Currently, charcoal industries in Indonesia use charcoal as the main product,
and the rest of approximately 70-80% is in the form of wasted gas that is released
to the atmosphere as air pollutant. Attempt to increase added value of the smoke
to be an environmentally friendly has been done, in various studies on the liquid
smoke, which is commonly called as wood vinegar or liquid smoke. The production
of liquid smoke can be integrated with charcoal making (Nurhayati
and Sofyan, 2005). The liquid smoke is widely used as antibacterial and
antioxidant by many food technologists.
Pyrolysis of coconut shells has been reported to produce liquid smoke with
phenolic level of 9.36%, carbonyls 8.34%, and organic acids 6.38%. The distilled
liquid smoke at lower than 100°C yields lower quantity of phenolics (3.90%)
and lower tar (0.29%) as compared with the distilled products at higher temperatures
(Darmadji, 2002). Solution of 2.5% liquid smoke made
of coconut shell extends the shelf life of fish ball from 16 to 32 hours at
room temperature (Zuraida et al., 2011).
Liquid smoke of oil-palm shells has been stated to inhibit the growth of Psedoumonas
fluorescens, Staphylococcus aureus, Escherichia coli and
Bacillus subtilis bacteria. The activity of biopreservative toward S.
aureus is exhibited at 0.6% concentration and toward E. coli at 0.8%
(Halim et al., 2006). Tamaela
(2003) and Yanti and Rochima (2009) also found that
liquid smoke of coconut shells is potential as an alternative in fish preservation.
The potency can be used for various processing and storage technology for fish
products, so that the potency of liquid smoke derived from oil-palm shells,
which is abundantly found in oil palm plantation, should be evaluated as fish
preservative of fresh catch and as fly repellent in fish salting-fermenting
MATERIALS AND METHODS
Materials: Materials used were crude liquid smoke of oil-palm shells
extracted from pyrolysis product at the temperature of 400°C (provided by
PT Global Deorub Industry), Staphylococcus aureus and Escherichia
coli (available from the Laboratory of Microbiology Culture Collection,
the Department of Biology, Bogor Agricultural University), fresh water pomfret
fish, patin fish (Pangasius hypophthalmus), Chrysoma megalocephoda
flies and brine shrimp larvae Artemia salina. The experiment was
done in 2011-2012. Chemicals used were Folin-Ciocalteau reagent, Tashiro indicator
solution, chloramphenicol in powder form, liquid media of Tryptic Soy Broth
(TSB) and solid medium of Nutrient Agar (NA).
Preparation of liquid smoke: Crude liquid smoke of oil-palm shells (20
L) was placed in a large container and distilled using a concentration boule
type TA62D at 80±5°C. The distillate in the form of liquid smoke
was then collected in a closed container. Total Acid Assay was measured by titration
(AOAC, 2005). Phenolics Determination was carried out
using Folin-Ciocalteau reagent (Waterhouse, 2002).
GC-MS analysis: Chemical compounds in the redistilled liquid smoke were
identified using gas chromatography-mass spectrometry (GC-MS) instrument equipped
with 60-meter HP5 column. Detector temperature, initial column temperature and
final column temperature were 250, 280 and 290°C, respectively. The carrier
gas was helium with flow rate of 23.7 mL min-1. at 17.56 psi pressure.
The liquid smoke sample injected was 1 μL. Toxicity assay was performed
on A. salina and calculated based on the percent mortality using probit
scale (Manilal et al., 2009).
Level of TVB-N Assay (Standar Nasional Indonesia 2354.8:2009): Samples
of fresh-cut pomfret fish (10 g each) that had been immersed in various concentrations
of liquid smoke were placed in beaker glass. To each beaker glass was added
90 mL perchloric acid 6%. The content of the beaker glass was stirred for 2
min and filtered. The filtrate (50 mL) was transferred into a distillation tube,
added a few drops of phenolphthalein indicator and antifoaming silicon. The
distillation tube was mounted on a steam distillation apparatus and 10 mL NaOH
20% was added. A collecting erlenmeyer was prepared, containing 100 mL H3BO4
3% and 3-5 drops of Tashiro indicator (violet solution). Steam distillation
was performed for approximately 10 min until 100 mL distillate was collected
and the final volume was approximately 200 mL (green solution). Distillation
of blank solution was performed in a similar way using 50 mL perchloric acid
6%. The distilled samples as well as the blank were titrated using solution
of HCl 0.02 N. The end point of titration was indicated by reforming the violet
color. The analysis was carried out every 8 h until the permissible total volatile
base nitrogen (TVB-N) value was reached. The experiment was repeated twice.
