The Usage of Chitosan from Shrimp Waste as Natural Preservative for Fish Cilok ( Traditional Food in Indonesia )

Cilok is one of the traditional food in Indonesia. Cilok is a popular treat among Indonesians due to its good taste and cheap price. To make it last longer, cilok is sometimes added with preservative. However, such attempt will endanger consumers if dangerous preservatives are used, such as formaldehyde and borax. Their usage in food preservation may negatively impact consumers’ health. The aim of this research was to develop an alternative, natural preservative for fish cilok. The preservative would be chitosan. The method used is an experiment that is by making a chitosan solution from the shrimp skin waste. In the experiment, chitosan was extracted from shrimp waste. A 1,5% chitosan concentrate was used. Its preservation potency was tested by giving three treatments towards fish cilok: fish cilok with 1,5% chitosan coating; fish cilok mixed with 1,5% chitosan solution and fish cilok without chitosan. The three treatments were observed and an organoleptic test was performed according to the preservation time in a room temperature on Day 1, 2, 3, 4 and 5. The parameters tested during the experiment were taste, color, odor and texture of fish cilok. The research results showed that fish cilok coated with 1,5% chitosan solution lasted longer than fish cilok without or added with chitosan.


INTRODUCTION
Cilok is one of popular treats in Indonesian community.It is children's favourite; one can find them easily in sideroads or schools.According to Yanuarlan (2015), the term cilok is an acronym of Sundanese words aci dicolok (skewered aci), a meatball shaped food made from tapioca starch (aci) and served using wood stick/skewer.
Cilok is not only made from tapioca starch, but also mixed with beef, chicken or fish meat.It is a favourite treat for both adults and children, since it is relatively cheap and easy to find.Fish cilok tastes best, but decays quickly.In order to make it last longer, some fish cilok makers or vendors use synthetic preservatives such as borax or formaldehyde which are dangerous to human health.
Cilok with borax as its preservative may endanger children's health.According to Safira (2014), one of children's favourite snacks is cilok.Children love its because it has delightful shape and chewy texture.One needs to be careful when buying cilok in roadside vendors because some of them might add dangerous food additive to cilok they sell.
Side effects resulted from consuming food which contains borax are irritations in digestion canals, shell and eyes, stomachache, headache, severe pain in upper part of stomach.Borax consumption for a long period may result in liver disease, body circulation system failure and death.Consuming 5-10 gram borax may result in shock and death on children.
Food preservatives should be made from natural substances which do not produce negative side effects on health.Therefore, new innovations in producing natural food preservatives should be attempted.One of them is the usage of chitosan.Chitosan is made from shrimp waste, especially shell and head parts because they contain chitin.
Chitosan is widely used in industries as a preservative alternative to formaldehyde and borax.Preservatives are additional substances added in food to reduce activities of microorganism, slowing down the decay process.Chitosan contains positively charged polycations which are able to reduce the growth of bacteria and molds (Harjanti, 2014).
Chitosan can be derived from chitin contained in shrimp shell.Shrimp shell contains beneficial nutritions such as proteins, calcium and chitin.Proteins and calcium can be used as additives in animal feed.Shrimp waste used in this research were head and shell parts which cover 35%-50% total weight of shrimp.
According to Marganov (2003), shrimp shell contains proteins (25%-40%), calcium carbonate (45%-50%) and chitin (15%-30%).Taolee (in Agustina, 2015) explains that chitosan is a derivation from chitin with structure of Chitosan is a polycationic polymer.Its hydroxyl and amine groups in the polimer chain allow chitosan to effectively bind heavy metal cations as well as cations from proteins and fats.Chitosan can also form an absorbing membrane during the binding of organic and anorganic compounds.These make chitosan more useful than chitin (Sanjaya et. al., 2007).As chitosan possesses a high economic value, therefore it is important to conduct a research for producing chitosan from shrimp shells, reducing the environmental contamination loads (Agustina et. al., 2015).
Chitosan is an organic compound derived from chitin.It is polyelectrolyte, non-poisonous, and biodegradable.Its hydroxyl and amine groups make it suitable as preservative and color stabilizer.Chitosan can be used as a preservative since it can slow down the growth of destructor microorganisms.Chitosan can be coated on food, minimizing the interaction between the product and its surrounding environment (Soegiarto et. al., 2013).
According to a study by Killay (2013), chitosan could be used as an anti-bacteria additive in comestibles.Such finding is supported by Hafdani (2011) who argues that chitosan is antimicrobial as it could delay the growth of phatogenic bacteria and decomposing microorganisms, including fungi.
Chitosan could also be used as a preservative for fish.Fish decays rather quickly.The decay process is caused by activities of enzymes, microorganisms and oxidation within fish body.A spoiled fish is indicated by unpleasantly sharp smell, inelastic flesh, cloudy eyes and sticky slime in gills and body (Arifin & Nugroho, 2016).
A good way for preserving fish is to soak it in water mixed with 1.5% chitosan.Another is to spray fish with 2.5% chitosan.Such attempts may prolong fish preservation for another 5 hours (Silvia et. al., 2014).
Chitosan can also be used to preserve fruits.A study by Trisnawati et.al. (2013) shows that chitosan can be used to preserve lanzones through coating.Coating can be done by dissolving chitosan powder in acetic acid solution.Next, soak fruits into the mix.Coating may reduce the loss of water, oxigen, flavour and dissolved ingredients in several products, making it effective in preserving food quality.Helander (2001) states that chitosan compound disrupts outer membranes of gram-negative bacteria.Chitosan compound, as an antimicrobial agent, can be added to comestibles since it is safe for human.Chitosan cannot be digested by human therefore it does not have calorie values and is discharged with feces.
A factor influential on the capability of chitosan as a preservative is the number of amine groups it contains.The number of amine group depends on the removed acetyl group.The removed acetyl group is called the degree of deacetylation.Therefore, the higher the degree of deacetylation is, the better the capability of chitosan as a preservative will be.The application of chitosan as a preservative is gained in the best condition of the degree of deacetylation (70.34%).The best soaking time is 45 minutes in 2% chitosan (Harjanti, 2014).
Chitosan has several benefits such as possessing a structure familiar to cellulose viber contained in fruits and vegetables.Various hypotheses regarding the working mechanism of chitosan as an anti-bacterial agent address its affinity which is strong with microbes' DNA, allowing it to bond wih DNA and then disrupt mRNA and protein synthesis (Killay, 2013).Therefore, a further research on the usage of chitosa as a natural preservative is needed.The aim of this research was to describe the creation process of chitosan and its effectiveness as a natural preservative for fish cilok.

