Effect of Chilling and Freezing on Fish Muscle

Abstract

Muscle samples of Mystus seenghala were stored at two different low temperatures, i.e at 4±1 o C (chilled) and at -12±2 o C (frozen) for 21 days. Weekly analysis was conducted to measure protein, lipid, ash, moisture, free fatty acid, pH and total plate count. The result clearly reveals that during storage, both the samples showed a highly significant (p<0.01) decreasing trend in protein, lipid, ash and moisture content. After 21 days, the percentage decrease was 54.35% and 22.70% for protein and 82.64% and 56.68% for lipid, 10.96% and 4.89% for moisture and 38.92% and 37.88% for ash content in chilled and frozen sample respectively. However, the free fatty acid and pH showed highly significant (p<0.01) increasing trend in both the samples. Similarly, the bacteriological studies revealed that the total plate count (TPC) in chilled and frozen samples also showed an increasing trend. It was found within acceptable limits (TPC=6.04±0.11 log cfu/g) in chilled sample upto 10 th day and up to 14th day (TPC=5.78±0.2 log cfu/g) in frozen sample. Thereafter, the microbial quality further deteriorates and became inedible for human consumption.

Full text

IOSR Journal of Pharmacy and Biological Sciences (IOSRJPBS)
ISSN: 2278-3008 Volume 2, Issue 5 (Sep-Oct. 2012), PP 05-09
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Effect of Chilling and Freezing on Fish Muscle
Roopma Gandotra, Shalini Sharma*, Meenakshi Koul and Sweta Gupta
Department of Zoology, University of Jammu, Jammu And Kashmir
*Email: shalini.shelly17@yahoo.com
Abstract: Muscle samples of Mystus seenghala were stored at two different low temperatures, i.e at 4±1oC
(chilled) and at -12±2oC (frozen) for 21 days. Weekly analysis was conducted to measure protein, lipid, ash,
moisture, free fatty acid, pH and total plate count. The result clearly reveals that during storage, both the
samples showed a highly significant (p<0.01) decreasing trend in protein, lipid, ash and moisture content. After
21 days, the percentage decrease was 54.35% and 22.70% for protein and 82.64% and 56.68% for lipid,
10.96% and 4.89% for moisture and 38.92% and 37.88% for ash content in chilled and frozen sample
respectively. However, the free fatty acid and pH showed highly significant (p<0.01) increasing trend in both
the samples. Similarly, the bacteriological studies revealed that the total plate count (TPC) in chilled and frozen
samples also showed an increasing trend. It was found within acceptable limits (TPC=6.04±0.11 log cfu/g) in
chilled sample upto 10th day and up to 14th day (TPC=5.78±0.2 log cfu/g) in frozen sample. Thereafter, the
microbial quality further deteriorates and became inedible for human consumption.
(Keywords: chiller, freezer, spoilage, acceptable limit, inedible.)
I. Introduction
Fish has been playing an important role in addressing nutritional and livelihood security of people in
the developing countries. Besides, it is good source of polyunsaturated fatty acids (PUFA’s), protein, minerals
and vitamins which are vital to our health. Although fish is highly nutritious, yet it is one of the most rapid
perishable foods because of its short shelf life. The extension of shelf life can be achieved by freezing, chilling,
salting, smoking, glazing etc. Consumers usually buy fish in bulk and store in refrigerator. Deterioration of fish
quality in refrigerator storage have great impact on the nutritious value of fish and the health of consumers.
Considering the importance from consumer view point, this study was designed to study the effect of two
different low temperatures (4±1oC and-12±2oC) on fish quality.
II. Materials And Methods
Fresh samples of Mystus seenghala were purchased from local market. They were immediately brought
to the lab in polythene bags along with crushed ice. The viscera of fish were removed; the fish was washed with
water and cut into pieces. These pieces were washed and immediately wrapped in aluminium foil, kept in air
tight plastic container and stored at 4±1°C (chilled storage) and at-12±2°C (frozen storage). Analytical
procedures for biochemical and microbiological changes were done on 0, 7th, 14th and 21st day of storage.
1.1. Biochemical composition:-
 Moisture content was measured by using hot air oven and aluminium moisture dishes.
 Ash content was measured by using Muffle furnace,
 Total lipid content was estimated by the method of Folch et al. (1957).
 Total protein content was estimated by following the method of Lowry et al. (1951).
 pH was measured by the method of Keller et al. (1974) using digital pH meter.
 Free fatty acid was determined by method of US Army laboratories (Natick) described by Koniecko (1979).
1.2. Bacteriological Profile
Total plate count (TPC) in the fish muscle was determined by method described by APHA (1984).
Readymade media (Hi-media) were used for the analysis. Serial ten-fold dilutions were made for inoculation.
The sample preparation was done near flame under laminar flow.
1.3. Statistical Analysis
Means and standard errors were calculated for different parameters, the data analysis was performed
using SPSS software. Differences between treatments were analysed by using independent measures one way
ANOVA. Post-hoc comparisons were conducted using Ducan’s test. The values were expressed as mean ±SE. p
value <0.05 were considered as significant and p value <0.001 were considered as highly significant.

