bacteria (LAB)
count were determined using plate count agar and De Man
Rogasa and Sharpe agar according to the method of AOAC
(2000). Spread plating on Potato Dextrose Agar acidified
with 10 g/100 g tartaric acid (Merck KGaA, Darmstadt,
Germany) was performed for mold and yeast counts
(MYC).
Chemical Properties Analysis
The pH value was measured at ambient temperature with a
pH meter (Satorious, Goettingen, Germany) which was cali-
brated at pH 4.0 and 7.0. The total acidity was analyzed by
titration using 0.01 N NaOH and phenolphthalein indicator
solution. The result was expressed as a percentage of citric
acid. The total soluble solid (TSS) was determined using a
hand refractometer. The activities of polyphenol oxidase
(PPO) and invertase were analyzed as described by Cano
et al. (1997) and Mao et al. (2007), respectively. The types
and concentrations of sugar were determined using a high
performance liquid chromatography (Agilent 1100 series,
Hewlett Packard Strasse, Waldbronn, Germany) equipped
with a refractive index detector (Stuckel and Low 1996). The
polyphenol content was analyzed according to the method
Journal of Food Safety 33 (2013) 515–525 © 2013 Wiley Periodicals, Inc.
IMPROVEMENT THE SHELF LIFE BY NISIN
of Balange and Benjakul (2009). Gallic acid was used as a
standard for polyphenol content. The 2,2-Diphenyl-1-
picrylhydrazyl (DPPH) radical-scavenging activity was
determined by DPPH assay, as described by Binson et al.
(2008), and expressed in term of μmol Trolox per mL. A
standard curve was prepared using Trolox.
Physical Properties Measurement
The clarity of the sample was estimated by measuring
the transmittance at 650 nm using a spectrophotometer
(Shimadzu, Kyoto, Japan) and expressed in term of percent-
age. The browning intensity was determined by monitoring
the absorbance at 420 nm.
Statistical Analysis
All analysis and measurements were performed in triplicate.
The experimental design for physical and chemical proper-
ties was a completely randomized design. The experimental
design for sensory evaluation was a randomized complete
block design. Data was subjected to analysis of variance. The
analysis was performed using an SPSS package (SPSS, Inc.,
Chicago, IL).
RESULTS AND DISCUSSION
Experiment 1 Determination of
Pasteurization Time
Currently, pasteurization is the mode commonly used for
heat treatment of palm sap. Pasteurization is the applica-
tion of heat to destroy human pathogens in food. During
the harvesting process, the contamination of microbes,
especially pathogenic microorganisms, was occurred. The
pathogenic microbes such as E. coli and St. aureus were gen-
erally found in the sap collected from palm trees such as
Palmyra palm and coconut palm (Atputharajah et al. 1986;
Karamoko et al. 2012; Santiago-Urbina et al. 2013). E. coli
and St. aureus were used as the indicators for evaluating the
efficiency of the pasteurization process in this study. E. coli
is a common inhabitant of the intestinal tract of humans
and animals and is considered an indicator of fecal con-
tamination in food. E. coli can be easily disseminated in
different ecosystems through the food chain. St. aureus is
an important foodborne pathogen because of its ability to
produce a wide range of extracellular protein toxins and
virulence factors that contribute to the pathogenicity of the
organism. Of particular relevance to the food processing
industry is the ability of some St. aureus strains to produce
heat stable enterotoxins that cause staphylococcal food poi-
soning. This ranks as one of the most prevalent causes of
gastroenteritis worldwide (Mhone et al. 2011).
517
IMPROVEMENT THE SHELF LIFE BY NISIN
TABLE 1. MICROBIAL LOADS OF PALM SAP PASTEURIZED AT
VARIOUS TIMES
Treatment Pasteurization time E. coli St. aureus
Control 3 + + of nisin would increase the sour taste due to the acid used to
5 + + dissolve nisin (Koiso 2010). Thus, the samples with added
7 + − nisin in the range of 0–40 IU/mL were selected for further
10 − −
12 − −
Nisin 10 IU/mL 3 + +
5 + −
7 − − of Pasteurized Palm Sap with Added Nisin
10 − −
12 − −
Note: +, positive detection; −, negative detection.
The inactivations of Gram-positive St. aureus and Gram-
negative E. coli by thermal pasteurization and the addition