This
research is experimental, conducted in a laboratory for producing chitosan from shrimp waste and creating fish cilok.Fish cilok is added with chitosan in order to discover effectiveness of chitosan as a natural preservative for fish cilok.
This research was conducted in a laboratory of FMIPA IKIP PGRI Jember for producing chitosan and test its effectiveness as a preservative.It was conducted during May -November 2018.This research needs to be done because to determine the effectiveness of chitosan as a natural preservative of cilok fish.The tools and materials used are presented in the following table.

A Procedure for Producing Chitosan
Chitosan is produced from a chitin isolation process.Chitin can be gained from a chitin isolation process from shrimp shells, consisting of two steps, demineralisation and deproteinisation.Demineralisation is conducted to eliminate organic compounds cointained in the shrimp waste.Shrimp shell generally contains 30-50% minerals (dry weight).Dominant minerals found in shrimp are calcium in form of CaCO3 and a small amount of Ca3(PO4)3.A scheme of chitosan extraction is presented in Figure 1.
Demineralisation can be conducted by adding HCI 1 N with the 1:7 ratio of the weight of materials and the volume of extractor for one hour in 90ºC temperature.Deproteinisation aims to eliminate proteins from shrimp waste.Its effectiveness depends on the power of the alkaline solution and the degree of temperature used.A 3,5% NaOH solution heated in 90ºC temperature for one hour can be used as an alternative to deproteinisation with a 1:10 ratio between dry shrimp waste and solution (Suptijah et. al.,1992).Suptijah, et. al., 1992) Chitosan is produced by eliminating acetyl group (-COCH3) from chitin by using a NaOH concentrated solution (50%) with a 1:20 ratio for one hour in 120-140ºC temperature (Suptijah et. al., 1992).The more acetyl groups are eliminated from the chitin polymer, the stronger the interaction among ions and hydrogen bonds from chitosan will be (Suptijah et. al., 1992).

