Influence of moringa oleifera leaf extract on microbial and quality parameters of Pangasius hypophthalmus mince under frozen storage (-18±2°c)

Abstract

Pangasius hypophthalmus is one of the most important commercial fishes with high nutritive qualities and excellent sensory properties such as tender flesh, sweet taste, absence of fishy odor and spines, delicate flavor and firm texture during cooking that help to gain its consumer preference. Moringa oleifera (commonly called drumstick) is one of such most important terrestrial plants which exhibit antibacterial, antifungal as well as antioxidant properties which influence its application in food preservation.In the present study, three different concentrations (5%, 10% and 15%) of Moringa oleifera leaves extract were used as antimicrobial and antioxidant agent to study the shelf life of minced meat prepared from Pangasius hypophthalmus during frozen storage at ×18±2°C for a period of 5 months. The treatments showed significantly (p < 0 . 05) improved values of the quality parameters such as TVBN, TMA and TBARS as compared to control throughout the frozen storage period. TPC was also found to be reduced significantly (p < 0 . 05) in treated samples. Presence of different phytochemicals that have effective antimicrobial properties of M.oleifera extract against wide range of bacteria which were responsible for accumulation of ammonia and volatile bases in fish flesh, decreased protein breakdown, thus lowering TVBN and TMA values. The antioxidant properties of polyphenols present in the M. oleifera leaf extract retarded the formation of malonaldehydes. From the present study, it can be concluded that 15% concentration of M. oleifera leaves extract is considered as most effective to maintain the quality of the minced Pangasius hypophthalmus during five months frozen storage at -18±2°C.

Full text

INFLUENCE OF MORINGA OLEIFERA LEAF EXTRACT ON MICROBIAL
AND QUALITY PARAMETERS OF PANGASIUS HYPOPHTHALMUS MINCE
UNDER FROZEN STORAGE (-18±2OC)
J. Chakraborty, S. Nath*, S. Chowdhury and K. C. Dora
Department of Fish Processing Technology, Faculty of Fishery Sciences, W B U A F S, Kolkata - 700 094, India
*e-mail:swarnadyutinath@gmail.com
(Accepted 18 July 2017)
ABSTRACT : Pangasius hypophthalmus is one of the most important commercial fishes with high nutritive qualities and
excellent sensory properties such as tender flesh, sweet taste, absence of fishy odor and spines, delicate flavor and firm texture
during cooking that help to gain its consumer preference. Moringa oleifera (commonly called drumstick) is one of such most
important terrestrial plants which exhibit antibacterial, antifungal as well as antioxidant properties which influence its application
in food preservation.In the present study, three different concentrations (5%, 10% and 15%) of Moringa oleifera leaves
extract were used as antimicrobial and antioxidant agent to study the shelf life of minced meat prepared from Pangasius
hypophthalmus during frozen storage at ×18±2ºC for a period of 5 months. The treatments showed significantly (p<0.05)
improved values of the quality parameters such as TVBN, TMA and TBARS as compared to control throughout the frozen
storage period. TPC was also found to be reduced significantly (p<0.05) in treated samples. Presence of different phytochemicals
that have effective antimicrobial properties of M.oleifera extract against wide range of bacteria which were responsible for
accumulation of ammonia and volatile bases in fish flesh, decreased protein breakdown, thus lowering TVBN and TMA values.
The antioxidant properties of polyphenols present in the M. oleifera leaf extract retarded the formation of malonaldehydes.
From the present study, it can be concluded that 15% concentration of M. oleifera leaves extract is considered as most effective
to maintain the quality of the minced Pangasius hypophthalmus during five months frozen storage at -18±2OC.
Key words : Moringa oleifera, Pangasius hypophthalmus, frozen storage, antimicrobial, antioxidant.
INTRODUCTION
Fish is highly perishable and is readily susceptible to
chemical and microbial deterioration (Gram and Huss,
2001) due to presence of polyunsaturated fatty acids and
essential amino acids in a large quantity. Pangasius
hypophthalmus is one of the most important commercial
fishes with high nutritive qualities and excellent sensory
properties such as tender flesh, sweet taste, absence of
fishy odor and spines, delicate flavor and firm texture
during cooking that help to gain its consumer preference.