of nisin in palm sap are depicted in Table 1. It was found
that the complete inhibition of E. coli and St. aureus was
detected when the pasteurization time reached 10 min.
The combination between nisin and thermal pasteurization
could reduce the pasteurization time to 7 min. St. aureus
was inactivated within 5 min of the pasteurization process
with the addition of nisin while a positive detection in
E. coli was still found. Nisin exhibits a broad spectrum of
inhibitory activity against Gram-positive bacteria including
their spore forms (Sanlibaba et al. 2009). However, it is not
generally active against Gram-negative bacteria due to their
outer membrane being a barrier to permeability. Further-
more, the amount of each initial pathogen might affect a
reduction in each microbe after pasteurization. According to
the result, the processing time of 10 min was selected for the
pasteurization of palm sap as considered by no pathogen
was then detected in the sample.
Experiment 2 Determination of
Suitable Nisin Concentration Range by
Sensory Evaluation
The influence of the addition of nisin on the sensory
attributes in comparison with the control sample is shown
in Table 2. All sensory attribute scores tended to decrease
with increasing concentrations of nisin. Significant changes
on all sensory attributes between the control sample and
the samples with 10 and 20 IU/mL nisin added were not
observed by the panelists. Significant differences in all the
sensory attributes between the control sample and the
nisin-treated samples were detected when the concentration
of nisin reached 30 IU/mL. This suggested that a high con-
centration of nisin caused an increase in sourness. Also,
concentrations >40 IU/mL were scored as unacceptable for
the sensory panel due to an increase in sourness. Generally,
the addition of nisin could extend the shelf life of food
518
P. NAKNEAN
without changing the taste of flavor. However, nisin is stable
under acidic conditions (at low pH levels) and normally
dissolved in acid before adding in food. High concentration
storage study.
Experiment 3 Study of the Quality Changes
during Storage
Changes in microbial loads of pasteurized palm
sap during storage. The microbiological quality of
pasteurized palm sap treated with nisin was determined
by monitoring the TVC, MYC and LAB counts in the
sample. For the control sample, the TVC was found to be
1.65 log cfu/mL after pasteurization while the LAB count
and MYC were not detected. The TVC, MYC and LAB count
of all the nisin-treated samples were not detected after
pasteurization. The results suggested that the combination
of nisin and pasteurization was effective in reducing the
microbial growth. In addition, nisin concentration affected
the microbial loads of the pasteurized palm sap during
storage, resulting in a positive effect on the extension of the
shelf life. There was an increase in the TVC, MYC and LAB
count of all samples during storage (Table 3). This result
indicated that some spoilage microorganisms may survive
and develop, leading to the limiting of the shelf life. After
treatment with pasteurization and nisin, the probable pres-
ence of injured cells or a fraction but not cultivable micro-
organisms in a sort of dormant state which, once restored,
could started growing again during the storage period
(Spilimbergo et al. 2013). Lower TVC, MYC and LAB count
was found in all nisin-treated samples during storage
compared to the control sample. Nisin (up to 30 IU/mL)
reduced the microbial loads with no further reduction
when using concentrations from 30 up to 40 IU/mL. The
Thailand Community Product Standard (2003) indicates
that TVC and MYC in pasteurized palm sap should not be
TABLE 2. SENSORY VALUES OF PASTEURIZED PALM SAP ADDED
WITH VARIOUS CONCENTRATION OF NISIN
Treatments/Sensory Overall
attributes Flavor Taste acceptability
Control 7.42a 7.14a 7.22a
Nisin 10 IU/mL 7.21a 7.34a 7.25a
Nisin 20 IU/mL 7.05a 7.26a 7.20a
Nisin 30 IU/mL 6.54b 6.23b 6.31b
Nisin 40 IU/mL 5.45c 5.61c 5.24c
Nisin 50 IU/mL 4.10d 3.47d 3.86d
Note: Means within the same column with different letters are signifi-
cantly different (P ≤ 0.05).
Journal of Food Safety 33 (2013) 515–525 © 2013 Wiley Periodicals, Inc.
P. NAKNEAN IMPROVEMENT THE SHELF LIFE BY NISIN