where, Vc is the volume of HCl for titration on sample, Vb
is the volume of HCl for blank titration, N is the normality of HCl solution,
W is sample weight (g), 14.007 is the atomic weight of nitrogen and 2 is the
Inhibition Assay on Bacteria was carried out as reported by Fitrial
et al. (2008). The NA medium containing bacteria (S. aureus
and E. coli, ±15 mL) was poured into the petri dish that already
contained solid NA medium (the first layer). The mixture was homogenized and
the second layer was left to solidify. In the first step, five wells were conditioned
aseptically, 6 mm diameter each. Into each well, the liquid smoke solution (8,
9 and 10% concentrations), a positive control (solution of 100 ppm chloramphenicol)
and negative control (sterilized distilled water) were transferred, 60 μL
each. Each of the bacteria, i.e. were tested with five replications. Diameters
of the measurable inhibition zone (replicated three times) were converted to
inhibition index. Preparation of Dried-fermented Fish followed Ariyani
et al. (2007) method. Patin fish was used in this experiment. In
the salting process, 30% of salt (based on the fish weight), was applied. Some
salts were put into the fish abdominal cavity and the rest of salts were dissolved
into a saturated solution and fermented for 48 h.
Assay for Repellency toward Chrysoma megalocephoda Flies (Yuliani
et al., 2005). The testing was performed using 25 flies that were
caged in a 60x40x30 cm glass tank. The treated fish were put into this glass
tank. Observation was started after 1 min. by counting the number of fly perch
on the dried-fermented fish every minute until 60 min. The experiments were
replicated three times for each concentration of 5, 10, 15 and 20% and the percent
repellency was calculated.
Statistical analysis: The observed data on inhibition index and fly
repellency were subjected to analysis of variance (ANOVA). If there was any
significant difference, the difference among treatment means was determined
by Duncans multiple range test at p = 0.05.
The redistilled liquid smoke of oil-palm shells at 80°C gave clear acidic
liquid, containing 63% of simple carboxylic acids and to a lesser extent were
phenolics, with no indication of toxic polyaromatic hydrocarbons. The liquid
was also a strong antibacterial toward S. aureus and E. coli,
which would be a beneficial property for fish preservative. The low TVB-N values
of fish muscles immersed in the liquid indicated good inhibition of protein
putrefaction until 24 h and 10% concentration of the gave significant effect
on avoiding fly infestation in salting-fermentation process.
Physical and chemical characteristics of the redistilled liquid smoke:
Oil-palm shells were initially pyrolyzed at 400°C, producing dark-color
liquid smoke with tar or Polycyclic Aromatic Hydrocarbons (PAHs). Preparation
of liquid smoke by redistillation at 80±5°C yielded clear yellowish
brown liquid (Fig. 1).
|| (a) Appearance of crude liquid smoke and (b) Redistilled
liquid smoke of oil- palm shells at 80°C
|| GC-MS analysis on liquid smoke of oil-palm shells with 90%
|MW = molecular weight
The liquid smoke of oil-palm shells has specific odor and volatile. There is
an indication that the redistillation of the liquid smoke at 80±5°C
is able to reduce PAHs content that has boiling point of more than 300°C.
Three parameters of liquid smoke were observed: organic acid content, total
phenolics and pH. The redistilled liquid smoke of the oil-palm shells exhibits
organic acids of 9.14%, total phenolics of 2.6% and pH 3.2. The GC-MS analysis
on the redistilled liquid smoke identified some chemicals as shown in Table
1. These compounds have more than 90% similarity with those in the GC-MS
instruments library. The highest constituent (41%) was acetic acid, followed
by phenol (26%). Other constituents with similarity more than 95% belong to
phenolics, i.e. 3-methylphenol, 2-methylphenol, 2-methoxyphenol and 3, 5-dimethylphenol.
Toxicity of the redistilled liquid smoke: Data of the toxicity test
toward brine shrimp larvae and the corresponding LC50 are exhibited
in Fig. 2. There are increasing numbers of dead larvae with
the increasing concentrations of the liquid smoke. Afterwards, the values are
derived in the form of relation between-log concentration of the liquid smoke
and percent of dead larvae. The LC50 value of 0.2147% is obtained
from a line equation y = -1.6662x + 4.9456 with R2= 0.8527.