Increasing market demand for this fish proves its
acceptance by the consumers almost all over the world
(Phan et al, 2009). With the availability of this fish in
large quantity, there is a good potential for development
of convenience products such as fish cutlets, fish fingers,
canned fish and fish curry in retort pouches (Rathod and
Pagarkar, 2013).
Lipid oxidation is a major cause of muscle food
deterioration leading to subsequent off-flavours,
unpleasant odours, texture, discolouration and decrease
in nutritious value (Frankel, 1998). To retain the good
quality characteristics for longer and extend the shelf life
during frozen storage of fish, chemical preservatives such
as butylated hydroxy anisole (BHA) and butylated
hydroxytoluene (BHT), have been widely used. Parallelly,
numerous studies are focused on using natural ingredients
to enhance fish quality and shelf life in order to avoid the
use of harmful synthetic preservatives (Khanedan et al,
2011). Many plant tissues are good sources of
phytochemicals, notably phenolic and flavonoids
(Gorinstein et al, 2005), that can act as the best alternative
to the mutagenic food additives.
Moringa oleifera (commonly called drumstick) is
one of such most important terrestrial plants which exhibit
antibacterial, antifungal as well as antioxidant properties
which influence its application in food preservation. Due
to the presence of many important substances like
ascorbic acid, estrogenic substances, beta sitosterol, iron,
calcium, phosphorus, copper, vitamin A, B and C, alpha
tocopherol, beta carotene, protein and essential amino
acids like methionine, cystine, tryptophan and lysine,
drumstick leaves can be considered as a dietary
supplement. Ethanolic extract of M. oleifera showed a
Biochem. Cell. Arch. Vol. 17, No. 2, pp. 463-469, 2017 www.connectjournals.com/bca ISSN 0972-5075

464 J. Chanraborty et al
broad spectrum antimicrobial property against many
pathogens including Staphylococcus aureus,
Escherichia coli, Bacillus sp., Pseudomonas
aeruginosa, Cornebacterium sp., Klebsiella
pneumonia and Acinetobacter sp. (Rajamanickam and
Sudha, 2013) due to presence of phytochemicals such as
flavonoids, saponins, tannins and other phenolic
compounds (Sato et al, 2004).
Along with its anti-inflammatory, anti-helminthic,
antifungal, pro-cogulant and flocculating properties,
Moringa leaves have been reported to be a good source
of natural antioxidants and thus, enhance the shelf life of
fat containing foods due to the presence of various types
of antioxidant compounds such as ascorbic acid,
flavonoids, phenolics and carotenoids (Siddhuraju and
Becker, 2003). This plant is of special interest in food
preservation because in addition to contributing taste and
aroma to foods, it also contains a variety of bioactive
substances, which are of considerable use in extending
shelf life. It can become a promising natural antimicrobial
agent with potential application in pharmaceutical and
food processing industry for controlling the pathogenic
bacteria as well as enhancing food safety and security.
Thus, the objective of present study is to determine the
shelf life of the washed mince of Pangasius
hypophthalmus treated with different concentration of
Moringaoleifera leaf extract under frozen (-18±2OC)
storage and to standardize the concentration during frozen
storage of minced meat of Pangasius hypophthalmus.
METHODOLOGY
Preparationof plant leaf extract (Moringa oleifera)
Fresh Moringa oleifera leaves obtained were
washed well with water to remove the adhering dust.
Then the leaves were air dried properly. 20 g of fresh
leaves were homogenized with 180 ml of ethanol-water
solution containing 50% sterile distilled water and 50%
ethanol as described by Peixoto et al (2011). The liquid
portion was separated from the residue by filtration using
Whatman no. 1 filter paper and this solution was used as
Moringa oleifera leaf extract in the experiment.
Preparation of fish mince from raw fish (Pangasius
hypophthalmus)
The fishes (Pangasius hypophthalmus) procured
from Howrah fish market were beheaded, descaled,
filleted and skinned manually. The fillets were fed into
silent cutter and the mince thus collected was washed
thoroughly by following three washing cycles using chilled
water. The meat was pressed using screw press to drain
off the excess water. Mince was prepared under good
hygienic and sanitary conditions to prevent any cross
contamination and was used for further studies.
