Effect of bloodmeal hydrolysate on bloodmeal-based thermoplastic mechanical properties

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1 Effect of bloodmel hydolyste on bloodmel-bsed themoplstic mechnicl popeties Mk Ly 1, John Vebeek 1, Guv Ahuj 1 1 School of Engineeing Univesity of Wikto Pivte Bg 3105, Hmilton, New Zelnd Emil: mcly@wikto.c.nz Abstct Novotein themoplstic (NTP) is bloodmel bsed plstic developed by the Univesity of Wikto by mixing bloodmel with wte, dditives nd ti-ethylene glycol (TEG - plsticise nd petoleum bsed poduct) so it cn be extuded nd injection moulded. In this esech, bloodmel ws hydolysed with lclse, typsin nd pepsin. The hydolystes wee used in NTP s substitute fo TEG t fou diffeent concenttions. Tensile stength, modulus nd glss tnsition tempetue decesed with incesing hydolyste, nd lso decesed with ech subsequent hydolysis step (Alclse, then typsin, then pepsin), consistent with plsticising effect nd the shote chin lengths of the hydolyste. While these tends wee pomising, the esulting mteil equied highe specific mechnicl enegy to extude t highe hydolyste contents nd ws vey bittle. The effect of slt content in the hydolyste nd ode of opetions in peping the mteil fo extuding on specific mechnicl enegy would need to be exploed futhe. Futue wok could look t fctionting the hydolyste to ecove the lowe molecul weight peptides, o to incese extent of hydolysis without incesing slt content in the hydolyste. Keywods: Themoplstic, bloodmel, hydolyste, plsticise, mechnicl popeties. Intoduction Renewble nd compostble bioplstics hve been successfully developed fom diffeent potein souces such s whet gluten, con, sun flowe, ketin, csein, soy, geltine nd whey (Vebeek nd vn den Beg 2009). A ecent pomising bioplstic is Novotein Themoplstic (NTP) poduced fom bloodmel. Bloodmel is highly ggegted, coss-linked nd insoluble poduct fom the met pocessing industy with glss tnsition tempetue ound 220 o C, too high fo it to be extuded s plstic without degding the potein. Bloodmel cn be extuded successfully unde eltively low tempetues when mixed with ue, sodium dodecyl sulphte, sodium sulphite nd ti-ethylene glycol (TEG) to educe hydogen-bonding, hydophobic intection nd coss-linking nd incese potein chin mobility (Vebeek nd vn den Beg 2010). It is compostble nd will lose hlf its mss within 12 weeks in compost (Vebeek et l 2011). NTP is in the pocess of being commecilized by the Univesity of Wikto spin-off compny Aduo Biopolymes. Potentil pplictions include endeble plstic components fo the met pocessing industy nd biodegdble pot plnt contines. TEG nd wte e used to plsticise NTP. Plsticises e typiclly low molecul weight molecules tht incese the spce between polyme chins llowing fo gete chin mobility. This lowes the glss tnsition tempetue, mking the mteil softe, moe flexible nd moe duble. Fo

