International Food Research Journal 25(3): 928-935 (June 2018)

Journal homepage: http://www.ifrj.upm.edu.my

Effect of salam [Syzygium polyanthum (Wigt) Walp.] leaves extract on the

microorganism population in chicken meat and shrimp and their sensory
1
Ramli, S., 1Lim, L.Y., 1Samsudin, N.I.P. and 1,2*Rukayadi, Y.

Department of Food Science, Faculty of Food Science and Technology, Universiti Putra
1

Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

2
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM
Serdang, Selangor Darul Ehsan, Malaysia

Article history Abstract

Received: 30 January 2017 Recently, there has been an increasing demand and interest in developing plant extracts as
Received in revised form: natural food sanitizer, owing to their antimicrobial properties. Hence, the aim of this study
5 March 2017 was to investigate the effect of salam (Syzygium polyanthum L.) leaves methanolics extract
Accepted: 6 March 2017
on the number of microflora on chicken meat and shrimp. Salam leaves extract at different
concentrations (0.0%, 0.1%, 1.00%) and exposure times (5, and 10 min) used to treat chicken
meat and shrimp by using dilution method. Result showed that the total plate count and
Keywords Staphylococcus aureus had been detected in untreated chicken and shrimp samples with 6.66 ±
0.12, 8.66 ± 0.15 and 7.25 ± 0.21, 6.54 ± 0.21, respectively. However, there was no Escherichia
Salam leaves
coli, Salmonella spp. and Vibrio cholerae detected in both samples. The number of total plate
Syzygium polyanthum L
Foodborne pathogens count (TPC) and S. aureus in chicken meat and shrimp were starting to reduce significantly at
Soaking treatment 0.01% concentration of salam leaves extract for 5 minutes of exposure time compared to initial
count. There was no significantly different between exposure times. The highest reduction in
number of microorganism population was at treatment with 1.0% extract for 10 min where
TPC was reduced from 6.66 ± 0.12 to 0.00 ± 0.00 log10 CFU/ml, and from 8.66 ± 0.15 to 4.88 ±
0.00 log10 CFU/ml in shrimp while S. aureus reduced from 7.25 ± 0.21 to 3.88 ± 0.01 and from
6.54 ± 0.21 to 4.92 ± 0.04 in chicken and shrimp, respectively. For the sensory acceptability,
overall acceptability were accepted by panellists until treatment 0.10% for 5 min and 10 min
of soaking time. In conclusion, salam leaves extract might be developed as natural sanitizer for
rinsing raw food materials such as chicken meat and shrimp.
© All Rights Reserved

Introduction most prominent food-borne pathogens which lead

to outbreaks of illnesses through consumption of the
Food contamination has become a serious contaminated fresh products. On the other hand, most
concern with the increasing number of outbreaks of of the Vibrio spp. are pathogenic for mankind and
food-borne illnesses (Rane, 2011). The alleviating usually responsible for causing alimentary infections
cases in food-borne illnesses have highlighted the in countries with warm coastal waters, where fish
importance of microbiological control in the food and shellfish are consumed raw or lightly cooked. It
industry. Microbial contamination in food that leads is important to point out that these pathogens have
to food-borne illnesses may be contributed by various been isolated in larger number from fish and shellfish
factors including inadequate food hygiene, place (Slavika et al., 2002). Wilfred et al. (2014), indicated
of preparation, cooking or processing procedure, the consumption of poultry meat has been associated
processing or serving utensils, raw materials, and with the incidence of outbreaks of foodborne illness.
time and temperature abuse (Annan-Prah et al., In addition, study by Akbar & Anal (2013), showed
2011). that most of S. aureus species easily be found in
According to Scientific Status Summary (2013), poultry meat.
types of food-borne bacterial pathogens commonly In order to resolve the problems of microbial
detected in raw meat and seafood are Salmonella contamination and spoilage in food, various food
sp., Staphylococcus aureus, and Escherichia coli. preservation strategies have been developed and
Besides, Vibrio cholerae is also commonly found applied to control the growth and propagation of
in seafood. Aside from this, Abadias et al. (2008) microorganisms including chilling, freezing, water
reported that Salmonella sp. and E. coli are the activity reduction, nutrient restriction, pasteurization

