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Aquatic Procedia 7 (2016) 285 295

2nd International Symposium on Aquatic Products Processing and Health

ISAPPROSH 2015

Nano-chitosan Utilization for Fresh Yellowfin Tuna Preservation

Yosmina Tapilatua,*, Prihati Sih Nugrahenib, Tamara Ginzela, Mattheus Latumahinac,

Ginno Valentino Limmond, Wiratni Budhijantoe
a
Centre for Deep-Sea Research, Indonesian Institute of Sciences, Jl. Y, Syaranamual Guru-guru Poka Ambon 97233, Indonesia
b
Fishery Department, Faculty of Agriculture, Gadjah Mada University, Bulaksumur Yogyakarta 55281, Indonesia
c
Fisheries Product Technology Department, Faculty of Fisheries and Marine Science, Pattimura University, Jl. Ir. M. Putuhena
Kampus UNPATTI, Poka Ambon 97233, Indonesia
d
Marine Science Center of Excellence, Faculty of Fisheries and Marine Science, Pattimura University, Jl. Ir. M. Putuhena
Kampus UNPATTI, Poka Ambon 97233, Indonesia
e
Chemical Engineering Department, Faculty of Engineering, Gadjah Mada University, Jl. Grafika No. 2 Yogyakarta 55281, Indonesia

Abstract

Chitosan utilization as a preservative agent for freshly caught fish has been studied intensively during the last ten years in
Indonesia, but the effort was concentrated more on ponds-raised fresh water fish. In this paper we report our preliminary results
of a study on using chitosan in the form of nanoparticle (nano-chitosan) to improve the shelf life of freshly caught wild young
yellowfin tuna (YFT- Thunnus albacares, Bonnaterre 1788). Pharmaceutical grade chitosan was dissolved in 0.2 mM aqueous
acetic acid and aqueous ammonium hydroxide was added to reach a certain value of pH at which 1 % concentration of nano-
chitosan particles was obtained. The YFT samples were immersed for 30 min in aqueous dispersion of nano-chitosan, and
incubated afterwards at two different temperature settings (4 C and 28 C). The fish deterioration was measured in terms of
Total Volatile Base (TVB) and bacterial Total Plate Count (TPC), with supporting data taken including organoleptic assessment,
pH and water content. The nano-chitosan did not seem to decrease the number of bacteria. However, the TVB data indicated that
nano-chitosan treatment significantly suppressed the bacteria activity and the effect was more pronounced at 28 C.
2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
2016 The Authors. Published by Elsevier B.V.
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the Science and Editorial Board of ISAPPROSH 2015.
Peer-review under responsibility of the science and editorial board of ISAPPROSH 2015

Keywords: Food preservative agent; fresh catch; nano-chitosan; Yellowfin tuna (Thunnus albacares, Bonnaterre 1788)

* Corresponding author. Tel.: +62 911 322 676; fax: +62 911 322 700.
E-mail address:yosmina.tapilatu@lipi.go.id

2214-241X 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Peer-review under responsibility of the science and editorial board of ISAPPROSH 2015
doi:10.1016/j.aqpro.2016.07.040
286 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295

Nomenclature

Chitosan cationic polymer produced by deacetylation of chitin

Nano-chitosan chitosan in the form of nano-sized particles dispersed in water
TPC Total Plate Count
TVB Total Volatile Base
YFT Yellowfin Tuna
A0B0 Experimental code for no nano-chitosan addition at 4 C
A0B1 Experimental code for no nano-chitosan addition at 28 C
A1B0 Experimental code for distilled-water-based nano-chitosan addition at 4 C
A1B1 Experimental code for distilled-water-based nano-chitosan addition at 28 C
A2B0 Experimental code for well-water-based nano-chitosan addition at 4 C
A2B1 Experimental code for well-water-based nano-chitosan addition at 28 C

