EXTRACTION AND EVALUATION OF CHITIN AND CHITOSON FROM BIOWASTE OF MACROBRICHUM ROSENBERGII, PENAEUS MONODON AND LITOPENAEUS VANNAMEI CULTURE PONDS, NEAR BAPATLA, ANDHRA PRADESH
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ABSTRACT
Chitosanisthemostimportantnaturalbasedcommoncationicpolymerandoneofthe crustaceansandotheranimal by-products,itcanbefoundintheexoskeletonof crustaceansviathedeacetylationprocessofchitin.Chitinisthefiberinshellfishsuchasshrimp,prawn,crabandlobster Theshrimpindustrygeneratesahugeamount of shells in terms of processing and these shells are commonlydiscarded as waste, which usually cause environmentalnuisance.This waste can be utilizedas an commercialsourceofchitinanditsderivativechitosan.Thechitinandchitosanareconsideredversatileandpromisingbiomaterial.Keepingthesignificanceofchitin andchitosan,thepresentinvestigationhastakenuptoevaluatetheyieldpercentageandqualityparametersofthefreshwaterprawnMacrobrachiumrosenbergii,tiger shrimpPenaeusmonodonandLitopenaeusvannamei Comparewiththe threespeciesM.rosebergiiwasbetterchitosanproducer
KEYWORDS:Chitin,Chitosan,M.rosenberiiandP monodonandLitopenaeusvannamei
INTRODUCTION:
The shrimp industry sector occupies a very significant role in the socioeconomicdevelopmentofourcountry Ithasbeenrecognizedasabetterincome and employment generator as it stimulates growth of a number of subsidiary industries andisalsoasourceof highlevelnutritiousfoodbesidesbeingaforeign exchange earner Crustacean's shells are commonly discarded as waste. However,theycouldbeusedtoextractamultiversatilepolymers, ofchitinand chitosan. Chitin is the most widespread amino polysaccharide in nature and is estimated annually to be produced almost as much as cellulose. It is a cationic aminopolysaccharide,composedofβ(1-4)linkedN-acetyl-D-glucosamineresidues.Itispresentintheexoskeletonofinvertebratese.g.crustaceans,molluscs, marinediatoms,insectsandinalgaeandfungiamongmicroorganisms(Noand Meyers,1989).InIndiaalone60,000to80,000tonnesofchitinouswastesareproduced annually, from which a lot of chitin can be recovered from crustacean biowaste(SureshandChandrasekaran,1998;Panchaksarietal.,2016).Chitosan and chitosan materials are nanoparticles, hydrogels and microspheres with diverseapplicationsinthefieldofnanotechnology,pharmaceuticals,tissueengineering,geneticengineering,biotechnology,environmentalpollutionprotection and food industries, biomedical products, photography (Panchaksari et al., 2016).Duetotheirabilityofvariousbiologicalpropertieslikebiodegradability, biocompatibilityinbothplantandanimaltissue,non-toxicity,antimicrobialand anti-inflammatory activity, and physico-chemical properties like polycationic electrolyte, solubility, adsorption and coagulation (Thakur and Thakur, 2014; Vakili etal.,2014).
Chitosan can be transformed into gels, beads, colloids, films and capsule (Sakthivel et al., 2015). They are seen in a wide range of applications from dietarytomechanicalsupport.However,theyhavealsocreatedproblemsindisposal as they are considered not biodegradable and take up several years to degrade back to the nature. In other words, they consume a large space and become a major issue as environmental pollution increasing the demands for biopolymers, which exhibit the characteristic of biocompatibility, biodegradable, and non-toxicity (Jayakumar et al.,2011). Karthik et al., (2016) reported thatthatchitosananditsproductshaveshowntohaveagreaterinfluenceonthe cell wall degradation. Chitosan being as a biopolymer extracted from shrimp shellscanbedevelopedtoactasasolutionforenvironmentalissue.Themajor economically important group of crustaceans includes shrimps, prawns, lobstersandcrabs.Nearly40-50%oftotalweightofcrustaceansgoesaswastewhile processingforhumanfoodandtheslowerdegradationofcrustaceanshellwaste hasbecomethemajorconcerninseafoodprocessingindustries(Xuetal.,2008). Further,ithasresultedinwastecollection,disposalandproblems(Kandraetal., 2012).
The amount of chitinand chitosan varies from organism to organism. Chitin is convertedintochitosanbydeacetylationwithalkalitreatmentathightemperature.Chitosanbeingasabiopolymerextractedfromshrimpshellscanbedevelopedtoactasasolutionforenvironmentalissue.Inaddition,chitosanalsogains itsfameinwastewatertreatmentandbiomedicalfieldduetoitsmetalabsorption andantibacterialpropertiesrespectively(Varmaand Vasudevan,2020).Tahami, (1994)concludedthatproperuseofcrustaceanwastesofvalueaddedbyproductswhicharepotentialapplicationsinthefieldoffoodandmedicine.Theversatilepropertiesofchitinsuchas,biorenewable,biocompatibility,biodegradablity, non-toxic, environmentally friendly and bio-functionality make it a promising
candidate. In coastal area ofAndhra Pradesh, only a limited quantity of shell waste is utilized for animal feed or chitin isolation (Panchaksari et al., 2016). Keepinginviewofsignificanceandapplicationsofchitosan,thepresentinvestigationhasbeentakenuptoevaluatethedifferenceinyield%andinthequality parameters among the fresh water prawn Macrobrachium rosenbergii and marineshrimpPenaeusmonodonand alsoLitopenaeusvannamei.