Determination of the shelf life of the washed mince
treated with Moringa oleifera leaf extract under
frozen (-18±2oC) storage condition
The study was conducted in a completely randomized
design for four treatments, prepared by manually mixing
the raw mince with Moringa oleifera leaf extract at
different concentrations (5%, 10% and 15%) and were
blended for 1 minute (Hazra et al, 2012) and keeping
another sample without any treatment. Samples were
then placed in Styrofoam trays and stored at ×18±2ºC
for 5 months. The treatments were (i) Pangasius mince
without any treatment i.e. control (C), (ii) mince treated
with Moringa oleifera extract at 5% concentration (5
ml extract/100 g mince) (S1), (iii) mince treated with
Moringa oleifera extract at 10% concentration (10 ml
extract/100 g mince) (S2), (iv) mince treated with
Moringa oleifera extract at 15% concentration (15 ml
extract/100 g mince) (S3).
Proximate composition of the raw Pangasius minced
meat was determined only once before frozen storage
that includes protein, fat, ash and moisture content.
Moisture of the experimental samples was measured by
Moisture Balance (Precisa, Dietikon, Switzerland). Total
nitrogen was estimated by Kjeldahl method (AOAC,
1995). Crude protein value was calculated by multiplying
the total nitrogen value by a factor of 6.25. Estimation of
total lipid was done by the method described by Bligh
and Dyer (1959). The ash content was measured by the
method of AOAC (1995). All the results were expressed
on wet weight basis.
The treatments were subjected in triplicate for Total
Plate Count (TPC) and physicochemical analyses at
fortnight interval starting from day 0. TPC was
determined by spread plating appropriate dilutions on Total
Plate Count Agar (Hi-media) (Nath et al, 2014) and
results expressed as log cfu/g. The physicochemical
indices used to analyse the shelf life of fish mince
wasTotal volatile basic nitrogen or TVBN (by the method
described by Nath et al, 2014), Tri Methyl Amine or TMA
(by the method described by AOAC, 1995) and Thio-
Barbituric Acid or TBA value (by the method described
by Tarladgis et al, 1960).
RESULTS AND DISCUSSION
The moisture, protein, lipid and ash contents of fresh
Pangasius hypophthalmusminced meat were estimated
to be 76.25%, 17.42%, 4.37% and 1.24% respectively
(Figure 1) which reveals that the fish had low moisture,
high protein and moderate fat content. Viji et al (2014)
reported that the proximate composition of the fresh

Influence of M. oleifera leaf extract on microbial and quality parameters of P. hypophthalmus 465
sutchi catfish meat was found to be 77 % moisture, 16.5%
protein, 4% crude fat and 0.97% ash, which supports the
findings of the present study. Rao et al (2013) reported
that the proximate composition of P. hypophthalmus fish
fillet was 17.24%, 78.2%, 2.84% and 1.3% for protein,
moisture, crude fat and ash content respectively. Similar
findings were also established by Rathod and Pagarkar
(2013), where moisture and protein contents of
Pangasius were reported to be 76.6% and 14.4%
respectively. High protein content (15.97%) in
Pangasianodon hypophthalmus was also reported by
Islami et al (2014). Proximate composition of fishes is
highly variable mainly due to species, catching season,
environment, diet, sex and age (Boran and Karaçam,
2011). Mushahida-Al-Noor et al (2012) affirmed that
different diets result in a wide variation in proximate
composition of Pangasius hypophthalamus i.e. 74.10%
to 79.15% moisture content, 15.50% to 16.60% protein,
4.08% to 8.08% lipid content and 1.20% to 1.24% ash
content.
The maximum acceptable count for fresh water fish
is 107 CFU/g as recommended by International
Commission on Microbiological Specification for Foods.
The changes in TPC in all the samples showed a gradual
increase from an initial value of 3.56 log CFU/g and finally
reaching to 6.92 log CFU/g, 6.67 log CFU/g, 6.46 log
CFU/g and 5.89 log CFU/g for control, S1, S2 and S3
respectively after 5 months of frozen storage (Figure 2).