2 potein bsed plstics, plsticises hve to be comptible with the mteil, so typiclly hve one o moe -OH goups, e.g. TEG, glyceol, ethylene glycol, tht llows hydogen bonding nd hydophobic intection with the potein (Bie et l 2014). In NTP, 20 pts of TEG pe 100 of bloodmel is used s plsticise, which in the finl injection moulded poduct is bout 13% by weight. NTP costs bout $1.16 pe kg to mnufctue, of which $0.82 is bloodmel nd $0.17 pe kg is the cost of TEG (TEG etils fo $900 pe 200L dum) (Sichone 2014). If the cost of plsticising the mteil could be educed, this would educe the cost of NTP. In ddition, TEG is petochemicl, so eplcing the TEG with plsticise fom biologicl mteil would lso educe the envionmentl impct in tems of non-enewble esouce consumption. Poteins cn be edily hydolysed into low molecul weight peptides fo impoving digestion, nd food functionl popeties such s solubility, emulsifiction, nd whip-bility (e.g. whey hydolystes). Some common poteses include lclse, typsin nd pepsin. Pepsin is gstic cid potese pesent in the stomch, with n optiml ph between 1 nd 3 nd tempetue ound 38 o C. Pepsin pefeentilly hydolyses peptide bonds fomed by tyosine, phenyllnine, lnine, leucine, cysteine, cysteine nd glutmic cid, but hs been found to cleve lmost ll peptide bonds except poline nd isoleucine. Exmples of use include minced chicken hed nd hemoglobin hydolysis (Suowk nd Fik 2006; Piot et l 2007). Alclse (lso clled subtilisin) is seine potese poduced fom bcillus subtilis, is specific fo the mide goup of seine, spgine, lnine, nd typtophn nd hs n optimum ph of 8 nd tempetue of 60 o C (Polg 1987). It is used fo potein stin emovl in lundy powdes, cosmetics, nd in contct lens clene. Typsin hydolyses the peptide bonds of lysine o ginine, nd hs n optimum ph of 8-9 nd tempetue of 38 o C (Rwlings nd Bett 1994). While hydolystes of collgen nd soy hve been mnufctued into plstics (Pei et l 2013, Lngmie 2008) nd hydolystes of bloodmel into fie-fighting foms (Bessle 2010), it ppes only soy hydolyste hs been ttempted s plsticise in soy isolte plstics (Vld et l 2006) nd ovolbumin hydolystes s plsticises in suspensions of lumin powdes (Schilling 2002). The im of this poject ws to exploe the effect of using potein hydolystes mde fom bloodmel s n ltentive plsticise to TEG. Mteils nd Methods Mteils Bloodmel ws collected fom the Wllce Copotion bovine endeing fcility (Wito, New Zelnd). Sodium dodecyl sulfte ws puchsed fom Meck, sodium sulphite fom BDH Lbs nd TEG fom Oic Chemnet. Alclse ws puchsed fom Novozymes, nd typsin nd pepsin fom Sigm. All othe chemicls wee puchsed fom Ajx Finechem nd wee nlyticl gde. Hydolyste poduction 1.13 kg of bloodmel (Wllce Copotion) ws dded to 30 lites of distilled wte nd the ph djusted to 8. The solution ws heted to 60 o C using submeged heting coil nd gitted using n ovehed stie. 250 ml of lclse ws dded nd the ection llowed to poceed fo 8 hous while keeping the ph constnt. 10 lites of solution (Alclse hydolyste) ws emoved nd centifuged to emove un-hydolysed bloodmel. Tempetue ws dopped to 38 o C nd 10.8 g of typsin ws dded to the emining 20 L of solution, ph mintined t 8, tempetue nd the ection llowed to poceed fo nothe 8 hous. 10 lites gin ws emoved fo centifuging (Typsin hydolyste). With the emining 10 lites, the ph ws djusted to 1.5, tempetue

3 loweed to 37.5 o C, 15 g pepsin ws dded nd the ection llowed to poceed fo nothe 8 hous befoe being neutlised nd centifuged (Pepsin hydolyste). Molecul weight distibution Molecul weight distibution of poteins in ech hydolyste wee nlysed using gel filttion chomtogphy. A Supedex :300 column (GE Helthce) ws connected to n AKTA FPLC (GE Helthce) nd 50 L of pe-filteed (using 0.45 m Minist syinge filte) smple loded nd pssed though the column t 0.5 ml/min. The unning buffe ws 0.02M phosphte buffe t ph 7. Potein concenttion nd conductivity in the effluent ws ecoded using n in-line UV spectophotomete t 280 nm nd n in-line conductivity mete. Avege molecul weight fo ech hydolyste ws clculted conveting volume to molecul weight using clibtion dt, multiplying molecul weight by the UV bsobence to obtin weighted molecul weight tht ws summed, which ws then divided by the sum of the UV bsobence. NTP poduction Afte centifugtion, ech hydolyste btch ws concentted to 50% solids by evpoting wte on heting pltes t 50 o C. Ech type of hydolyste ws dded to stndd NTP fomultion t fou diffeent concenttions (Tble 1), esulting in 12 btches. Ue, sodium sulphite, sodium dodecyl sulphte nd hydolyste wee dded to wte in beke ccoding to the ecipe in Tble 1, nd heted to o C on hot plte fo 15 minutes while being mixed. This ws dded to bloodmel nd the mixtue mixed gin fo 5 minutes using both blende nd mnul mixing. The ctul mss of wte used ws djusted to ccount fo the wte content in the hdyolyste. The esulting mixtues wee double bgged in zip-lock bgs nd efigeted until used fo extusion. Tble 1. Recipe used fo ech btch of NTP. Amount Ingedient Mss (g) pph(bm) Bloodmel Ue Sodium sulphite 9 3 Sodium dodecyl sulphte 9 3 Wte Hydolyste ddition (dy weight) Extusion of blended fomultions A twin scew Themopism TSE-16-TC extude ws used to extude ech NTP fomultion. The tempetue pofile fom the feede to the die ws 70, 100, 100, 100 nd 120 o C fo ech zone espectively (Figue 1). Extude scew speed ws 150 pm nd feed te fom the hoppe to the extude ws 60 Hz. Mss flow te fom the extude, toque nd pessue wee mesued ech minute fo ech btch. Specific mechnicl enegy (SME in kj/kg) ws clculted by: P SME M RPM RPM