TVB-N values: TVB-N values of pomfret fish that have been preserved
for 30 min using the redistilled liquid smoke are shown in Fig.
3. The concentrations of the liquid smoke were 0, 8, 9 and 10% and the TVB-N
values were determined every 8 hours at room-temperature storage. Increasing
TVB-N values are caused by the action of degrading bacteria on the fish muscular
tissues that produces mainly ammonia, trimethylamine and dimethylamine. With
prolonging the storage time, the TVB-N values also increasing. Analysis on hour-8
shows that immersion in 10% concentration of liquid smoke produces the lowest
TVB-N value (4 mg/100 g) among the three other treatments, as well as those
observed at hour-16. Analysis on hour-24 shows that TVB-N value for the control
(38 mg/100 g) has exceeded the maximum permissible value, whereas those treatments
with the 8-10% concentration of liquid smoke are still below the threshold.
All treatments have exceeded permissible TVB-N values at hour-32.
||Relation between -log concentration of liquid smoke and percent
of dead larvae
||Total volatile base nitrogen (TVB-N) values of pomfret fish
preserved with various concentrations of liquid smoke 8%, 9%, 10% and control
Until 24 h, pomfret fish that have been immersed in the liquid smoke showed
TVB-N values that are lower than that without liquid smoke treatment, indicating
that the fish are well preserved by the redistilled liquid smoke.
Antibacterial properties: Two species for assay on antibacterial activity
were used, namely S. aureus (representing Gram positive) and E. coli
(representing Gram negative). Antibacterial activity of the liquid smoke
can be seen from the clear zone formed surrounding the corresponding well (Fig.
4). The clear zone formed on S. aureus is more distinct as compared
with that on E. coli. This is related to the effects of the liquid smoke
on the bacterial growth. Based on the diameter of formed clear zone, the liquid
of oil-palm shells has antibacterial activity in strong category. The strength
of antibacterial activity is designated as follow: inhibition area >20 mm
is very strong, inhibition area of 10-20 mm is strong, inhibition area of 5-10
mm is moderate and inhibition area <5 mm is in weak category (Davis
and Stout, 1971).
|| Antibacterial activity of redistilled liquid smoke derived
from oil-palm shells toward (a) S. aureus and (b) E. coli
||Inhibition indices of liquid smoke of oil-palm shells as compared
with commercial antibiotics
|Value with different letter in the same column is significantly
different at p<0.05
Data of the formed clear zone diameter were further converted into inhibition
index (Table 2). The values of inhibition index exhibited
by the liquid smoke of concentration 0, 8, 9 and 10% are also compared with
that of 100 ppm chloramphenicol. The average of inhibition index by the liquid
smoke toward S. aureus shows higher value than that toward E. coli
for the same concentration. The value is also compared with inhibition by common
antibiotics chloramphenicol 100 ppm. Test on Duncans multiple range gives
three categories of results, designated as a, b and c. The same letter means
that the treatments do not give significantly different results. On S. aureus,
the use of 8, 9 and 10% liquid smoke give the same inhibition effect (letter
b); however, it is significantly different with that of chloramphenicol 100
ppm (letter c) and the blank (letter a).
||Fly repellency on the dried-fermented fish with different
application of redistilled liquid smoke
|| Significance of fly repellency based on Duncans multiple
|Value with different letter in the same column is significantly
different at p<0.05
The 8% concentration of liquid smoke (letter b) gives inhibition differently
with that of 9 and 10% concentration (letter c) toward E coli. The use
of chloramphenicol 100 ppm. does not inhibit the growth of this particular bacteria,
just similar to the blank (letter a).
Repellency to flies: Observation on repellency of the liquid smoke toward
flies is shown in Fig. 5. Increasing repellency is linearly
correlated with the increasing concentration of the applied liquid smoke. The
best repellency of the liquid smoke applied on the fermented salted fish was
analyzed using Duncans multiple range (Table 3). The
first step was to determine the highest value of all treatments. The highest
value was 99.69, obtained from concentration of 10% liquid smoke. The next step
considers the letters corresponding to the treatment. All of the average of
repellency is followed by different letter. This means that every treatment
has significant effect, differently from one to another. Therefore, the average
of the highest repellent (10% concentration of liquid smoke) is the best treatment.
Physical and chemical properties and toxicity: From the appearance aspect,
color of the redistilled liquid smoke is no longer dark. The redistillation
turns the previous dark color liquid smoke to bright yellowish-brown. This physical
property shall give good effect for its utilization in foodstuff.