The TPC in control sample was significantly (P<0.05)
higher than the treatments over a storage period of 5
month suggesting that the M. oleifera leaf extract
possesses antimicrobial properties that resulted in growth
inhibition of bacteria. This can be supported by the findings
of Sato et al (2004) who reported that the leaves of M.
oleifera contain a number of phytochemicals such as
flavonoids, saponins, tannins and other phenolic
compounds that have antimicrobial activities against
microorganisms. During the study, a sudden decrease in
TPC value was encountered in all the samples on the
15th day of storage which may be due to the effect of
cold shock of freezing temperature on the microorganisms
present in the minced meat. This corroborates with the
findings of Ranken (2000), who showed that the TPC
value of the chicken meat ball samples reduced during
the first 20 days of frozen storage due to the effect of
freezer temperature on the microbes in extending their
lag phase. After 20 days of storage, a significant increment
of the microbes occurred due to the adaptability of the
microbes to the freezer temperature (Sinhamahapatra et
al, 2013). The results of the present study are in
agreement with the findings of Hazra et al (2012) also
reported that there is a significant (p<0.05) reduction in
TPC in cooked ground buffalo meat treated with Moringa
leaf extract at 1%, 1.5% and 2% levels (log 2.95±0.29
CFU/g, 2.72±0.17 CFU/g and 2.65±0.19 CFU/g
respectively) as compared to control (log 2.96±0.22 CFU/
g) during refrigerated storage. Likewise, Adeyemi et al
(2013) also reported that after eight weeks of storage of
smoked dried African catfish (Clarias gariepinus), the
microbial load in the control was found to be >3.0×108
CFU/g, where as in the samples treated with 1%, 2%
and 3% M. oleifera marinade (MOM), the microbial load
was 5.0×107 CFU/g, 6.0×107 CFU/g and 7.0×106 CFU/g
respectively stored under room temperature. Falowo et
al (2016) reported that the raw ground beef meat treated
with Moringa leaf stalk extract showed comparatively
lower Total Viable Count (5.28±0.53 log CFU/g) than
the control (5.80±1.05 log CFU/g) after 3 days of storage
at 4OC. In the present study, S3 exhibited the lowest TPC
at the end of the 5 months storage period under frozen
storage (-18±2°C) suggesting that 15% concentration of
Moringa oleifera leaves extract possesses the best
antimicrobial properties against fish mince (Figure 2).
TVBN serves as an indicator for the assessment of
the freshness of fish. Enzymes from spoilage
microorganisms, particularly proteolytic enzyme, can
metabolize the amino acids of the fish muscle producing
a wide variety of volatile compounds resulting in off
flavours and odors. The total chemical compounds of
trimethylamine or TMA (produced by spoilage bacteria),
dimethylamine or DMA (produced by autolytic enzymes
during frozen storage), ammonia (produced by the
deamination of amino-acids and nucleotide catabolites),
and other volatile basic nitrogenous compounds associated
with seafood spoilage are measured in TVB-N test (Huss,
1995). The TVB-N value between the ranges of 30–35
mg/100 g for flesh is generally regarded as the limit of
acceptability for ice stored cold-water fish for human
consumption (Castro et al, 2006). In the present study,
the initial TVBN content was 10.20 mg % that finally
raised to 29.12 mg %, 28.53 mg %, 25.77 mg % and
24.85 mg % for control, S1, S2 and S3 respectively at
the end of 150 days of frozen storage (-18 ±2°C) (Figure
3), thus, remained within the acceptable limits of 30–35
mg/100 g. The TVBN in control sample was significantly
(p<0.05) higher than the treatments over a storage period
of 5 month suggesting a significant influence of Moringa
extract in reducing TVBN value which may be due to
the presence of different phytochemicals that have
effective antimicrobial properties against wide range of
bacteria, which are responsible for accumulation of
ammonia and volatile bases in fish flesh. The increase in

TVBN values during the ûrst month could be attributed
to bacterial activity and endogenous enzymes of ûsh
(Ibrahim and El-Sherif, 2008). Hossain et al (2005)
reported that in Thai Pangas (Pangasius sutchi) TVBN
value was found to be 24.25 mg/ 100g at 20th day of ice
storage which was within the range of acceptable value.
But, at the end of 25th day, the TVBN value increased to
40.1 mg/ 100 g which exceeded the recommended level.