4 Whee P is the ted powe of the extude (1.25 kw), M is the mss flowte of extudte (kg/s), nd e the ctul nd ted toque of the extude (Nm), nd RPM nd RPM e the ctul nd ted evolutions pe minute of the extude scews. ws 12 Nm, RPM ws 150 nd RPM ws 500. Figue 1. Extude scew configution. Extuded mteil fom ech fomultion ws then pelletised using ti blde gnulto (Cstin Mchiney) nd 4 mm mesh sceen nd wee double bgged in zip lock bgs nd efigeted befoe injection moulding. Ech pelletised fomultion ws injection moulded into stndd dog bone specimens (ASTM D638-03) nd impct bs (12.8 x 6.0 x 3.4 mm) using BOY-35A injection moulde. The tempetues fo the zones fom the feed to the nozzle wee 100, 115, 120, 120, nd 120 o C. The mould tempetue ws set t 65 o C. Injection moulde scew speed ws 200 pm with n injection pessue of 150 b, nd esidence time of 40 seconds in the mould. Specimens wee conditioned fo seven dys t 50% eltive humidity nd 27 o C befoe tensile nd impct testing. Tensile testing Tensile specimens wee nlysed ccoding to the ASTM D method fo tensile stength, modulus nd elongtion t bek. An Inston 33R-4204 tensile teste ws used, fitted with 5 kn lod cell, opeting with coss hed speed of 5 mm pe minute. A 50 mm extensomete ttched to the middle of the specimen to mesue stin. Five mesuements wee cied out fo ech fomultion nd vege vlues nd stndd devitions obtined. Boken specimens wee then oven died to obtin moistue content. Impct testing Impct specimens wee notched nd impct tested on impct teste. Specimen width nd thickness ne the notch, nd notch size wee mesued fo ech specimen befoe testing. The specimen ws plced on the impct teste with notch fcing wy fom the hmme which ws plced t set distnce fom specimen. Impct cceletion ws set t 2.90 m pe second. Enegy equied to bek the specimen in kj/m 2 ws noted nd eclculted with the smple dimensions. Dynmic mechnicl nlysis A Pekin Elme Dynmic Mechnicl Anlyse ws used to obtin glss tnsition tempetues of injection moulded smples. Impct bs wee mounted in the DMA in single cntileve bending system, enclosed by cemic hete. Smples wee heted to 180 o C fom oom tempetue t

5 te of 2 o C/minute. Dt ws collected fo 1Hz, 10Hz nd 30Hz t dynmic displcement of 0.03mm. The stoge modulus (E), loss modulus (E ) nd loss fcto (tn δ) wee ecoded by mchine pogm intefce. A pek in tn δ epesented tnsition tempetue. Pecentge cystllinity X-y diffction (XRD) (Pnlyticl Empyen) ws used to detemine the pecentge cystllinity of the tensile specimens using pmetes listed in Tble 2. Tble 2. Pmetes fo XRD. Pmetes Vlue Scn type Single scn Wvelength (Å) X y tube Empyen Cu LFF HR Anode mteil Cu Voltge 45 Cuent 40 Solle slits (d) 0.04 Msk (mm) 6.6 Filte Lge bet filte-nickel Scn mode Continuous Scn nge 4-60 XRD gphs obtined (Figue 2) wee bseline coected between ngles of 5 nd 35 2Ɵ, Gussin cuve fitted unde the mophous hlo egion between 10 nd 35 2-thet, cystllinity obtined by subtcting the cuve e fom totl pek e, nd pecentge cystllinity by dividing cystlline e by totl pek e. Duplictes wee done fo ech specimen. Figue 2. A typicl XRD gph fo NTP tht hs been bseline coected nd Gussin cuve fitted unde the hlo. Results nd Discussion Bloodmel hydolyste Convesion of bloodmel into hydolyste ws bout 70% by mss. Bloodmel hydolyste molecul weights nged between 80 kd down to vey smll sized peptides, with chcteistic peks ound ml (45 kd to 12.5 kd), mjo pek t 20 ml (4.3 kd) fo lclse hydolyste nd 20.8 ml (2.8 kd) fo typsin nd pepsin hydolystes, ml (0.5 to 0.4 kd) fo lclse nd typsin nd 26.7 ml (0.170 kd) fo pepsin (Figue 3). Alclse hydolyste hd n vege molecul weight of 8.9 kd, typsin hydolyste 5.5 kd nd pepsin hydolyste went bck up to 8.8 kd. This could be due to the pepsin dded; the pek t 15 ml on the pepsin hydolyste chomtogm coesponds to 35 kd which is close to the molecul weight fo pepsin (34.5 kd). The esulting