A Procedure for Making Fish Cilok
According to Suprapti (2003), a general overview of making fish cilok is presented in Figure 2.
Fish fillets, after 5 minutes untouched, are blended until smooth and put them into a bowl.Mix them with ingredients, whipped egg whites and tapioca flour until a homogeneous, shapeable dough is formed.Make small balls (cilok) from the dough.Then, boil cilok until they are floating.After that, drain them.A Procedure for Testing Chitosan as a Food Preservative In this research, a test on chitosan as the fish cilok preservative was conducted by giving three treatments as presented in Table 2.  Research data were collected via documentation and organoleptic tests (Harjanti, 2014).Documentation was done by taking photos of fish cilok during the experiment.The organoleptic tests which were performed consisted of specifications on the whole appearance such as taste, colour, odor and texture.The tests were conducted towards the three treatments: 1) the fish cilok with 15% chitosan coating; the fish cilok mixed with 1,5% chitosan and 3) the fish cilok without chitosan.Observations on each treatment had been done for 5 days consecutively.

Chitosan
In this research, shrimp heads and shells were used to produce chitosan.Dried shrimp heads and shells were blended, resulting in shrimp shell powder.The amount of the shrimp heads and shells was 300 gram while the amount of HCI 1N used in the demineralisation was 2,1 liters.
The demineralisation resulted in pink shrimp head and shell powder.The powder then was used in the next phase, deproteinisation.The deproteination phase is carried out by reacting the result of demineralization with strong base (NaOH), so that the protein will dissolve in NaOH solution.This deproteination reaction aims to break the bonds between protein and chitin by adding sodium hydroxide.In this process, the powder was mixed with 3 litres of 3,5% NaOH.
The deproteinisation resulted in white shrimp head and shell powder.The powder was then used in the next phase, deacetylation.According to Dompeipen, et.al (2016), Isolation of chitosan compounds was obtained by performing a deacetylation reaction on chitin.Deacetylation is the process of converting an acetyl group (-NHCOCH3) to chitin into an amine group (-NH2) with the addition of strong bases such as NaOH.
The deacetylation reaction of chitin is basically an amide hydrolysis reaction of α-(1-4) -2-acetamide-2-deoxy-D-glucose.The OH-ion concentration is very influential on the process of releasing the acetyl group from the chitin acetamide group.In this process, the powder was mixed with 6 litres of 50% concentrated NaOH.During the chitosan creation process, the amount of chitin produced was 50 gram while the amount of chitosan produced was 30 gram.Therefore, the rendemen in the chitosan creation was 10%.

Fish Cilok
The type of fish used for making cilok was Treadfin Bream (Nemipterus nematophorus).In this research, the dough was mixed with 1 kg fish fillets.Next, the dough was divided into three smaller portions.Cilok which is made of three parts has the same color, smell, texture and taste, namely white, chewy texture, and fishy taste.
Small balls (cilok) from the three smaller portions with 3 cm diagram were shaped.The first portion was used to create cilok with 1,5% chitosan coating.The second portion was used to create cilok mixed with 1,5% chitosan and the last portion was used to create cilok without chitosan.
Cilok fish in the first treatment, after cooking then draining and waiting until the temperature is not too hot.Furthermore, fish cilok is dipped in 1.5% chitosan solution for 2 minutes.The fish is then drained and stored at room temperature.In this first treatment, chitosan protects cilok fish from the outer surface of the cilok, so it can protect the cilok from oxidation and protect it from bacterial contamination.
Cilok fish in the second treatment, before cilok is cooked, cilok is mixed with a 1.5% chitosan solution of 55 cc.Cilok then cooked.After cooking, cilok is drained and stored at room temperature.In

Table 1 .
The Tools and Materials for Making Chitosan and Fish Cilok

Table 2 .
A Procedure of Chitosan Test