TABLE 3. CHANGES IN MICROBIAL LOADS

Storage times (week)
(log cfu/mL) OF PASTEURIZED PALM SAP Nisin concentration
DURING STORAGE Microorganisms (IU/mL) 0 2 4 6 8 10
TVC (log cfu/mL) 0 1.65 2.28 3.04 4.35 5.71 6.85
10 nd 1.75 2.19 2.65 3.33 4.72
20 nd nd 1.81 2.18 2.67 3.81
30 nd nd nd nd 2.18 2.67
40 nd nd nd nd 2.09 2.68
M&Y (log cfu/mL) 0 nd 1.75 2.54 3.49 5.39 8.17
10 nd nd 1.61 1.93 2.49 4.73
20 nd nd nd 1.65 1.96 3.63
30 nd nd nd nd 1.63 1.98
40 nd nd nd nd 1.72 1.95
LAB (log cfu/mL) 0 1.54 1.79 2.59 3.33 4.55 5.71
10 nd nd 2.21 2.63 3.11 4.54
20 nd nd nd 2.27 2.65 3.68
30 nd nd nd nd 2.17 2.66
40 nd nd nd nd 2.13 2.62

Note: LAB, lactic acid bacteria; M&Y, mold and yeast; nd, not detected; TVC, total viable count.

more than 5 × 102 cfu/mL and 100 cfu/mL, respectively.
According to these criteria, the shelf life of the samples as
considered from microbiological standard was approxi-
mately 2, 6 and 8 weeks for the control sample and sample
added 10 and 20 IU/mL nisin, respectively, while the
samples added 30 and 40 IU/mL nisin were assured for at
least 10 weeks of storage. The utilization of nisin as an anti-
microbial agent was reported in cheese, milk, dairy prod-
ucts, canned foods and hot baked flour products (Zapico
et al. 1998; Samelis et al. 2003; Black et al. 2005). Some
attempts to use it in juices have been reported (Walker and
Phillips 2008; Pathanibul et al. 2009; Li et al. 2012; Zhao
et al. 2013). Nisin exhibited antimicrobial activity toward a
wide range of Gram-positive bacteria, especially foodborne
pathogens (Li et al. 2012). The permeabilization of cell
membrane and inhibition of cell wall synthesis are the two
kill mechanisms acted by nisin (Pathanibul et al. 2009). The
C-terminal region of nisin binded to the cytoplasmic mem-
brane of vegetative cells, penetrated into the lipid phase
of the membrane, and formed the pores, which allow the
efflux of potassium ions, Adenosine-5’-triphosphate and
amino acids. This resulted in a dissipation of the proton
motive force and hence cell death (Zapico et al. 1998; De
Arauz et al. 2009; Sanlibaba et al. 2009). Therefore, the
addition of nisin could be use as an alternative method to
improve the safety and increase the shelf life of pasteurized
palm sap.
Change in pH and Total Acidity of Pasteurized Palm
Sap during Storage. The change in the pH and total
acidity of pasteurized palm sap during storage is shown in
Table 4. Initially, all nisin-treated samples had a lower pH
and higher total acidity than the control sample and the pH
decreased significantly (P < 0.05) with increasing nisin con-
centration. This effect could be attributed to the low pH of
the nisin solution (Zhao et al. 2013). The pH of the samples
with 30 and 40 IU/mL of nisin added tended to slightly
decrease throughout storage times for 10 weeks. A continu-
ous decrease in the pH and an increase in total acidity were
observed for the control sample and the samples with 10
and 20 IU/mL of nisin added during storage. This result
was related to the acid production caused by the activity
of microorganisms. Hence, the concentration of nisin in the
range of 30–40 IU/mL could decrease the growth of micro-
organism in pasteurized palm sap during storage. This
resulted in the reduction of acid formation.
Change in TSS of Pasteurized Palm Sap during
Storage. TSS decreased during storage from an initial
value of approximately 18.00°Brix to 11.50, 14.00, 15.00,
17.00 and 17.20°Brix for the control sample and the samples
added 10, 20, 30 and 40 IU/mL nisin, respectively. The
fermentation of the sugar was responsible for the reduction
in TSS of palm sap during storage. The samples treated with
nisin in the range of 30–40 IU/mL maintained a high TSS
throughout the storage for 10 weeks. This was because of
the antimicrobial properties of nisin.
Change in the PPO Activity of Pasteurized Palm
Sap during Storage. According to previous works
(Loetkitsomboon 2004; Taipaiboon 2004), the brown color
of pasteurized palm sap is related to the oxidation of phe-
nolic compounds catalyzed by PPO activity. PPO (tyrosi-
nase, E.C. 1.14.18.1) is an enzyme containing copper, which
catalyzes two distinct reactions involving molecular oxygen
with various phenolic substrates (Jittanit et al. 2011). It
was found that PPO activity was partially inactivated after
processing as shown by the reduction of PPO activity in all