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III. Result and Discussion:
3.1. Total Protein Content
At 4±10C, the initial value of total protein content of muscle of Mystus seenghala was found to be
18.01±0.06% and on 21st day this value was found to be least i.e. 8.22±0.2%. At -12±20C, it was found to be
18.81±0.06 % on the first day of frozen storage and on the last day i.e., 21st day of experimentation the value
was further decreased to 14.54±0.04%. The overall decreased was found to be highly significant (p≤0.01). In
present investigation, decreasing trend was observed. In chilled sample, there was 54.35% decrease, whereas in
frozen samples, there was 22.70% decrease after 21st day of storage. The present results were found to be in line
with those of Kandeepan and Biswas (2007). They found 23% decrease in total protein content of buffalo meat
at 4±10C (Chilled Storage) and 3.5% decrease at -10±10C (Frozen Storage) after 7 days. According to them, the
lower protein content of chilled meat at 4±10C was due to increased microbial growth resulted from higher
water activity (aw) and enzymatic autolysis and on frozen storage the protein content was decreased due to
protein denaturation and proteolysis induced by enzymatic activities of psychrotrophic microbial growth.
Siddique and Ali (1979) attributed the decrease in protein content of prawn during ice storage to the leaching
effect of the amino acids and water soluble proteins leaching out with melting ice. Similarly, Kolodjiejska et al.
(1987) while working on biochemical changes in fish muscle during low temperature storage found a
remarkable rate of denaturation and autolysis of fish protein. Zamir et al. (1998) calculated 21.79% decrease in
total protein content after 7 days of storage of crab meat at 7±20C. Arannilewa et al. (2005) calculated 27.94%
decrease in protein content of Tilapia (Sarotherodun galiaenus) after 60 day of frozen storage. Siddique et al.
(2011) while assessing the effect of freezing time on nutritional value of Puntius sophore, P. sarana and P.
gonionotus registered similar trends during the frozen storage at -50C of 20 days. They observed 9.44% decrease
in P. sophore, 9.94% decrease in P. sarana and 5.34% in P. gonionotus in protein content during frozen storage
of 20 days.
3.2. Total Lipid Content
At 4±10C, the total lipid content was found to be decreased from initial value of 4.84±0.015% (0 day)
to the final value of 0.84±0.04% (21st day). At -12±20C, it was estimated to be 4.94±0.03% (o day zero) and
reaches up to 2.14±0.06 % (21st day). There was 82.64% decrease in total lipid content when stored at 4±10C
and when stored at -12±20C, there was 56.68% decrease in total lipid content after 21st day. The overall decrease
found to be highly significant (p ≤0.01). These results get strong support from the findings of Kandeepan and
Biswas (2007) who conducted a similar experiment on buffalo meat. They found 47.94% decrease in chiller
(4±10C) and 17.80% decrease in freezer in total lipid content during the storage period of 7 days. They
attributed this marked decrease in chiller to the exposure of strong light, as in display cabinets, which
accelerated oxidation of fats causing discoloration. In freezer they extended the storage up to 75 days. During
this prolonged storage, the lipid oxidation occurred mainly due to losses in triglyceride fraction. Agnihotri
(1988) reported deterioration in meat lipids took place due to intermediary activities of endogenous meat
enzymes leading to hydrolysis of fat. Zamir et al. (1998) attributed the loss in lipid of crab meat stored at
refrigerator temperature 7±20C for one week due to the oxidative rancidity. Arannilewa et al. (2005) calculated
25.92% decrease in total lipid content in Tilapia after storing it in freezer compartment of the refrigerator for 60
days and associated the changes in fat content during frozen storage with the oxidation of fat. Siddique et al.
(2011) found that total lipid content decreased during frozen storage of three species of Puntius. They calculated
35.34%, 19.24% and 21.78% decrease in total lipid content in P. sophore, P. sarana and P. gonionotus
respectively.
3.3. Moisture Content
At 4±10C, the initial moisture content in raw fish muscle was 88.64±0.1%. On the final day of storage,
it was observed to be 78.92±0.02%. At -12±20C, the initial moisture content in raw muscle stored in freezer was
88.04±0.04% and on 21st day of the storage, the value decreased to 83.03±0.02%. The overall decrease found to
be highly significant (p ≤0.01). Here, the percent decrease in moisture content of raw fish stored at 4±10C was
calculated to be 10.96% decrease on 21st day, while it was calculated to be 4.89% decrease at -12±20C.These
results are in agreement with those observed by Kandeepan and Biswas (2007), when they stored buffalo meat
in chiller and freezer compartment of refrigerator. Here, the percent decrease in moisture loss was found to be
5.11% in chiller and 1.57% in freezer after seven days of storage. They advocated that the more decrease in
moisture content was due to evaporation of moisture from meat in chiller, whereas the decrease in moisture
content was due to sublimation of surface water of the meat in the freezer. In freezer, the storage was extended
up to 75 days, the significant moisture losses in later storage periods was due to the myofibrillar distortion
resulting in the poor water detention ability of the meat. According to Kirschnik et al. (2006), moisture content
was constant for 14 days in samples of tail meat of the giant river prawn, (Macrobrachium rosenbergii) stored
without direct contact in ice, while in samples stored in direct contact with ice it increased approximately 6%,