1. Introduction

Chitosan [(C6H11NO4)n] is a deacetylated product of chitin [(C8H13NO5)n], with the molecular weight of around
800 kDa. It is a cationic polymer with (2 000 to 3 000) monomers, and known to have the following important
properties: biodegradable, non-toxic and edible (Kerry, 2012). More importantly, chitosan acts as an antimicrobial
agent due to its positively charged polycation (El Ghaouth et al. 1994). Its nano particle form (nano-chitosan) was
proven to be more compact and more potent antibacterial agent, as reported by Ramezani et al. (2015), when
comparing the effectiveness of chitosan and nano-chitosan coatings on silver carp fillets (Hypophthalmicthys
molitrix) in refrigerated storage at 4 C.
Utilization of chitosan as a preservative agent for freshly caught fish has been studied intensively during the last
10 yr in Indonesia, but the effort was concentrated more on ponds-raised groups, such as Nile Tilapia (Oreochromis
sp.) (Mahatmanti et al. 2010) and Catfish (Pangasius hypopthalmus) (Suptijah et al. 2008). On the other hand,
previous studies on chitosan utilization on tropical marine fishes are rare and mostly applied to pre-treated products,
such as salted Indian Scad (Decapterus sp.) (Swastawati et al. 2008). There was also one very preliminary microbial
test of its application on smoked Skipjack (Katsuwonus pelamis) (Killay, 2014).
Yellowfin Tuna (YFT, Thunnus albacares) is a major component of the central and western Pacific (CWPO)
tuna landings. A combination of small and medium scale fisheries in the Philippines and eastern Indonesia land
contributes to approximately 20 % of the annual yellowfin catch globally in 1994 to 1995 (Itano, 2000). Tuna
fisheries provide vital support to coastal economic development in Indonesia, creating employment in the catching
sector and in onshore processing, as well as many thousands of indirect jobs. It is included in the commercially
important big-pelagic fish groups caught in Maluku Province waters. Indonesian Ministry of Marine Affairs and
Fisheries Division of Fishing data mentioned that there are about 371 units of tuna fishing boats licensed in Fishing
Ground Zone 714 (Banda Sea and Tolo Bay) alone, with approximately 10 700 t YFT produced in 2010 (Besewni et
al. 2011).
Freshly caught wild marine fishes such as this group are highly perishable that strong preservation methods are
required. The warm tropical climate and the geographic hindrance as an archipelago make the preservation efforts
more intriguing. The conventional preservation method (cold storage) is usually energy intensive and not affordable
for low-income fishermen. Other existing preservation agents were proven either expensive (eg. Lactic acid), or
potentially hazardous for public health (eg. formaldehyde). An innovation to the preservation method is thus deemed
necessary, in particular in a condition where fishing period has to be prolonged due to the long distance between the
fishing ground location and the unloading site.
To date, no information available on the possibility of innovating the cold storage method to preserve YFT. The
objective of this paper was to describe the preliminary result of a study carried out to observe the effect of utilizing
nano-chitosan to preserve the quality of freshly caught wild young YFT at 4 C (standard cold storage temperature
 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295 287

for fisheries products) and at 28 C. The observation of this effect at the latter temperature was done to assess the
possibility of complete removal of ice blocks.

2. Material and methods

2.1. Sample origin and pre-treatment

The experiment was done on laboratory scale, with freshly caught wild young YFT purchased from long line
fishermen in Latuhalat, Ambon. The total length of samples bought was measured in situ directly after purchase.
Samples were eviscerated and remaining body liquids were drained as thoroughly as possible to reduce bias due to
microbial contaminants from the internal organs. Individual weight of each sample was measured before and after
the evisceration process. Samples were placed in individual clean transparent plastics and arranged in a clean
storage box so as to minimize the piling effect and treated immediately with nano-chitosan upon arrival to the
laboratory. No ice was added to reduce microbial contamination source, but the storage box environment was
conditioned to be as cool as possible (between 10 C and 18 C) during transport.