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IMPROVEMENT THE SHELF LIFE BY NISIN P. NAKNEAN

TABLE 4. CHANGES IN pH, TOTAL ACIDITY, RESIDUAL PPO ACTIVITY AND INVERTASE ACTIVITY OF PASTEURIZED PALM SAP DURING STORAGE

Storage times (week)

Nisin concentration
Parameters (IU/mL) 0 2 4 6 8 10
aA bB cC cD dE
pH 0 6.6 5.8 4.5 3.8 3.6 3.5dF
10 6.2bA 6.0aA 5.7abB 5.2bC 4.5cD 4.0cE
20 6.0cA 6.0aA 5.9aA 5.8aAB 5.4bC 4.8bD
30 5.8dA 5.8bA 5.8aA 5.7aAB 5.7aAB 5.6aB
40 5.6eA 5.6cA 5.6bA 5.6aA 5.5aA 5.5aA
Total acidity (%) 0 0.038dE 0.045aE 0.060aD 0.090aC 0.180aB 0.290aA
10 0.040cdD 0.044abCD 0.048bC 0.055bC 0.100bB 0.150bA
20 0.042bcC 0.042bC 0.048bB 0.048cdB 0.050cB 0.104cA
30 0.044abC 0.044abC 0.044cC 0.047dB 0.050cB 0.055dA
40 0.046aC 0.046aC 0.046bcC 0.050cB 0.050cB 0.055dA
Residual PPO activity (%) 0 55.23aA 49.57aB 36.42aC 9.12cD 5.78dE 3.21cF
10 52.25bA 46.21bB 40.14bC 30.63bD 22.34cE 10.65bF
20 48.63cB 43.14cC 35.75cD 60.62aA 24.11bE 13.36bF
30 46.21dB 40.63dC 35.21cD 60.62aA 27.52aE 24.21aF
40 42.75eA 36.25eB 35.51cB 28.22bC 25.14bD 23.45aD
Invertase activity (unit/min/g) 0 24.14aB 22.63aC 19.98aD 21.36aC 24.96aB 28.14aA
10 24.53aA 22.96aB 17.93aC 16.32bC 18.96bC 22.68bB
20 23.06aA 21.36aB 19.52aCD 18.69aD 17.98bcD 20.36bBC
30 24.68aA 22.12aB 18.52aC 17.22abC 16.93cC 16.56cC
40 24.42aA 22.28aB 18.84aC 16.23bD 16.21cD 15.24cD

Note: Different superscript letters (lowercase) in each column mean statistically significant differences (P < 0.05).
Different superscript letters (capital letter) in each row mean statistically significant differences (P < 0.05).

samples. The PPO activity of the control sample decreased
by approximately 45% from the original value measured in
fresh palm sap (Table 4). With regard to the effect of adding
nisin, a lower initial residual PPO activity was observed in
all the nisin-treated samples when compared to the control
sample (P < 0.05). An increase in the concentration of nisin
caused a decrease in initial PPO activity. The reduction in
PPO activity seemed to correspond to the decrease in the
pH which was affected by the low pH of the nisin solution.
During storage, a gradual decrease in PPO activity was
observed in all samples. The residual PPO activity of the
control sample was approximately 3.21% after 10 weeks of
storage, followed by 10.65, 13.36, 24.21 and 23.45% for the
samples with an added 10, 20, 30 and 40 IU/mL of nisin,
respectively. The reduction in the PPO activity of pasteur-
ized palm sap suggested that the PPO activity was sensitive
to an acidic pH and hence more prone to loss of PPO activ-
ity. The optimum pH for PPO activity is normally around
neutral and ranged from 5 to 7 (Sapers 1993). Thus an
increase in acidity as affected by the activity of microbes
could reduce the PPO activity in palm sap, particularly in
the control sample and the samples added 10 and 20 IU/mL
nisin. The reduction in PPO activity in juices during storage
was also found in carrot juice and apple juice (Park et al.
2002; Gui et al. 2006).
Change in Invertase Activity of Pasteurized Palm
Sap during Storage. The presence of invertase in palm
sap was because it occurs naturally and is also synthesized
by microorganisms (Taipaiboon 2004). The initial invertase
activity of fresh palm sap was 55.21 × 10−3 unit/min/g.
A decrease in invertase activity was observed in all samples
after pasteurization. This could be because of the denatur-
ation of the invertase enzyme caused by thermal processes.
Regarding the effect of the adding of nisin, an increase in
the nisin concentration did not influence the initial inver-
tase activity (P ≥ 0.05). The change in invertase activity
of the pasteurized palm sap during storage is demonstrated
in Table 4. A continuous decrease in invertase activity
was observed during storage in the samples with 30 and
40 IU/mL nisin added. On the other hand, the invertase
activity decreased in the first 4, 6 and 8 weeks of storage and
increased thereafter for the control sample and the samples
with 10 and 20 IU/mL nisin added. The increase in invertase
activity was probably due to some species of yeast secreting
the invertase enzyme. It is generally known that the primary
sources of invertase are yeasts such as Sa. cerevisiae,
Sa. carlsbergensis and fungi such as Aspergillus oryzae and
A. niger (Singh et al. 2006). This finding corresponded with
the findings for microbial loads. In contrast to PPO, inver-
tase exhibits relatively high activity over a broad range of
pH (3.5–5.5) (Amaya-Delgado et al. 2006; Andjelkovic et al.
2010), thus high acid condition created by microorganisms
does not greatly inhibit the invertase activity. This study
indicated that there was considerable activity of invertase
enzyme retained in pasteurized palm sap and this could play
an important role in sucrose degradation, causing an
increase in the reducing sugar.