*Corresponding author.
Email: [email protected]
929 Ramli et al./IFRJ 25(3): 928-935

or synthetic antimicrobials (Davidson, 1993). Materials and Methods

However, most of these procedures may result in the
loss of organoleptic properties of foods and reduce Dried salam leaves
consumer acceptability (Pradeep, 2011). Besides Samples, the dried salam leaves (Syzygium
that, nowadays consumers also put a lot of concern in polyanthum L.), were purchased from herbal market,
the use of synthetic additives due to its adverse health Pasar Baru, Bandung, Indonesia. The samples were
effect. Therefore, instead of synthetic antimicrobials, identified and deposited in Laboratory of Natural
natural antimicrobials are more preferable by Products, Institute of Bioscience (IBS), Universiti
consumers and thus natural antimicrobials such Putra Malaysia (UPM), Serdang, Selangor, Malaysia.
as organic acids, essential oils, plant extracts, and The dried salam leaves were packed in sealed plastic
bacteriocins become considerable alternatives to bags and kept at room temperature.
ensure food safety (Cleveland et al., 2001; Burt,
2003). The antimicrobial activity of plant extracts Chicken and shrimp
are studied and found to be having phytochemical Raw chicken meat (boneless fillets) and raw
constituents such as phenolic compounds, protein- shrimp (white leg type) were purchased from Tesco
like compounds, flavonoids and tannins (Pradeep, Hypermarket, IOI City Mall, Putrajaya, Malaysia.
2011). Both samples were purchased on the day of analysis
Salam leaves, which is synonym to Syzygium to ensure their freshness, and to avoid contamination
polyanthum L., is a deciduous tropical tree belonging during storage.
to the Myrtaceae family (Kato et al., 2013). This plant
grows wildly on lowlands and is widely distributed Extraction of salam leaves
in the temperate, subtropical and tropical regions in The salam leaves were extracted using the
the world (Perumal et al., 2012). Salam leaves has methods reported by Rukayadi et al. (2008) with
been used traditionally as medicine or therapeutic slight modifications. One hundred grams of salam
agents include effective against ulcer, hypertension, leaves powder were placed in 500 ml conical flask
diabetes, hyperuricemia, diarrheal, gastritis, skin and soaked in 400 ml of absolute methanol (99.8%)
diseases and inflammation (Sumono and Wulan, (Sigma-Aldrich, Saint Louis, MO, USA). The
2008). Salam leaves had been found to have mixture underwent sonication for a total duration
antimicrobial functions. It was found to be able to of 45 min with the sonicator water bath (Delta
inhibit the growth of microorganisms such as Bacillus Ultrasonic Cleaner D150H, Taiwan). Mixture was
cereus, Bacillus subtilis, E. coli, Salmonella sp., S. then filtered using Whatman filter paper No. 2
aureus and Pseudomonas fluorescens (Setiawan, (Whatman International Ltd., Maidstone, England)
2002). Other than that, salam leaves are also found to attached to an the aspirator pump. After that, the
be able to cure diarrhea due to the presence of tannin liquid solution underwent evaporation with a rotary
and also able to treat diabetes and skin infection vacuum evaporator (Heidolph Instruments, Germany)
(Dalimartha, 2007). It was also reported to have at 50ºC and 150 rpm for 30 min to obtain methanol-
potential antioxidant properties (Perumal et al., 2012; free salam leaves extract. The extract was kept at 4ºC
Darusman et al., 2013). until further analysis.
Researcher had reported the effectiveness of
garlic extract to reduce microbial growth on poultry Preparation of salam leaves extract for treatment
meat such as E. coli, Salmonella typhimurium, S. In this preparation, 3 g salam leaves extract
aureus and Bacillus cereus (Yadav et al., 2002). was diluted with 30 ml 10% dimethyl sulfoxide
Clove extract was found to inhibit the growth of (DMSO) (Sigma-Aldrich, Saint Louis, MO, USA)
S. typhimurium, Aeromonas hydrophila and toxin with the ratio (1:10) to make up a 10% salam leaves
production by E. coli in chicken meat during extract solution. The 10% salam leaves extract
refrigerated storage (Singh et al., 2004). solution was then serially diluted in the sterile de-
However, no study has been reported on ionized water (DIW) (B Braun Medical Industries,
the treatment of daun salam extract against the Penang, Malaysia) to make up extract solutions
microorganisms in chicken meat and shrimp. of three different concentrations (1%, 0.1% and
Therefore, the aim of this study was to determine 0.01%) which were used on the raw chicken meat
the antimicrobial activity of salam leaves on natural and shrimp. concentration of salam leaves extract
microorganism population in chicken meat and solution were prepared and ready to be used for the
shrimp as a basic screening in order to developing application on raw chicken meat and shrimp. This
natural food sanitizer. methodology was referred to Yusoff et al. (2015)
 Ramli et al./IFRJ 25(3): 928-935 930