2.2. Nano-chitosan preparation and application

Nano-chitosan at the concentration of 1 % was prepared freshly from pharmaceutical grade chitosan, according to
methods proposed by Liu et al. (2004). Glacial CH3COOH (3.5 mL) (for analysis, Merck, Germany) was mixed with
200 mL clean unsterile water, inside which 20 g chitosan powder of 70 % to 75 % deacetylation degree (PT Biotek
Surindo) was added progressively. The mix was homogenized with a stirrer bar and water was added progressively
to completion at 2 L of volume. A 25 % NH3 solution (for analysis, Merck, Germany) was diluted to 4 % and
pipetted to adjust the pH to be in between pH 6.3 to pH 6.5. The obtained solution was kept at room temperature
until use. Two kinds of water were used to prepare nano-chitosan solution in this experiment, namely distilled water
(DW) and clean water taken from a well (WW). The purpose was to see the difference in nano-chitosan strength
when preserving fresh fish, between the solution prepared with water produced in the laboratory scale and the one
prepared from water used for ice blocks production. The latter water source, located in Latuhalat, nearby the
fishermen landing area was used to imitate as closely as possible the in situ condition of local fishermen preparation
(ice blocks) prior to fishing trips.
Samples used throughout this experiment were immersed completely in the 1 % nano-chitosan solution for 30
min. Solution volume was prepared as to enable the sample immersion entirely and separately, because it was to be
used only once for every sample and not in a repetitive manner. The samples were retrieved from the solution
immediately after the immersion period and dried away on the bench at room temperature for 5 min. Storage was
carried out afterwards in individual plastic wrapping in two different settings, namely in refrigerator (to mimic YFT
treatment after caught, preservation at 4 C 1 C) and in air conditioned room temperature ( 28 C) (see Fig. 1 for
experimental flow) for 24 h. Observation was made every 6 h. Negative controls were made from young YFT not
immersed in any of the two solutions prepared for this experiment and put at the similar temperature settings.
288 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295

Fig. 1. Experimental design of 1 % nano-chitosan application to young YFT samples

2.3. Total Volatile Base (TVB) analysis

The TVB analysis was done according to National Standardization Agency of Indonesia (SNI) protocol SNI-01-
4495-1998. This protocol was used due to the unavailability of the most recent one at the time of the experiment. In
brief, fish extracts for TVB determination were prepared by homogenizing 2 g fish sample with 10 mL 7.5 %
Trichloroacetic acid (TCA) and left on the bench for 1 min. The sample was filtered and the filtrate was collected.
Filtrate (1 mL) was pipetted into the Conway outer chamber. The Conway system was closed afterward, and 1 mL
potassium carbonate (K2CO3) (Merck, Germany) was subsequently added into the chamber. The chamber was re-
closed, and 1 mL boric acid (H2BO3) with the concentration of 1 % was added into the inner chamber. Vaseline was
applied on the plate cover and the plate containing filtrate was gently mixed manually over the bench for one min.
The plate was incubated afterwards at 30 C for 24 h. After the storage period was over, one drop of BCGMR
indicator was added into the sample. Titration using 0.01 N HCl was carried out afterward at the inner Conway
chamber until the filtrate color turned to pink. The analysis was carried out in duplicates. The threshold used was 30
for fresh marine fishes. The TVB value was calculated using Eq. (1)

(V 1  V 2) u N HCl u14.007 u100 (1)

TVB 
W
where, TVB = Total Volatile Base (mg N %);
V1 = Volume of sample (mL);
V2 = Volume of blank solution (mL)
N HCl = Normality of HCl titrated (N)
W = sample weight (mg)
 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295 289

2.4. Total Plate Count (TPC)

The Total Plate Count (TPC) analysis was done according to the SNI protocol SNI 2332.3:2015, with some
modifications as followed. Homogenized fish sample (2 g) in 10 mL physiological solution (0.9 % w/v NaCl) was
prepared as the initial dilution (101). The serial dilution was made by adding 0.1 mL of previous dilution into 0.9
mL physiological solution. The resulting dilutions (10 1 to 106) were spread streaked onto Plate Count Agar (PCA)
plates using autoclaved cotton buds. After one hour incubation at the laminar flow, inverted plates were incubated at
35 C for 48 h. Formed colonies were counted after the storage period, and the resulting numbers were multiplied by
the dilution factor to obtain the number of Colony Forming Unit (CFU) per g of fish sample. This test was carried
out in duplicates, with the required acceptable limit is maximum 5 105 CFU g1.