Manthey (1988) established that lower storage
temperature may result in lower increase in TVBN value
during the storage period. This can be supported by the
finding of Anderson (2008), who reported that in frozen
hilsa fish stored at –20°C, TVBN value increased from
5.60 to 27.20 mg/100 g at the end of 75 days. Suvanich
et al (2000) stated that the increase in TVBN values in
of minced fish at the end of frozen storage was may be
due to the breakdown of endogenous compounds into
non-protein N-compounds (Vareltzis et al, 1997). But,
studies on storage of different frozen fish species, it was
suggested that TVB-N values might change depending
on the spoilage flora and analysis method (Dalin et al,
2013). TVBN value may also depend on the species,
catching methods, season and region, age and sex of the
fish (Nasopoulou et al, 2012). In the present study, S3
exhibited significantly p<0.05) lowest values of TVBN
at the end of the 5 months storage period under frozen
storage (-18±2°C) suggesting that 15% concentration of
Moringa oleifera leaves extract is most effective in
maintaining the acceptable level of TVBN values in fish
mince (Figure 3).
TMA is the most commonly used volatile amine in
the fish industry for evaluating freshness and spoilage in
fish, since it is produced from bacterial utilization of
trimethylamine oxide (TMAO), a naturally occurring
osmoregulatory substance found in the fish species.
Formation of Trimethylamine (TMA) is caused by
reduction of Trimethylamine oxide (TMAO) by bacterial
activity and partly by intrinsic enzymes and often used
as index of freshness of marine fish (Villareal and Pazo,
1990). The maximum permissible limit of TMA is 5 mg
N/100g flesh beyond which seafood will develop an
objectionable odour and taste (EOS, 2005). In the present
study, the initial TMA content was 0.57 mg N/100 g which
finally increased to 4.69 mg N/100 g, 4.11 mg N/100 g,
3.82 mg N/100 g and 3.58 mg N/100 g for control, S1, S2
and S3 respectively after 5 months of frozen storage (-
18 ±2°C) (Figure 4) retaining all the values within limit.
TMA accumulation is a result of bacterial breakdown of
TMAO and this occurs to a significant level only during
logarithmic phase of microbial growth (Huss, 1988).
During frozen storage, the increase in TMA content of
all the samples is due to action of spoilage bacteria and
endogenous enzyme, which imparts an unpleasant “fishy”
odor (Kilinc and Cakli, 2004). Natseba et al (2005)
reported that freezing inhibits bacterial activity and thereby
inhibits TMA accumulation. Hozbor et al (2006) found a
strong correlation between the microbiological changes
in sea salmon stored in ice and other quality indices like
TMA-N, TVN-N and Histamine, indicating that, with the
decrease in microbial activity, there is simultaneous
decrease in TMA value. In the present study, it is clearly
noticed that all the treatments showed significantly
(p<0.05) lower values of TMA than the control throughout
the storage period among which S3 (15% concentration
of Moringa oleifera extract) was lowest which may be
due to the ability of Moringa extract to reduce microbial
load, thereby decreasing protein breakdown and hence
lower values of TMA were resulted. This finding can be
supported by the report of Siddhuraju and Becker (2003)
stating that the plant has broad spectrum antimicrobial
activity against both gram-positive and gram-negative
bacteria due to the presence of antibiotic compounds in
it.
TBA is the secondary breakdown product of lipid
oxidation and is widely used as an indicator of the degree
of lipid oxidation (Ucak et al, 2011). The level of tissue
malonaldehyde, a secondary degradation product of lipid
present, is often measured in order to assess the extent
of lipid peroxidation that has occurred in biological systems
(Khayat and Schwall, 1983). A TBA value in the range
of 1–2 mg malonaldehyde/kg of fish sample is usually
taken as the limit of acceptability (Lakshmanan, 2000)
beyond which (2 mg malonaldehyde/kg) the product will
smell and taste. During frozen storage (-18±2OC), the
initial TBA value was 0.23 mg MDA/kg which finally
increased to 1.30 mg MDA/kg for control, 1.24 mg MDA/
kg for S1, 1.13 mg MDA/kg for S2 and1.09 mg MDA/kg
for S3 sample after 5 months (Figure 5), all remained
within optimum limit. This may be due to the antioxidant
properties of polyphenols present in the Moringa oleifera
extract that retarded the formation of malonaldehydes
as reported by Sarah et al (2010). O’Byme et al (2002)
showed that polyphenols in Moringa extract act as chain
breaking peroxyl radical scavengers which lead to the
inhibition of lipid peroxidation and also prevent low density
lipoprotein oxidation. Hazra et al (2012) reported that
the cooked ground buffalo meat treated with 1%, 1.5%
and 2% Moringa oleifera leaves extract showed
significantly (p<0.05) lower TBA values as compared to
control during refrigerated storage (4±1OC) among which
the lowest value observed in 1.5% extract treated sample.