The redistilled liquid smoke of oil-palm shells mainly contains organic acids
and phenolics. Lower organic acids are weak acids with pKa of approximately
5. However, the organic acids are more acidic as compared with the phenolics,
particularly due to resonance stabilization of the carboxylic anions (RCO2-).
These acidic compounds play important role in creating the low pH of the liquid
smoke (pH 3.2). In addition, the low pH value represents a high quality of liquid
smoke particularly in its utilization as food preservatives (Wijaya
et al., 2008; Theron and Lues, 2011). Furthermore,
compounds of PAHs family are not identified, indicating that the redistillation
of the liquid smoke at 80°C is effective in eliminating the carcinogenic
PAHs content. GC-MS analysis on liquid smoke is also performed in a previous
study. Budijanto et al. (2008) on his investigation
on the safety of liquid smoke derived from coconut shells for food products
shows that there are 40 constituents identified by GC-MS analysis. There are
phenolic components, such as phenol, 2-methoxy phenol, 3,4-dimethoxy phenol,
2-methoxy-4-methyl phenol. Dihydroxy benzoic acid, methoxybenzoic acid and hydroxyl
benzoic acid are present as minor components.
Level of organic acids, total phenolics and pH of our study are different with
the study by Darmadji and Triyudiana (2006) which also
used liquid smoke redistilled below 100°C, producing 12.34% organic acids
and 1.1% total phenolics. Therefore, the redistilled liquid smoke from oil-palm
shells gives lower organic acids (9.14%) but higher total phenolics (2.6%).
The difference is due to cellulose-hemicellulose and lignin contents in the
raw materials, from which the pyrolysis process would determine the liquid smoke
quality. There seems to be a clear correlation between lignin and phenolics
contents. The liquid smoke from oil-palm shells is also less acidic as compared
with the liquid smoke from bamboo (3.08), Leucaena wood (2.9) and corn
cobs (3.0) (Swastawati et al., 2007). Acids level
is also affected by distillation temperature (Darmadji,
Three main components of the liquid smoke from our study are carboxylic acids,
phenolics and carbonyls. Hemicelluloses are wood component that firstly undergo
pyrolysis to produce furfural, furan, acetic acid and its homologues. Hemicelluloses
consist of pentosans (C5H8O4) and hexosans
(C6H10O5), with the average proportion depend
on the raw materials. Pyrolysis of the pentosans produces furfural, furan and
their derivatives, as well as carboxylic acids. Together with cellulose, pyrolysis
of hexosans also produces acetic acid and its homologs. Decomposition of hemicelluloses
happens at 250-300°C. Lignin in pyrolysis process will produce phenolics
that will contribute to smoke aroma in smoked products. Phenolics are produced
from lignin decomposition at 300-450°C (Girard, 1992).
Further process in the pyrolysis of cellulose will produce acetic acid and carbonyl
compounds, such as acetaldehyde, glioxal and acreolin. Pyrolysis of lignin will
produce phenol, guaiacol and syringol, together with their homologs and derivatives
Phenolics contribute to the quality for modifying color, flavor, aroma and
odor (Sengul et al., 2009). Level of total phenolics
in the liquid smoke of oil-palm shells 0.26% (b/b) is more than 20 times higher
than that of liquid smoke of teakwood, pine wood and bamboo (Wijaya
et al., 2008). Phenolics with low boiling points are good as bacteriostatics.
Organic acids are stronger in inhibiting bacterial growth as compared with phenolics;
however, combination of these two categories of organic acids will give rise
to higher inhibition. Phenolics, as healthy phytochemicals, are commonly used
in food industries as antioxidant and antibacterial (Esekhiagbe
et al., 2009).
Toxicity of the liquid smoke using BSLT method revealed that no larvae survived
on the concentrations of 0.3, 0.4 and 0.5%. LC50 in this study is
0.2147%. A compound is designated to have acute toxicity if the LC50
is below 0.1% (Carballo et al., 2002). Therefore,
based on this assay, the redistilled liquid smoke of oil-palm shells is not
considered as toxic material.