2.5. Supporting parameters and statistical analysis

Fish sample pH was measured from TPC initial dilution (see section 2.4) using pH meter (EZDO pH 5011).
Water content was analyzed using the method described in SNI 01-2354.2-2006. Briefly, drying oven was set up for
heating at 105 C. Empty plates were kept in the oven for 1 h minimum at the same temperature. The preheated
empty plates were then displaced into a desiccator for about 30 min and weighed. Pounded fish samples (2 g) were
placed into the conditioned plates and dried at the oven for 16 h to 24 h at 105 C. The plates were removed from the
oven into a desiccator after the drying period was over, and kept there for another 30 min and then weighed. The
water content was calculated using the following equation,

B C
Watercontent(%) u100% (2)
B A

where, A : weight of empty plate (g)

B : cumulative weight of plate and sample before heating (g)
C : cumulative weight of plate and sample after heating (g)

Sensory evaluation was carried out by two newly trained panelists using the fresh fish organoleptic assessment
document as described in SNI 01-2729.1-2006 form (Appendix A). This protocol was used due to the unavailability
of the most recent one at the time of the experiment. Whole fish samples were examined physically for general
appearance of skin/texture, color and appearance of flesh, odor and color and form of eyes. Defect points from nine
(excellent grade) to seven (threshold) were at the acceptability range, and points lower than seven indicated rejection
from panelists.
Univariate analysis of variance (ANOVA) were carried out using PASW Statistics 18 software (IBM SPSS),
at 95 % confident level, to see the effects of nanochitosan treatment, temperature difference and storage time in the
context of the interaction between nanochitosan treatment and temperature to TVB results obtained.

3. Results and discussions

Total lengths (TL) of fishes used as samples in this experiment were from 36.9 cm to 43.3 cm, with the average
of 39.8 cm. This measurement indicated that these were young YFT, because the length of adult female YFT with
50 % maturity caught at Maluku waters was estimated at 104.6 cm (Itano, 2000). Before the evisceration process, the
samples weighed from 1.031 kg to 1.275 kg (average weight 1.137 kg). About 4 % to 14 % total body weight loss
was measured after samples evisceration. The body weight of samples stored ranged from 0.928 kg to 1.114 kg at
the average weight of 1.010 kg.
Nano-chitosan preparation indicated the instability of the solution. At 1 % concentration, the solution tended to
have residues at the bottom of the container after 24 h. Proper mixing should be carried out often to make sure that
the nano-chitosan was properly dispersed in water prior to utilization.
290 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295

3.1. The TVB analysis

The TVB test was one of the methods used notably to determine fish freshness. It is based on volatile bases
found in fish flesh. The TVB value higher than 30 mg N % indicates that the fish flesh quality was reduced.

Fig. 2 .Total Volatile Base (TVB) analysis of freshly caught wild young YFT. Values shown were the average of duplicates. Details regarding
samples code (A1B0 - A2B1) can be referred in Figure 1.