MATERIALSANDMETHODS:
Samplecollectionandpreparation: This study was conducted in Laboratory of Department of Zoology and Aquaculture,AcharyaNagarjunaUniversity FreshwaterprawnMacrobrachium rosenbergii were collected from fresh water ponds near Bapatla and shrimp Penaeus monodon and Litopenaeus vannamei alsocollectedfromcultureponds nearBapatla,GunturDistrict,AndhraPradesh(Fig-1;2;3). Exoskeletonwas separated,washed,shadedried,andcrushedintopowder Thebiowastewith4% alkalitoseparatetheproteinandtreatmentwith4%acidtoremovethecalcium carbonate.The resulting chitin product can be further deacetylated by concentrated50%alkalitoproducechitosan.
Demineralizationanddeproteinization: Demineralization was carried out by dissolving 10 g of dried shell powder in 300ml of 2mol/dm3 HSO4 solution with a ratio of 1:30, at room temperature. 2 The solution was kept for 4 hour Acid was drained off and the sample was washedwithdistilledwateruntilneutralpHwasreached.Afterthedemineralization the sample was dissolved in 300ml of 2mol/dm3 NaOH solution, at room temperature, for deproteinization. The sample was again kept for 4hour and washeduntilneutralpHwasreached(Pauletal.,2014).
Deaectylation: Degreeofdeacetylationreferstotheremovalofacetylgroupfromthechain,this is determined by potentiometric titration.The obtained chitin was dissolved in 55% NaOH solution, with a ratio of 1:30 and boiled at 110°C for 4hour After coolingthesamplewaswashedwith40%NaOHandfilteredtoobtainchitosan (Pauletal.,2014;Burrowsetal.,2007).
SolubilityofChitosan:
Chitosandissolvescompletelyin1%aceticacid.Weighafewgramsofchitosan andadd35ml1%aceticacid.Itwaskeptinamagneticstirrerfor30mins.The sample was taken out and insolubles were removed by filtration through WhatmannNo.1filterpaperandweighed.
Percentageof
Yield: Percentage of yield for chitin and chitosan was calculated from the weight of chitinandchitosanproducedasapercentageofstartingdryrawmaterial.(Zaku etal.,2011)
Litopenaeus vannamei givenTableNo.1andyieldofChitinandChitosanwas givenTable No. 2. In this study one kg of prawn Macrobrachium rosenbergii sample produced 585g of biowaste, Penaeus monodon produced 459g and Litopenaeus vannamei goes to 525g of biowaste. In case of chitin yield M. rosenbergii goesto61.15g, P monodon goes to51.32g and L. vannamei goesto 55.06g inthesample.
Table1:Yieldofbio-wastefromexperimentalorganisms
ExperimentalSpecies Totalsample weight(g) WetBio-waste (g) Dryweightof crude(g)
Macrobrachium rosenbergii 1000 585 125.1 Penaeus monodon 1000 459 109.9 Litopenaeus vannamei 1000 525 115.5
Table2:YieldofChitinandChitosanfrom1Kg.ofeachexperimental organisms.
SpeciesName WetBioWaste(g) CrudeBio-Waste indry(g) Chitin(g) Chitosan(g)
Macrobrachium rosenbergii 585 125 61.15 40.6 Penaeus monodon 459 109.9 51.32 38.8 Litopenaeus vannamei 525 115.5 55.06 36.9 525
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Fig.4:YieldofCrudebio-waste,ChitinandChitosanfromthe M. rosenbergii, P. monodon and L. vannamei
Thepurificationprocessiseasierforshrimpshellswhicharethinner Theshells ofthespeciesaresamesizeandgrouped,thencleaned,driedandalsogroundinto smallshellpieces. Chitinwithadegreeofdeacetylationof75%oraboveisgenerally known as Chitosan (Knaul,1999). An increase in either temperature or strengthofsodiumhydroxidesolutioncanenhancetheremovalofacetylgroups fromchitin,resultinginarangeofchitosanmoleculeswithdifferentproperties andhenceitsapplications(Baxter,1992).Arbiaetal(2013) reportedthatyieldof chitinfromcrab'sshellis60%to70%.Alietal (2019)observedthatbetteryield (78%) from mud cab due to impurities left in the chitin and incomplete deproteinization. Recentlythecommercialvalueofchitinhasincreasedbecause ofthebeneficialpropertiesofitssolublederivatives,whicharesuitableinchemistry, biotechnology, agriculture, food processing, cosmetics, veterinary, medicine, dentistry, environment protection and paper or textile production (SynowieckiandAl-Khateeb,2003;TharanathanandKittur,2003).Generation ofthisenormousamountofwasteandmoreimportantlytheincreasingcommercialvalueofthesolublederivativesofchitinnecessitatesthedevelopmentofa suitableprocessforsolubilizationofchitinouswasteanditsconversionintouseful polymers.