Potency as antibacterial in fresh fish preservation: Redistilled liquid
smoke in this study act as bacteriostatics (inhibit the growth without lethal
effect) on E. coli and bacteriocidal/bacteriolitic (lethal effect) on
S. aureus. Inhibition on bacterial growth by an antibacterial agent can
be due to the inhibition of synthesis of cell wall, proteins, nucleic acids
and disruption of the functions of cell membranes (Jawetz
et al., 2010). Resistance of E. coli which is higher than
that of S. aureus toward antibacterial agents may be related to the structure
of cell membranes. E. coli is a member of Gram negative bacteria with
layered membrane structure, among which are lipoproteins, lipopolysaccaharides
and peptidoglicans (Madigan et al., 2010).
Organic acids are food preservatives that function as acid-taste elicitor and
reduce bacterial growth by reducing pH within the food to pH level that can
inhibit bacterial growth. Principle of the inhibition by organic acids is that
the undissociated part of the acids (anion moieties) can penetrate into the
cell wall of the bacteria and disrupt the normal physiological function of the
cells (Theron and Lues, 2011).
Phenolics in the liquid smoke of pelawan (Tristania obovata)
wood actively attack on bacterial vegetative cells, can penetrate and disrupt
cell walls and precipitate proteins in the microbe cells (Panagan
and Syarif, 2009). Inhibition by phenolics may be due to interaction through
hydrogen bonding with some important proteins as enzyme constituent (Saravanakumar
et al., 2009).
Fish putrefaction is commonly indicated by high TVB-N value. The increase in
TVB-N values is due to bacterial action as proved by inclination of bacterial
population. Immersion treatments in liquid smoke solutions are able to reduce
the rate of volatile base formation as compared with the control. The higher
the concentration of the liquid smoke, the better the ability to inhibit the
formation of the volatile bases. The maximum TVB-N level for fresh fish in Japan
and Australia is 30 mg N% (Siagian, 2002). Dwiyitno
and Riyanto (2006) in the experiment using liquid smoke of coconut shells
for preserving fresh mackerel is able to maintain TVB-N value below the maximum
level until 12-hour storage with treatments using 7.5 and 10% concentrations.
Based on BSLT, the LC50 for liquid smoke of oil-palm shells is 0.22%,
meaning that it is safe to be incorporated in foodstuff. Safe limit for phenol
in food products is 0.0006-0.5% or 0.06-5000 mg kg-1 (Girard,
1992). Therefore, immersion in the redistilled liquid smoke (0.18-0.26%)
will not exceed the determined safe limit. Phenol content will also decrease
in further fish processing such as washing and heating for products to be consumed.
Potency as fly repellent: Fly infestation on dried-fermented fish during
sundrying may be due to some volatile bases such as ammonia and hydrogen sulfide.
These two compounds are originated from protein degradation caused by proteolytic
enzymes during autolysis and fermentation processes (Ariyani
et al., 2007). However, immersion in liquid smoke solution has elicited
odorous phenolics dislikable to flies. In addition, the presence of 30% salt
is indirectly functioning as insecticide (Ariyani et
al., 2007). Some phenolics detected in the redistilled liquid smoke
of oil-palm shells, namely cresol, creosol, guaiacol, syringol, eugenol and
xylenol, may gave specific aroma. Furfural as the only member of aldehydes that
is also detected in this particular liquid smoke has been reported as a disinfectant
(Madigan et al., 2010). Furfural gives pleasant
aroma that reduces aroma of the phenolics that have rather sharp odors. This
compound gives specific aroma of the liquid smoke that function as repellent.
Action of the liquid smoke is not lethal as compared to insecticides which are
working as contact insecticide. Hence, insects that do not like poison with
repellent action can quickly avoid the baits (Yuliani et
Liquid smoke of oil-palm shells redistilled at 80°C gives clear yellowish-brown
in color with specific aroma of volatile constituents. With chemical specifications
of organic acids 9.1%, total phenolics 2.6% and pH 3.2, the redistilled liquid
smoke is potential as a safe preservative for fresh fish. By 30 min immersion
in the liquid smoke solution, the fish freshness can be longer than that without
liquid smoke treatments, i.e. up to 24 h at room temperature storage. The redistilled
liquid smoke of oil-palm shells is bacteriostatics in nature and shows strong
inhibition toward bacterial growth. The best treatment as antibacterial is toward
S. aureus which represents Gram positive. Immersion in the solution of
redistilled liquid smoke functions as antibacterial and fly repellent for dried-fermented
fish. The fly-repellency of the treated dried-fermented fish is excellent, with
the repellency above 95%. Thus, the redistilled liquid smoke of oil-palm shells
can be applied as safe fish preservative and can be combined with other preservation
methods such as cooling and salting.