Prior to storage (T0), the TVB number indicated the fresh condition of the samples used, varied from 13 mg to 15
mg N % (Fig. 2). Samples immersed in 1 % nano-chitosan solution made by both types of water (A1B0-A2B1)
indicated an increase in TVB number after 6 h of storage, but still below the freshness threshold of international
standard. This was maintained until the end of the storage period, where TVB numbers ranged between 18 and 24.
Only one sample immersed in DW at 28 C (A1B1) that showed consistent increase of TVB number, from 15 mg N
% to 28 mg N %.
On the other hand, after six hours of storage, negative control incubated at 28 C (A0B1) showed decrease in
freshness, with TVB number peaked at 30 mg N %. The sharp increase was possibly caused by the autolysis process
and bacterial activity during the storage period. Despite the evisceration phase prior to storage, the bacteria existing
internally and externally worked actively to convert the existing protein and/or its derivative compounds in the
samples flesh tissues into volatile bases such as ammoniac, histamine, hydrogen sulfide (H 2S) and trimetilamine
(Karungi et al. 2003) which produced the rotten-egg-like odor. The TVB number was decreased at 12 h of storage
and stabilized at 26 mg N % 6 h after. This activity was confirmed by the TPC result and the organoleptic
assessment (cf. section 3.2 and 3.3), where the bacterial total number reached 7.9 105 CFU g1 at 6 h of storage
and organoleptic assessment indicated changes in samples appearance in all four parameters (eyes, flesh, texture and
odor) below the acceptable limit (7). The sample without nano-chitosan treatment stored at 4 C (A0B0) had TVB
number that started to increase only after 12 h of storage, but did not trespass the freshness threshold. The overall
TVB data indicated that fish sample immersed in WW based 1 % nano-chitosan and stored at 4 C (A2B0) gave the
optimal result, with TVB number only as high as 19 mg N % during the experiment.
The ANOVA test indicated that there was no statistically difference of treatments effect on TVB results
(p = 0.116), but there were significant effects of temperature and storage time on these results (p < 0.05). However
there was a statistically significant interaction between the effect of treatments and temperature on TVB result
(p < 0.05). The nano-chitosan solution seemed to be effectively penetrated into the flesh muscle tissue in the
 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295 291

immersion period, which led to the bonding activity with autolysis-induced enzymes. As a consequence, the
autolysis process seemed to be somewhat blocked until the end of the storage period.

3.2. The TPC analysis

Bacterial count carried out indicated that samples immersed in 1 % nano-chitosan made with DW and WW
(A1B0 and A2B0) had lower number of contaminants when kept at refrigerated storage (4 C 1 C) compared to
those stored at 28 C 1C (A1B1 and A2B1) after 24 h (Table 1). They were also lower than the one without nano-
chitosan treatment (A0B0) at the same temperature setting. This result confirmed the antibacterial properties of
nano-chitosan as reported by Ramezani et al. (2015). The immersion of samples in this solution prevented flesh
oxidation and water absorption and thus inhibit bacterial growth, as observed by Fan et al. (2009) when
investigating the effect of chitosan coating on silver carp quality and shelf life during frozen storage. Moreover,
nano-chitosan solution is known to naturally destroy bacterial cell walls, render them prone to lysis which led to
lethal consequences (Liu et al. 2004).
When fishes were stored at 28 C 1 C, the 1 % nano-chitosan solution could prolong the bacterial inhibition 6 h
longer compared to those without nano-chitosan treatment. The samples stored at 28 C 1 C without nano-
chitosan treatment (A0B1) showed consistent results compared to their high TVB results (see section 3.1), in that
the colonies formed had higher count than the accepted limit (5 105 CFU g1) starting at 6 h of storage. The
bacterial count was continuously increased until the end of storage period, reaching 1.8 107 CFU g1.

3.3. Supporting parameters

The organoleptic assessment is an approach of using human sensory receptors as the main method to assess the
quality of a product. This test plays an important role as an initial tool to detect any anomalies and/or changes
occurring in the products appearance, including the observation of color and form of eyes, color and appearance of
flesh, appearance of texture and odor. Prior to storage (T0), the samples observed had between 8 and 9 defect points
for all parameters. Sample without nano-chitosan treatment was rejected (defect points below 7) after 6 h storage,
when stored at 28 C 1 C (A0B1). This result went in line with TVB and TPC ones (cf. section 3.1 and 3.2).

Table 1. The TPC average value (duplicates) of YFT immersed in 1 % nano-chitosan solution. Bolded numbers indicated values equal to or
higher than the accepted limit of bacterial count for fresh seafood products (5 x 105 CFU g1)

Storage period Average value of TPC (CFU g1)

(h) A0B0 A0B1 A1B0 A1B1 A2B0 A2B1
3 3 2 2 3
0 4.2 10 4.2 10 1.5 10 1.5 10 4.2 10 4.2 103
5 5 4 4
6 1.3 10 7.9 10 1.3 10 TNTC 1.2 10 1.5 103
12 1 105 3 106 1.5 105 1 105 5 104 3 105
5 7 6 6 5
18 2.01 10 1.33 10 2.7 10 1.7 10 2 10 1.7 106
24 1.6 106 1.8 107 5 104 1 108 1 106 2 107