Effect Of Chilling And Freezing On Fish Muscle
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revealing absorption of water through exposed surface of meat and decrease in solid content (crude protein)
during early stages of storage in ice.
3.4. Ash Content
Fish muscle at 0 day of storage at 4±10C, the ash content was found to be 1.49±0.09% and the final
value on 21st day was found to be 0.91±0.03%. At -12±20C, it was about 1.61±0.07% on day zero and on 21st
day it was estimated about 1±0.02%.
The overall decrease found to be highly significant (p ≤0.01). Okeyo et. al. (2009) observed that the ash
context of the frozen raw Nile perch decreases with storage time. They calculated 12.69% decrease after 22 days
of ice storage. However, Kandeepan and Biswas (2007) registered 14.87% decrease in chiller and 20.66%
decrease in freezer after 7 days of storage. According to Arannilewa et al., 2005, the ash content remains almost
the same throughout the sixty days frozen storage of Tilapia. It changed from 26.13±2.20 (recorded on 0 day) to
26.80±1.44 (recorded on 60th day).
3.5. Free Fatty Acid (FFA)
At 4±10C in present studies, it was observed to be 0.5±0.01% on day 0 and 12.27±0.01% on 21st day.
At -12±20C, it was 0.57±0.02% on day zero and 5.61±0.05% on 21st day of storage. Here, more FFA content
(12.27±0.01%) was observed in chilled muscle as compared to FFA content (5.61±0.05%) in frozen muscle
after 21 days. At 4±10C, the raw fish sample was found to be near the acceptable limit (5%) on 7th day
(4.89±0.04%) and at -12±20C, the sample crossed the acceptable limit on 21st day (5.61±0.05%). Rodriquez et
al. (2007) observed increasing FFA during frozen storage farmed coho salmon (Oncorhynchus kisutch).
According to Pacheo-Aguillar et al. (2000) during post mortem period, lipid (glycerol-fatty acids esters) present
in the fish muscle undergo hydrolysis, resulting in the release of fatty acids. This is also supported by Okeyo et
al. (2009), who stated that the accumulation of FFA could be due to lipases and phospho-lipase activity in
digestive organs in muscle of Nile perch.
Table: 1 Changes in raw muscle of Mystus seenghala stored under chilled condition at 4±10 C.
Table: 2 Changes in raw muscle of Mystus seenghala stored under frozen condition at -12±20 C.
-Mean±SD with different superscript in a row differs significantly (P<0.05).
3.6. pH
In present studies, fish muscle that was stored in chiller (4±1o C) showed rapid increase in pH, while
when in freezer (-12±2o C), there was comparatively slow increase in pH. It increased from 6.8 to 7.4 in chilled
Days
Total
Protein
(%)
Total
Lipid (%)
Moisture
(%)
Ash (%)
FFA(%)
pH
TPC(log10
cfu/g)
0
18.01±0.06a
4.84±0.2a
88.64±0.1a
1.49±0.09a
0.5±0.01a
6.8a
2.74±0.2a
7
15.97±0.02b
3.03±0.25b
83.02±0.015b
1.12±0.025b
4.89±0.04b
7.0b
6.04±0.11b
14
11.93±0.03c
1.95±0.02c
80.81±0.09c
1.04±0.04c
8.17±0.7c
7.2c
7.30±0.07c
21
8.22±0.2d
0.84±0.04d
78.92±0.02d
0.91±0.03d
12.27±0.01d
7.4d
9.90±0.02d
Days
Total
Protein (%)
Total
Lipid (%)
Moisture (%)
Ash (%)
FFA(%)
pH
TPC(log10
cfu/g)
0
18.81±0.06a
4.94±0.03a
88.04±0.04a
1.51±0.07a
0.57±0.02a
6.8a
2.70±0.09a
7
17.02±0.02b
3.83±0.1b
86.82±0.1b
1.30±0.1b
1.52±0.13b
6.9b
4.30±0.11b
14
15.78±0.03c
2.74±0.11c
85.07±0.7c
1.12±0.74c
2.32±0.03c
7.0c
5.78±0.2c
21
14.54±0.04d
2.14±0.06d
83.73±0.07d
1±0.02d
5.61±0.05d
7.2d
7.77±0.1d