with slight modification. terms of colour (observed with eyes), odour (smell
with nose), texture (touch with finger) and the overall
Media for enumeration of microorganism by spread acceptability on each treatment samples. The rating
plate method for each analysis of samples were given in a range of
Media were chosen based on Microbiology scale from extremely like (scale of 9) to extremely
Manual (12th ed, Merck, Darmstadt, Germany) dislike (scale of 1). Scores ≥5 were “acceptable”.
which included Plate count agar (Merck, Darmstadt,
Germany), Tryptic soy agar (Merck, Darmstadt, Statistical analysis
Germany), Mac Conkey agar (Hardy Diagnostics, Data were analysed by using MINITAB 16 for the
Santa Mania, USA), X.L.D. medium (OXOID Ltd., analysis of variance (ANOVA), one-way, unstacked,
Basingstoke, Hampshire, England) and T.C.B.S. whereby Tukey’s test was employed to determine the
Cholera medium (OXOID Ltd., Basingstoke, significance difference (p<0.05) between different
Hampshire, England). treatments. Results were expressed as means ±
standard deviation (SD) of duplicate analyses, unless
Treatment of chicken and shrimp samples with salam otherwise stated.
leaves extract
Ten grams of raw chicken meat and shrimp Results
samples were immersed in 25 ml of tap water, DIW
and different concentrations (1.0%, 0.1% and 0.01%) Yields of salam leaves extraction
of salam leaves extract solutions at room temperature A 100 g of dried weight of salam leaves extracted
for 5 and 10 min exposure time. A sample without with methanol solvent yielded 3.06 g extract (3.06%).
soaking with any solution served as the positive
control, for both chicken meat and shrimp. Effect of salam leaves extract on number of
microorganisms in treated chicken meat and shrimp
Enumeration of microorganisms by spread plate Chicken meat and shrimp samples were treated
method with different extract concentrations (1.0%, 0.1%,
All treated samples were aseptically removed 0.01%, tap water and DIW) and different soaking
using a flame-sterilized spatula and drained on times (5 min and 10 min). TPC and selective agar
absorbent paper for drying. The treated samples were media were used to enumerate the number of natural
placed into the stomacher bag (Baglight, BagSystem, microflora and food-borne pathogens in chicken meat
Interscience, France) with 90 ml of 0.1% peptone and shrimp samples after treatment. The results were
water. The mixture was homogenized using a interpreted in log10 CFU/ml and summarized in Table
stomacher machine (BagMixer 400-P, Interscience, 1 and Table 2, respectively.
France) for 2 min at 250 rpm. The enumeration of
microorganisms was conducted by spread plate Sensory evaluation of raw chicken meat and shrimp
methods. The colony forming unit per gram (CFU/g) treated with salam leaves extract
of samples were calculated after 24 h of incubation Results for sensory evaluation are presented in
at 37ºC. Table 3 and Table 4, respectively.

Sensory evaluation Discussion

The sensory evaluation acceptability test was
performed as according to Brasil et al. (2012), Potential contamination in chicken meat and
with slight modification. A preliminary test was shrimp by the foodborne pathogens can be caused
conducted to determine whether the treated food through the unhygienic practices in handling,
would be acceptable to consumers. The sensory cooking or post cooking storage of products.
evaluation was carried out for the visual sensory During washing process of raw foods material is
test of each treatment of the raw chicken meat and also one of the causes for the foodborne pathogens
shrimp samples. All samples were treated with tap to growth with rapidly. However, those numbers
water, DIW and different concentrations (1.0%, 0.1% of microorganisms in raw foods can be reduced by
and 0.01%) of salam leaves extract solutions for 5 using the antimicrobial compounds in the sanitizers.
and 10 min exposure time. A group of 30 random Therefore, sanitizers which possess antimicrobial
panellists were invited to perform the evaluation on property become important and common in washing
the treatment samples. The evaluation was conducted raw food materials (Nychas, 1995).
based on the 9-point hedonic scale for acceptance in In this experiment, salam leaves extract was
931 Ramli et al./IFRJ 25(3): 928-935