Samples treated with DW and WW based nano-chitosan solution and stored at 4 C 1 C (A1B0 and A2B0)
were still on the acceptability threshold (equal to 7) after 6 h of storage. The eyes assessed to have points below 7 h
after 12 h storage, but flesh and texture appearances as well as odor were still at the acceptability limit. The texture
and odor were relatively unchanged after 18 h of storage, but eyes color and form and flesh appearance started to
indicate values below 7, or rejected level. These results were in contrast when compared to those treated with nano-
chitosan but stored at 28 C 1 C (A1B1 and A2B1), where samples freshness were notably decreased (values
below 7) after 18 h of storage.
Sensory evaluation results indicated that the combination of 1 % nano-chitosan solution with refrigerated
temperature (4 C 1 C) could be effective to ensure fresh seafood products appearance. The nano-chitosan
solution functioned as edible coating that prevent bacterial contamination and thus prolongs the storage period.
292 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295

Fig. 3. Organoleptic assessment of samples (a) eye color and form; (b) flesh appearance; (c) texture appearance; (d) odor.

The pH value was used as one of the indicators to determine fresh seafood product freshness level. In the case of
fishes in particular, the decomposition process was induced by autolysis and bacterial attack that leads to pH
changes (Ghaly et al. 2010). Prior to storage (T0) the pH value ranged from pH 5.8 to pH 6.6 (Table 2). However
these values varied in different pattern for all samples during the storage period. The negative control stored at 4 C
1 C (A0B0) indicated sharp pH decrease after 6 h of storage, but a slight increase was observed after 12 h. The
pH value was stabilized at 5.8 after 18 h until the end of the experiment. On the other hand, when compared to those
immersed in nano-chitosan and also stored at 4 C 1 C (A1B0 and A2B0), the pH changes during the experiment
were not too important, and only fluctuated within 0.1 to 0.4. The YFT immersed in DW based nano-chitosan
(A1B0) indicated the same pattern of pH changes as A0B0, but the one immersed in WW based solution (A2B0)
tended to became more basic at 12 h of storage and changed afterwards into more acidic state after 18 h until the end
of experiment. The decrease of pH into a more acidic state was likely caused by the influence of acetic acid used as
nano-chitosan solvent (Suptijah et al. 2008). Nano-chitosan solution used in this experiment had pH set between pH
5.3 and pH 5.8. The immersion in nano-chitosan solution also seemed to inhibit bacterial activity, to the point that
protein degradation was also delayed. However, when the pH value increase, it seemed that the existing bacteria
started to regain their activities and protein degradation started to occur, which led to the accumulation of the
nitrogen bases (Chaijan et al. 2005).

Table 2. The pH values measured of YFT immersed in 1 % nano-chitosan solution.

Storage pH value
period (h)
A0B0 A0B1 A1B0 A1B1 A2B0 A2B1
0 6.6 6.6 6.0 6.0 5.8 5.8
6 5.7 6.1 5.9 6.2 5.9 6.2
12 5.9 6.4 6.3 6.1 6.2 6.0
18 5.8 6.0 5.8 6.0 5.9 6.7
24 5.8 6.2 5.7 6.1 5.7 6.1
 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295 293