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P. NAKNEAN IMPROVEMENT THE SHELF LIFE BY NISIN

the highest sucrose content being detected at the end of

Change in Type and Concentration of Sugar of
Pasteurized Palm Sap during Storage. Changes in the
sucrose, glucose and fructose content during the storage of
pasteurized palm sap are presented in Table 5. Initially, the
addition of various concentrations of nisin influenced the
sucrose content (P < 0.05). An increase in nisin concentra-
tion caused a decrease in sucrose content, suggesting that
the low pH of the nisin solution might promote sucrose
inversion during pasteurization. The results indicated that
as the storage time increased, the sucrose content of the
samples with 30 and 40 IU/mL nisin added decreased
gradually. However, the control sample and the samples
with 10 and 20 IU/mL nisin added showed a sharp decrease
in sucrose content during storage. The reduction in sucrose
content correlated well with the increase in the glucose and
fructose content during storage. The formation of glucose
and fructose was mainly affected by sucrose inversion.
Similar results were also observed in the storage of apple
juice concentrate (Babsky et al. 1986), banana juice (Yousaf
et al. 2010) and sugar cane juice (Yusof et al. 2000; Singh
et al. 2006).
Among all the treatments, the control sample contained
the highest reducing sugar and lowest sucrose content at
the end of storage. The sucrose inversion was normally
governed by the action of invertase and the high acidic
condition. From the results, the greatest pH reduction was
observed in the control sample. The high invertase activity
found in the control sample favored sucrose inversion
which is responsible for the loss of sucrose. Therefore, the
addition of high levels of nisin, particularly at 30–40 IU/
mL, could minimize the loss of sucrose. This was clear from
storage in the samples with 30 and 40 IU/mL nisin added.
The use of nisin could retard the growth of microbes, result-
ing in the pH of the sap being maintained as noted above.
This result was concomitant with the lower invertase activ-
ity during storage in the sample with high concentrations
of nisin (30–40 IU/mL) compared to the control sample
and the samples containing low concentrations of nisin
(10–20 IU/mL).
Change in Polyphenol Content of Pasteurized Palm
Sap during Storage. Polyphenol compounds, a large
group of aromatic phenols, are present in plants. They are
involved in enzymatic browning, as well as the forming of
haze in liquids. This clouding is caused by the condensation
of the polyphenol together or in complex formation with
proteins. That is why the polyphenol content needs to be
reduced below a certain level in order to prevent clouding.
However, phenolics have been shown to have antioxidative
properties. The antimutagenic and anticarcinogenic effects
of phenolics have also been demonstrated (Balasundram
et al. 2006). The presence of polyphenol compounds in
palm sap is because they occur naturally and are dissolved
from Kiam wood or Payorm wood when the sap is collected
(Phaichamnan et al. 2010). Changes in the polyphenol
content of pasteurized palm sap during storage are shown
in Fig. 1a. There were no significant differences in the initial
polyphenol content among the samples (P ≥ 0.05). A sig-
nificant decrease in the polyphenol content was found for
all samples during storage (P < 0.05). At the end of storage,
the lowest polyphenol content was observed in the control