6 molecul weights e lowe thn hemoglobin (64 kd) nd bovine seum lbumin (66 kd), which mkes up the mjoity of potein in bovine blood. The Supedex 200 column used hs column volume of 24 ml, theefoe thee my hve been some hydophobic intection between the column nd some of the peptides etding thei pssge though the column, esulting in them exiting between 25 nd 40 ml. Figue 3. Gel filttion chomtogm fo lclse, typsin nd pepsin hydolystes. Pocessbility of NTP with hydolyste NTP with hydolyste dded genelly ws ble to be extuded nd injection moulded, with the exception of NTP with 20 g lclse hydolyste. On this smple, the injection moulde blocked nd only one ptil tensile specimen ws ble to be poduced. With incesing hydolyste, foming becme n issue while peping the mixtues fo extusion, nd the mteil ws incesingly hde to extude with the extudte coming out in smll lengths. This ws suppoted by the specific mechnicl enegy (SME) esults (Figue 4) which incesed with incesing hydolyste. Pepsin nd typsin hydolystes gve highe SME thn lclse t the highe hydolyste levels. b Figue 4. ) Specific mechnicl enegy equied to extude the NTP with hydolystes, nd b) moistue content (% by weight) of the extudte. The highe SME could be cused by the slight eduction in wte content in the extudte (Figue 4b) due to the highe potein content in the pe-extuded mixtue comped to wte nd othe

7 dditives (lthough wte content fo NTP extudte with pepsin nd typsin hydolyste t 80 g pe btch ws simil to the 20 g btch extudte). Wte lso cts s plsticise nd would explin the incese in SME. In ddition, bloodmel ws dded fte the hydolyste nd othe egents hd been mixed, so thee could hve been pefeentil intection of the ue, sodium sulphite nd sodium dodecyl sulphte with the poteins in the hydolyste, the educing thei intection with bloodmel, lso cusing n incese in SME. Anothe possibility is the incesed slt content in the hydolyste fom mintining the ph duing the hydolysis ection incesing the potein hydophobic intection in the mteil. Futue expeiments would need to exploe whethe o not the slt content in the hydolyste contibuted significntly to extudte SME, eithe by deslting the hydolyste o by educing the mount of ph djustment cied out which would educe hydolysis yield. Othe expeiments could lso exploe keeping totl potein to wte nd othe egent tios constnt nd lso exploe the effect of ode of egent mixing in NTP/hydolyste poduction. NTP mechnicl popeties Incesing hydolyste concenttion in NTP loweed tensile stength nd secnt modulus (Figue 5), with the lclse hydolyste giving the highe tensile stength nd modulus, followed by typsin nd pepsin (The esult fo lclse t 20 g pe btch should be ignoed s the btch blocked the injection moulde nd only one specimen could be poduced fo testing). This is consistent with the ovell eduction in vege potein molecul weight in the NTP/hydolyste cusing eduction in mechnicl popeties. Conventionl NTP in compison hs tensile stength of ound 9 MP, modulus of 620 MP, toughness of 3.2 MP nd stin t bek of 0.7 (Shmsuddin 2013). With incesing hydolyste we hoped to see n incese in stin t bek, toughness nd impct stength due to gete chin mobility, but toughness nd impct stength mginlly decesed with incesing hydolyste while stin t bek ws 0.01 indicting n extemely bittle mteil. Glss tnsition tempetues (T g ) (Figue 6) obtined fom the pek in tn δ fom DMA esults emined constnt t ound 78.5 o C fo NTP with lclse hydolyste while they decesed with incesing hydolyste ddition fo typsin nd pepsin, with pepsin giving the getest decese in T g. fom 81.5 to 77 o C. This showed tht the pepsin nd typsin hydolystes did hve plsticising effect, with the pepsin hydolyste hving the getest effect. In compison, conventionl NTP ws ound 61 o C (Shmsuddin 2013) to 66 o C (Bie 2014). A gete plsticising effect might be seen if the smlle peptides fom hydolysis could be septed fom the highe molecul weight peptides, fo exmple, by pssing the hydolyste though low molecul weight cut-off ultfilttion membne, nd concentting nd using the pemete in the NTP. Unfotuntely, T g of the hydolystes wee unble to be obtined s the powdes fte oven dying wee vey hygoscopic nd pidly bsobed moistue fom the i, conveting bck into viscous syup. Pecentge cystllinity in NTP/hydolyste smples (Figue 6b) wee genelly consistent ound 20-23% while conventionl NTP ws ound 19-20% fo powdeed smples (Shmsuddin 2013) to 26% (Bie 2013). Cystllinity in potein plstics cn be ttibuted to -helices nd -sheets (Bie et l 2013). An incese in cystllinity fo NTP/hydolyste might be expected s in pevious wok, Fouie Tnsfom Inf Red nlysis using Synchoton light showed tht NTP with TEG s plsticise hd lowe -sheets thn NTP without TEG (Bie et l 2013). Altentively, the pesence of low molecul weight peptides could educe ovell cystllinity by incesing the mophous egions in the plstic. XRDs of the hydolystes could not be obtined fo the sme eson s T g could not be obtined.