The increase in TBA value during the storage may be
466 J. Chanraborty et al

attributed to the partial dehydration of fish and to the
increased oxidation of unsaturated fatty acids. The
increase in TBA values of Moringa oleifera treated
samples was significantly (p<0.05) lower than the control
sample throughout the storage period of 5 months
suggesting that Moringa leaves extract can extend the
shelf life of fish samples by inhibiting lipid oxidation in
fish. The present result is in agreement with the findings
of Pari et al (2007) stating that Moringa oleifera leaves
extract can potentially be used as preservatives as well
as antioxidants in food industry due to its antibacterial
and antioxidative activities. TBARS values first increased
Influence of M. oleifera leaf extract on microbial and quality parameters of P. hypophthalmus 467
during frozen storage due to the accumulation of products
from lipid oxidation especially Malonaldehyde (MA) and
malondialdehyde (MDA) and then a lower increase rate
due to interaction of MA and MDA with protein, amino
acid, glycogen etc. resulting in lower amount of free MDA
(Goulas and Kontominas, 2007). Under frozen
temperatures (–5.150 to –18.15°C), there is a decrease
in activity of spoilage microorganisms and enzymes
present in fish products, delaying the formation of
metabolites from protein degradation (Bazarra et al,
2015). However, low temperatures do not prevent lipid
oxidation totally due to the action of endogenous
Fig. 1 : Proximate composition of raw Pangasius minced meat. Fig. 2 : Changes in TPC of Pangasius minced meat treated with
Moringa oleifera extract under frozen condition (-18±2°C).
Fig. 3 : Changes in TVBN of Pangasius minced meat treated with
Moringa oleifera extract under frozen condition (-18 ±2°C).
Fig. 4 : Changes in TMA-N of Pangasius minced meat treated with
Moringa oleifera extract under frozen condition (-18±2°C).
Fig. 5 : Changes in TBARS of Pangasius minced meat treated with Moringa oleifera extract under frozen condition (-18±2°C).
*Results are mean of three
determinations (n=3) with s.d, #
Values vary significantly (p<0.05)
among the treatments.
**C= Control, S1= 5ml M. oleifera
extract/100g mince, S2= 10 ml M.
oleifera extract/100g mince, S3= 15
ml M. oleifera extract/100g mince.

468 J. Chanraborty et al
lipoxygenases even under frozen conditions (Karlsdottir
et al, 2014), which explains the deterioration of lipid during
frozen storage. The present study reveals that among the
treated samples, 15% concentration always showed
significantly (p<0.05) better result than the other treated
samples during measuring product stability and rancidity
by TBA assay.
From the present study, it is observed that, although,
all the samples (both control and treatments) showed a
trend of deterioration during 5 months frozen storage
period at -18±2OC, whereas the rate was much lower in
case of M. oleifera leaves extract treated samples than
control. Among the treatments, the quality of the minced
meat sample treated with 15% concentration of Moringa
leaves extract (S3) was found to be the best in terms of
TPC, TVBN, TMA-N, TBARS and protein solubility at
the end of frozen storage. Hazra et al (2012) reported
that among 1%, 1.5% and 2% levels of aqueous solution
of crude extract of drumstick, 1.5% concentration was
the most effective for treating cooked ground buffalo meat
during refrigerated storage at 4±1OC. According to
Adeyemi et al. (2013), 3% (W/V) Moringa oleifera
marinade (MOM) was found to be better than 1% and
2% MOM when treated with smoked dried African
catfish (Clarias gariepinus) stored at ambient
temperature for two months. Hence, from this present
study, it can be concluded that 15% concentration of
Moringa oleiferaleaves extract is considered as most
effective to maintain the quality of the minced Pangasius
hypophthalmus during five months frozen storage at
-18±2OC.
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