TABLE 5. CHANGES IN SUGAR CONTENT OF PASTEURIZED PALM SAP DURING STORAGE

Storage time (week)

Nisin concentration
Parameters (IU/mL) 0 2 4 6 8 10
aA aA aB bC bD
Sucrose content (%) 0 13.50 12.46 10.05 6.23 3.12 1.04cE
10 13.14aA 12.80aA 10.52aB 7.40abC 4.23bD 2.63bcE
20 12.84abA 12.02aA 10.53aB 8.79aC 6.53aD 3.79bE
30 11.50bA 10.20bAB 9.50aBC 8.52aCD 7.34aDE 6.89aE
40 11.04bA 10.43bAB 9.64aBC 8.21aCB 7.38aDE 6.55aE
Glucose content (%) 0 1.93aB 2.15aB 2.89aB 3.25aAB 4.84aA 5.87aA
10 1.89aB 1.95aB 2.46aB 2.95aB 3.83aAB 4.61aA
20 1.97aC 1.99aC 2.23aC 2.39aC 3.64aB 5.64aA
30 1.94aC 1.98aC 2.31aC 2.46aC 3.55aB 4.25aA
40 1.86aB 1.98aB 2.35aB 2.48aB 2.50bB 4.40aA
Fructose content (%) 0 1.75aD 2.03aCD 2.63aC 3.14aB 4.75aA 5.72aA
10 1.76aC 1.89aC 2.35aBC 3.05aB 3.62abB 4.51aA
20 1.70aC 1.92aC 2.21aC 2.43aC 3.62abB 5.51aA
30 1.79aB 1.98aB 2.26aB 2.41aB 3.24abB 4.20aA
40 1.72aB 1.89aB 2.25aB 2.43aB 2.51bB 4.32aA

Note: Different superscript letters (lowercase) in each column mean statistically significant differences (P < 0.05).
Different superscript letters (capital letter) in each row mean statistically significant differences (P < 0.05).

Journal of Food Safety 33 (2013) 515–525 © 2013 Wiley Periodicals, Inc. 521
IMPROVEMENT THE SHELF LIFE BY NISIN
1.2
1
Polyphenol content (µg/ml)
0.8
0.6
0.4
0.2
Control 20 IU
10 IU 30 IU
0 0
0 2 4 6
Storage time (week)
FIG. 1. CHANGE IN POLYPHENOL CONTENT (A) AND DPPH RADICAL SCAVENGING ACTIVITY (B) OF PASTEURIZED PALM SAP DURING STORAGE AS
AFFECTED BY THE ADDITION OF NISIN
sample. Lee et al. (2007) also found a decrease in polyphe-
nol content in banana juice during storage. The decrease in
the total polyphenol content could be due to the oxidation
degradation of phenolic compounds and the polymeriza-
tion of phenolic compounds with proteins (Phaichamnan et
al. 2010). In addition, the PPO was normally considered to
be the source of the main enzymes responsible for the decay
of phenols in processed juice and their derived foods (Cao
et al. 2012; Keenan et al. 2012).
Change in DPPH Radical Scavenging Activity of
Pasteurized Palm Sap during Storage. The free
radical scavenging of the sap was determined by a DPPH
free radical scavenging assay. DPPH is a stable free radical
with a maximum absorption at 517 nm that accepts an elec-
tron or hydrogen atom in order to become a stable diamag-
netic molecule. In the presence of a substance capable of
donating a hydrogen atom, its free radical nature is lost and
hence the reduction in the DPPH radical activity was deter-
mined by the decrease in its absorbance at 517 nm. This
assay is generally used to evaluate the free radical scaveng-
ing potential of an antioxidant molecule. The change in the
DPPH radical scavenging activity of pasteurized palm sap
during storage is demonstrated in Fig. 1b. Initially, the addi-
tion of nisin did not affect the DPPH radical scavenging
activity. In addition, a continuous decrease in the DPPH
radical scavenging activity was found in all samples during
storage. The lowest DPPH radical scavenging activity was
detected in the control sample at the end of storage. The
reduction in the DPPH radical scavenging activity during
522
P. NAKNEAN
a 16 b
DPPH radical scavenging activity
12
8
4
Control 20 IU 40 IU
40 IU
10 IU 30 IU
8 10 0 2 4 6 8 10
Storage time (week)
storage could be attributed to the continuous precipitation
of phenolic compounds. This was due to the formation of
haze and the loss of polyphenol content which was affected
by the PPO activity.
Change in Clarity of Pasteurized Palm Sap during
Storage. The color and clarity of juices are related to
consumer acceptability. The turbidity of pasteurized palm
sap during storage was normally caused by the presence
of active hazes formed by phenolic-protein complexes
(Phaichamnan et al. 2010). Changes in clarity during
storage are shown in Fig. 2a and expressed in term of trans-
mittance value. A continuous increase in the transmittance
values was observed in all samples, except the control
sample and the sample with 10 and 20 IU/mL nisin added,
up to the end of storage. During storage, the undissolved
particles caused by the interaction between protein and
polyphenol compounds were developed as sediment, result-
ing in increments in clarity (Kermasha et al. 1995; Siebert
et al. 1996). The increase in clarity reached a maximum
after 4, 6 and 8 weeks and then tended to decrease until the
end of storage for the control sample and the samples with
10 and 20 IU/mL nisin added, respectively. Results sug-
gested that, the suspended solid such as yeast and bacteria,
giving a milky white appearance, might be developed as evi-
denced by the increase in microbial load during storage.
Change in Browning Intensity of Pasteurized Palm
Sap during Storage. Color plays an important role in
the appearance and acceptability of foods. The natural color
Journal of Food Safety 33 (2013) 515–525 © 2013 Wiley Periodicals, Inc.
P. NAKNEAN IMPROVEMENT THE SHELF LIFE BY NISIN