8 b c d e Figue 5. Mechnicl popeties of NTP/hydolyste: ) tensile stength, b) secnt modulus, c) stin t bek, d) toughness (o enegy to bek) nd e) impct stength. b Figue 6. ) Glss tnsition tempetue obtined fom the pek in tn δ fom DMA. b) NTP/hydolyste cystllinity fom XRD.

9 Conclusions Themoplstics could be successfully poduced with NTP using bloodmel hydolystes s substitute fo TEG. Tensile stength, modulus nd glss tnsition tempetue decesed with incesing hydolyste, nd lso decesed with ech subsequent hydolysis step (Alclse, then typsin, then pepsin), consistent with plsticising effect nd the shote chin lengths of the hydolyste. While these tends wee pomising, the esulting mteil equied highe specific mechnicl enegy to extude t highe hydolyste contents nd ws vey bittle. The effect of slt content in the hydolyste nd ode of opetions in peping the mteil fo extuding on specific mechnicl enegy would need to be exploed futhe. Futue wok could look t fctionting the hydolyste to ecove the lowe molecul weight peptides, o to incese extent of hydolysis without incesing slt content in the hydolyste. Biogphy Mk Ly (Pesente). Mk is senio lectue in Engineeing t the Univesity of Wikto, New Zelnd nd membe of the potein plstics/biocomposites esech goup led by D John Vebeek. Mk hs PhD in biopocessing nd his esech inteests e in potein plstics nd potein ecovey fom pocess wstes. Refeences Bie JM, Vebeek CJR nd Ly MC (2014), Theml nd mechnicl popeties of bloodmel-bsed themoplstics plsticized with ti(ethylene glycol). Mcomolecul Mteils nd Engineeing, 299(1): Bie JM, Vebeek CJR nd Ly MC (2014), Themlly esolved synchoton FT-IR micoscopy of stuctul chnges in bloodmel-bsed themoplstics. Jounl of Theml Anlysis nd Cloimety, 115(1): Bie JM, Vebeek CJR, nd Ly MC (2014). Plsticize migtion in bloodmel-bsed themoplstics. Jounl of Applied Polyme Science, 131(4): 1-9. Bie J (2014), pesonl communiction, Aduo Biopolymes. Bessle D (2010), Potein bsed foming gents nd methods of mking theeof. US Ptent: US A1. Lngmie F, Mokejs P, Kolomznik K nd Mldek M (2008), Plsticizing collgen hydolyste with glyceol nd low-molecul weight poly(ethylene glycols), Themochimic Act, 469(1 2): Pei Y, Yng J, Liu P, Xu M, Zhng X nd Zhng L (2013), Fbiction, popeties nd biopplictions of cellulose/collgen hydolyste composite films. Cbohydte Polymes, 92(2):

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