Table 1.Number of microorganism presence in untreated chicken meat (initial bacterial load) and
treated chicken meat samples (log10 CFU/ml)

Mean values ± standard deviation with different lowercase letter in the same column have significant
difference (P<0.05).
1
ST = soaking time
2
DIW = deionized water
3
TFTC = too few to count (colonies < 30)

obtained through extraction. Extraction was done The highest reduction of S. aureus population also
by using absolute methanol due to its capability to occurred in treatment 1.0% extract from initial load
produce high yield of extract containing high chemical of log10 CFU/ml 7.25 ± 0.21 to 4.11 ± 0.01, and the
compounds from the plant sample (Caunii et al., reduction in population decreased with the decreasing
2012). Table 1 shows the number of microorganisms concentration of extract solutions. On the contrary,
detected in chicken meat before and after soaking chicken meat samples soaked in tap water and DIW
treatment. The number of microorganisms in total gave only slight reduction in bacterial population
plate count (TPC) detected was log10 CFU/ml 6.66 for both 5 min and 10 min soaking. For TPC, the
± 0.12, S. aureus was log10 CFU/ml 7.25 ± 0.21 reduction only from log10 CFU/ml 6.66 ± 0.12 to 6.61
and none were detected for E. coli and Salmonella ± 0.03 and 6.55 ± 0.0 whereas the population of S.
sp. respectively. In this finding, S. aureus was the aureus reduced from log10 CFU/ml 7.25 ± 0.21 to
highest number of species found in the samples, and 7.13 ± 0.03 and 7.15 ± 0.00 respectively.
this correlated with findings from Akbar and Anal Table 2 shows the number of microorganisms
(2013) who also found S. aureus to be predominant in detected in raw shrimp before and after soaking
poultry meat. The number of total plate count (TPC) treatment. The number of natural microorganisms
and S. aureus in chicken meat were starting to reduce in total plate count (TPC) detected was log10 CFU/
significantly at 0.01% concentration of salam leaves ml 8.66 ± 0.15, whereas for S. aureus was log10
extract for 5 minutes of exposure time compared to CFU/ml 6.54 ± 0.21 and E. coli and V. cholerae not
initial count. However, reduction of the bacterial detected. Results also showed that the total surviving
count was not significantly different between the bacterial population (TPC) on shrimp was reduced
soaking times. after treated with different concentrations of salam
Table 1 also shows the highest reduction of leaves extracts (1.0%, 0.1% and 0.01%), tap water
bacterial population (TPC) occurred in 1.0% and DIW. Basically, the reduction trend in shrimp
concentration from initial bacterial load log10 CFU/ samples after treatment was the same as the trend
ml 6.66 ± 0.12 to ‘Too Few To Count’ (TFTC) and in chicken meat samples. The number of total plate
the population was decreased with the decreasing count (TPC) and S. aureus in shrimp were starting to
concentration of extract solutions. This finding reduce significantly at 0.01% concentration of salam
trend was correlated with Boziaris et al., (2010) leaves extract for 5 min of exposure time compared
who indicated that plant extract delayed the growth to initial count. The highest reduction for both total
of microorganisms in a concentration-dependent bacterial population and S. aureus occurred in 1.0%
manner where the higher the concentration, the higher concentration at 10 min soaking from initial bacterial
the growth in inhibition and reduction. It showed that load of log10 CFU/ml 8.66 ± 0.15 to 4.88 ± 0.00 and
the reduction number of microorganisms in chicken log10 CFU/ml 6.54 ± 0.21 to 4.92 ± 0.04, respectively.
meat was strongly correlated with the concentration On the other hand, there was no bacterial count
of salam leaves extract. from untreated chicken and shrimp samples for
 Ramli et al./IFRJ 25(3): 928-935 932

Table 2. Number of microorganism presence in untreated shrimp (initial bacterial load) and
treated shrimp samples (log10 CFU/ml).

Mean values ± standard deviation with different lowercase letter in the same column have significant
difference (P<0.05).
1
ST = soaking time
2
DIW = deionized water

Table 3. Result for sensory evaluation on the application of salam leaves extracts on raw
chicken at different concentrations at different soaking times

Mean values ± standard deviation with different lowercase letter in the same column have significant
difference (P<0.05).