Water content is an important measure of fresh seafood product, because it will determine the quality of edible
flesh for later packing, shipment and consumption. The main constituent of fish flesh is water, roughly between
70 % to 80 % of the weight depending on the species. The water in fresh fish muscle is tightly bound to the proteins
in the structure and thus can only be expelled after prolonged chilled or frozen storage. This was because the
proteins are less able to retain all the water in these conditions. Murray and Burt (2012) noted that edible flesh from
tuna group (Thunnus sp.) fishes landed or imported in quantity in Britain contain 71 % water content. Water
retention is thus essential in preserving the fresh fish quality.
In this experiment, all samples showed water content changes throughout the storage period (Table 3). The YFT
treated with DW-based nano-chitosan in refrigerated storage (A1B0) reached 4 % increase compared to the one
stored at 28 C 1 C (A1B1) after 24 h with 3 %. Whereas the edible flesh of YFT treated with WW-based nano-
chitosan had a 2 % decrease of water content (from 73 % to 71 %) after 24 h refrigerated storage. However this
decrease did not affect the flesh quality much as the TVB value was at 22 mg N % (see section 3.1). It seemed that
albeit the occurring degradation, most of the proteins were still able to retain some water. Those that did not treated
with nano-chitosan (A0B0 and A0B1) reached 5 % to 6 % of water losses during the storage, because the proteins
were less able to retain water content as a consequence of their degradation. Nano-chitosan solution could thus
facilitate the water retention properties of the existing proteins at YFT edible flesh.

Table 3.Water content (%) of YFT immersed in 1 % nano-chitosan solution.

Storage period Water content (%)

(h) A0B0 A0B1 A1B0 A1B1 A2B0 A2B1
0 75 75 68 68 73 72
6 71 70 69 69 69 70
12 70 68 70 69 70 71
18 69 64 66 71 71 70
24 70 69 72 71 71 72

4. Conclusion

The utilization of 1 % nano-chitosan solution for storing freshly caught wild young YFT at refrigerated
temperature (4 C 1 C) seemed to prolong storage period, due to maintained freshness compared to those
untreated and stored at similar temperature. This preliminary result indicated the potential of innovation in the
refrigerated storage condition with nano-chitosan solution to maintain whole YFT quality before commercialization.
However, this was only applied to post-mortem eviscerated fishes landed at the unloading sites. Further studies will
include nano-chitosan solution application in situ during fishing period, in order to observe the efficacy of its
application to prolong storage from fishing ground. Current studies carried out by our research group include the test
of sodium trypolyphosphate addition into nano-chitosan solution to improve the stability of the latter.

Acknowledgment

This preliminary study was a part of and funded by an MP3EI research project entitled: Development of nano-
chitosan production technology from shrimp waste for fish preservation in Maluku archipelago area. This is
contribution number 004 from MP3EI nano-chitosan research team.
294 Yosmina Tapilatu et al. / Aquatic Procedia 7 (2016) 285 295

Appendix A. Determination of defect points (four parameters) of fresh fish samples, as modified and
unofficially translated from SNI 01-2729.1-2006 document.

Characteristics of whole fish Defect characteristics Defect points

Color and form of eyes a) Bright, bulging with protruding lens; transparent eye cap 9
b) Bright, flat lens; transparent eye cap 8
c) Slightly bright, flat lens, grayish eye pupil, slight clouding eye cap 7
d) Slight sunken lens, greyer eye pupil, slight clouding eye cap 6
e) Slight sunken lens, grey eye pupil, slight clouding eye cap 5
f) Sunken lens, pupil turn to milky white color, cloudy eye 3
g) Sunken eye covered with yellow slime 1
Color and appearance of flesh a) Very bright flesh cut, species specific, no reddish lateral line, intact abdominal wall 9
b) Bright flesh cut, species specific, no reddish lateral line, intact abdominal wall 8
c) Slight bright flesh cut, species specific, no reddish lateral line, intact abdominal wall 7
d) Slight dullness flesh cut, reddish lateral line, softened abdominal wall 5
e) Dull flesh cut, clear reddish lateral line, softened abdominal wall 3
f) Very dull flesh cut, very clear lateral line, very soft abdominal wall 1
Odor a) Very fresh, species specific 9
b) Fresh, species specific 8
c) Natural 7
d) Faint ammoniac and sour odor 5
e) Strong ammoniac odor, faint H2S odor, strong sour odor 3
f) Strong sour odor 1
General appearance of texture a) Firm, elastic 9
b) Slightly firm, elastic 8
c) Slightly firm, slightly elastic 7
d) Moderately soft and some loss of elasticity 5
e) Some softening 3
f) Limp and floppy 1

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