ChitosanisinsolubleinmostorganicsolventsandinwateratneutralpH.However,itdissolvesinacidicsolutions.Chitinanditsderivativechitosanareofcommercial interest due to their excellent biocompatibility, biodegradability, nontoxicity, chelating and adsorption power With these characteristics especially chitosan has many attractive applications in biotechnology, food and pharmaceutical industry, in cosmetics, environmental engineering, in agriculture and aquaculture(Varmaand Vasudevan,2020).Thelowerlimitofyield(50%)from chitinmaybeduetotheproteincontentsandimpuritiesthatwereremovedduring deacetylation and precipitation process. Diya et al (2014) reported yield of chitosanfromwholecrabshellis30-36.7%.Highchitosanyieldfromchitin in thepresentstudy(66.3-75.6%)maybeduetothe betterchitinyield,asitvaries between different crustaceans according to species and seasons (Sakthivel et al.,2015).Thequalityofchitosandependsonthesourceofchitinanditsmethod of isolation and chitosan with different extents of deacetylation are commerciallyavailabletoday Theapplicationsofthechitosandependonthecharacteris-
tics, such as appearance of polymer, turbidity of polymer solution, degree of deacetylationandmolecularweight.Utilizationofsuchchitinouswastesforthe productionofsomeusefulproductsisbeingconsideredtheformationofauseful product such as chitin and chitosan through biological (Gagne and Simpson, 1993)andchemicaltreatmentforuseinsewagetreatment,animalfeed,foodpreservation, and formulations of biofungicides (Muzzarelli, 1997; Gohel et al., 2005).
Thechitinandchitosanareusedinthepreparationofmaterialslikewounddressing,antiviralandantifungalagents,DialysismembranesBiomedicalbeads,Fabrics and gauzes (Subashinghe,1999).Chitosan is a wound healing accelerator , anditseffectivenessinprotectingwoundfrombacterialinvasionbysuppressing bacterialproliferation.Itmayactaseffectivelyagainsttyphoidproducingmicroorganism(YadavandBhine,2004). Qualityofchitosanisdeterminedfromseveralparameters,thedegreeofdeacytylationisaqualityparameterthatindicates anacetylgroupwhichcanberemovedfromyieldofchitosan.Highdeacetylation degreeofchitosanmeansthattheacetylgroupcontainedinthechitosanisweak. Deacetylation degree of chitosan varies between 56-99% an average of 80% dependingonthesourceandmethodofpreparation(Hussain,etal.,2013).Other qualityparametersofchotosanarethecolourofachitosan,whereintheapplicationofchitinandchitosanalsodependentonitscolour Chitosanwithwhitecolourorlookscleanhasbetterqualitysocommonlyusedinthefieldofhealthand foodindustries.
Chitin and chitosan are attracting a great deal of attention because of their distinctivebiologicalandphysicochemicalcharecterstics.Ithasbeenobservedthat the percentage of chitosan yield from shrimp waste collected form Penaeus semisulcatus isfoundtobe32.25%(Khanafarietal.,2008),chitosanyieldfrom biowaste of Penaeus carinatus and Penaeus monodon was found to be 34% (Yateendra.,etal.,2012),18.6%(Alimuniar andZainuddin1992),30%(Sibi., et al., 2013), 17% (Mohanasrinivasan, 2014), chitosan yield from biowaste of Penaeus monodon,chitosanyieldformthebiowasteof Penaeus monodon was foundtobe67.47%and46% (Ansharpatria,2013;Divya etal.,2014). Inthe present study biowaste of 585g M.rosenbergii goes to 40.6gms chitosan, P.monodon produced 459g biowaste and this biowaste produced 38.8g of chitosanandbiowasteof525g Litopenaeusvannameigoesto36.9gmschitosan. Itisobviousthattheamountofchitosanyieldisproportionaltotheamountchitin obtainedfromthebio-wasteofshellfish,theamountofchitinyieldinterndepend ontheamountofbio-wasteobtainedfromshellfish.Chitosaniswidelyusedin manyapplicationssuchaschelatingagent,watertreatmentadditive,drugcarrier, bio-degradablepressure-sensitiveadhesivetape,wound-healingagents,inmembranesandhasotheradvantagesfornumerousimportantapplications(Cheunget al., 2015) and also antibacterial activity against Urinary Tract Infection (UTI) pathogens(Abhirametal.,2021).
Acknowledgments:
TheauthorsarethankfultoDST-FIST,GovernmentofIndiaforprovidingfor Financialassistance.
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