E. coli, Salmonella sp. and V. cholerae, indicating for inhibition of pathogens. Among the compounds
that the samples from hypermarket were fresh and identified which have antibacterial activity include
clean at the time the experiment was performed. hexadecanoic acid and phytol (Preethi et al., 2010).
Enterobacteriaceae is a hygiene indicator of food Besides that, Jananie et al. (2011) also stated that
(Zeitoun et. al., 1994). Enterobacteriaceae family is phytol (0.66%) possess antimicrobial activity.
Gram-negative bacteria that include many of more According to Irawan et al. (2012), salam leaves
familiar pathogens such as Salmonella, E. coli, extract contained carbohydrate, tannins alkaloid,
Klebsiella, Shigella and Proteus. Hence, the initial steroid, triterpenoid, and flavonoid and saponins.
population of Enterobacteriaceae can be an indicator Flavonoids have been reported to possess antibacterial,
of adequate hygiene conditions of food. antioxidant, anti-inflammatory, antiallergic,
Finding showed that the effect of salam leaves antimutagenic, and vasodilatory activity. Tannin is
extract was positive which induced a reduction in believed to be responsible for the antibacterial activity
microbial population. Besides that, studies from of salam leaves. The mechanisms of inhibition of the
Setiawan (2002) also proved that salam leaves bacteria growth involved precipitation forming and
extract possesses antimicrobial activities to inhibit denaturing of the bacteria protein (Nur Amalina,
the growth of microorganisms such as E. coli, V. 2014).
cholerae and Salmonella sp. Roughly, mechanism Since salam leaves has been proved to have
of antimicrobial action from plant extract can be antimicrobial functions, as also demonstrated in
explained as the membrane disruption pathway this study, sensory evaluation was also carried out
by phenolics and metal chelation by flavonoids in in order to determine the effect of salam leaves
inhibiting the growth of microorganisms. According extract application in the food system on the sensory
to Lau (2015), salam leaves contain different active properties, even at low concentrations. The variations
compounds where each of them functions differently in colour, odour and texture of the food samples after
933 Ramli et al./IFRJ 25(3): 928-935

Table 4. Result for sensory evaluation on the application of salam leaves extracts
on raw shrimp at different concentrations at different soaking times.

Mean values ± standard deviation with different lowercase letter in the same column have significant
difference (P<0.05).

treatment do account for variations in food quality and microorganism population in chicken meat and
consumer acceptance rather than caused by spoilage shrimp was positive. The reduction of the number
bacteria (Solomon et al., 2014). Sensory evaluation of microorganisms by salam leaves extract was
results were interpreted and presented in Table 3 dependent on extract concentration. The higher the
for chicken meat and Table 4 for shrimp. Sensory concentration of extracts resulted the higher the
evaluation was conducted in visual testing basis microbial reduction. In chicken meat and shrimp,
with 30 random panellists. Four attributes including the numbers of microorganisms were starting to
colour, texture, odour and overall appearance were reduce significantly at 0.01% concentration of
evaluated in a 9-point hedonic scale. daun salam extract for 5 minutes of exposure time
Based on the data in Table 3 and Table 4, overall compared to initial count. There was no significantly
acceptability were accepted by panellists until different between exposure times. For the sensory
treatment 0.10% for 5 min and 0.1% for 10 min of acceptability, overall acceptability was accepted by
soaking time. This meaning the treatment by tap panellists until treatment 0.10% for 5 min and 10 min
water, DIW, 0.01% and 0.1% extract at 5 and 10 of soaking time. Overall, these results suggested that
min were not significantly different and this types salam leaves extract exhibited antimicrobial activity
of solutions does not affected colour, odour, texture and might be further developed as a natural food
and overall acceptability of the samples. In this test, sanitizer.
tap water was act as a control. The highest overall
acceptability of treated chicken meat at 5 min and 10 Acknowledgement
min treatment was by soaking in 0.01% extract.
Based on Table 4, for raw shrimp samples, the The study was supported by Penelitian
highest preference of treated sample by panellists Fundamental - FRGS Fasa 2- 2014 to Yaya Rukayadi
was 0.1% extract at 5 min soaking which scored at with number FRGS/2/2014/SG05/UPM/02/2.
6.23 ± 1.55 and 0.01% at 10 min with 6.10 ± 1.63.
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