2
Control 20 IU 40 IU
60 a b 10 IU 30 IU

1.8
50
Transmittance (%)

Browning intensity
40
1.6

30

1.4
20
Control 20 IU 40 IU

10 IU 30 IU

10 1.2
0 2 4 6 8 10 0 2 4 6 8 10
Storage time (week) Storage time (week)

FIG. 2. CHANGE IN CLARITY (A) AND BROWNING INTENSITY (B) OF PASTEURIZED PALM SAP DURING STORAGE AS AFFECTED BY THE ADDITION
OF NISIN

of palm sap is normally a translucent white or pale brown
shade. However, an increase in brown shading was observed
during harvesting due to an enzymatic browning reaction
and the pigment of wood dissolved in the palm sap
(Loetkitsomboon 2004; Taipaiboon 2004). In addition, a
Maillard reaction could be developed during pasteurization,
leading to increments in the brown color. Moreover, the
PPO activity still remained after mild pasteurization as
noted above. It was found that the addition of nisin did not
affect the initial browning intensity of the pasteurized palm
sap. During storage, the browning intensity of all samples
increased with the storage time (Fig. 2b). The results suggest
that both enzymatic browning and a Maillard reaction took
place during storage. A slow increase in the browning inten-
sity was found in all nisin-treated samples during storage.
This result might be because the addition of nisin caused a
decrease in pH, resulting in less appropriate conditions for
PPO activity. In addition, a marked increase in the brown-
ing intensity of the control sample was observed within the
first 3 weeks of storage, suggesting that there was high PPO
activity in the control sample. Subsequently, a slow increase
was found until the end of storage. This slow increase could
be because the PPO activity was inhibited by the acidic
conditions caused by the fermentation of sugar.
CONCLUSION
This study demonstrated the possible use of nisin as a
natural preservative ingredient to extend the shelf life of
pasteurized palm sap. Initially, the addition of nisin caused a
decrease in pH, PPO activity, sucrose content and increase
in total acidity of pasteurized palm sap while the invertase
activity, clarity and browning intensity was not affected.
In addition, the decrease in pH and sucrose content and
increase in browning intensity were minimized by the
addition of nisin during storage, particularly the samples
with 30 and 40 IU/mL of nisin added. Moreover, palm sap
treated by using a combination of added nisin (30 IU/mL)
and mild pasteurization (75C, 10 min) guaranteed at least
10 weeks of storage, whereas the microbial load was reduced
below the standard level. Future research could focus on the
study of the market potential of the storage life of pasteur-
ized palm sap stored under an ambient temperature for
easier transportation.
ACKNOWLEDGMENT
The financial support provided by Srinakharinwirot
University is appreciated.
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