Effect Of Chilling And Freezing On Fish Muscle
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sample and from 6.8 to 7.1 in frozen sample during 21 days. These results are in line with those of Kandeepan
and Biswas (2007) while conducting experiment on chilled and frozen buffalo meat. Erkan and Ozden (2008)
stated that the increase was due to an increase in volatile bases from the decomposition of nitrogenous
compounds by endogenous or microbial enzymes. Obemeata et al. (2011) observed that the increase in pH was
higher in the 4oC stored sample of fish-Tilapia, than in the -18 oC stored samples, indicating that biochemical
and microbial changes are occurring faster in the fish of 4 oC stored fish.
3.7. Total Plate Count (TPC)
As recommended by International Commission on Microbiological Specification for Food,
ICMSF, (1986), an increase of total plate count (TPC) up to levels exceeding the value of 6 log CFU/g is
regarded as microbial spoiled fish muscle not fit for human consumption. In present investigation at 4±10C, the
bacterial load in raw fish muscle for period of 21 days showed considerable increase. The TPC in raw fish
muscle on day zero was rather low i.e. 2.74±0.2 log cfu/g and on 21st day of storage, it was increased further to
the final value of 9.90±0.02 log cfu/g. At -12±20C, the TPC in fish muscle on day 0 was found to be low in
2.70±0.09 log cfu/g and at the end of storage i.e. 21st day, it was found to be 7.77±0.2 log cfu/g. In present
investigation, it was clearly depicted that the microbial growth was more rapid with increasing storage
temperature. It has been reported that at -12±20C, the TPC (5.78 log cfu/g) was found to be within the
permissible limit on 14th day of storage whereas at 4±10C, the TPC (6.04 log cfu/g) crossed the permissible
limit on 7th day of storage. Lawire (1998) attributed the microbial growth to the growth promoting effect of
moisture on microbes in meat stored in chiller. Bao et al. (2007) reported a faster microbial growth in chilled
than in superchilled samples of Arctic Charr fillet under the effect of dry ice and superchilling. Liu et al. (2010)
reported increase in mesophilic bacterial load was found to be 85% in tray packed tilapia fillets stored at 00C.
They calculate initial load of 3-4 log CFU/G/g, reaching 7.4 log CFU/G/g on day 13.Obemeata et al. (2011)
showed an increase in bacterial count from 7.9×103 to 7.6×107 cfu/g when Tilapia fish was stored at 40C and
from 7.9×103 to 5.4×101cfu/g when Tilapia was stored at -180C. They stated that freezing of fish at -180C
created an unfavourable environmental condition for the growth and the survival of the micro-organisms, while
freezing at 40C allows the rapid proliferation of the micro-organisms.
IV. Conclusion
Freezing of fish muscle at -12±2o C causes comparatively lesser spoilage than chilling at 4±1o C. This
study reveals that as the storage period increases, there occurs degrading changes in biochemical and
bacteriological composition that has direct effect on shelf life and market value of the fish. Freezing of fish
creates unfavourable environmental conditions which slow down the bacterial growth and biochemical
decomposition of fish muscle, thereby increasing the shelf life; while chilling at 4±1oC allows the comparatively
rapid proliferation of bacteria, protein denaturation, lipid hydrolysis and oxidation; thereby reducing the shelf
life. Hence, for storage purposes freezing is recommended, since frozen fish muscle will have the enhanced
shelf life.
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