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Industrial Crops and Products
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reparation and properties of peanut protein films crosslinked with citric acid
arendra Reddya, Qiuran Jianga, Yiqi Yanga,b,c,∗
Department of Textiles, Clothing & Design, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United StatesDepartment of Biological Systems Engineering, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United StatesNebraska Center for Materials and Nanoscience, 234, HECO Building, East Campus, University of Nebraska-Lincoln, Lincoln, NE 68583-0802, United States
r t i c l e i n f o
rticle history:eceived 2 October 2011eceived in revised form 1 February 2012ccepted 2 February 2012
eywords:eanut proteinsilms
a b s t r a c t
Solution cast films made from peanut proteins extracted from peanut meal and crosslinked with citricacid were found to have good dry and wet tensile properties but poor biocompatibility. Plant proteinsare abundantly available, are non-immunogenic and can be used to develop films, fibers, nanoparticlesand other types of materials for various applications. Previous attempts on developing films from peanutproteins have used high amounts of glycerol resulting in poor film properties. In this research, peanutproteins were made into films using 3–15% glycerol and the effect of film forming conditions on the tensileproperties and thermal behavior was studied. Films were crosslinked with cytocompatible citric acid as
the crosslinking agent. Ability of the crosslinked films to be used as substrates for tissue engineering wasstudied using mouse fibroblast cells. It was found that films developed in this research had much highertensile strength and modulus than films previously developed from peanut proteins. Crosslinking withcitric acid improved the dry and wet strengths of the films without affecting the water vapor permeability.Peanut protein films did not support the attachment and growth of mouse fibroblast cells suggesting thatpeanut proteins were cytotoxic.
. Introduction
Plant proteins such as zein, wheat gluten and soyproteins haveeen widely used to develop films, fibers and nano and microparti-les for various applications (Liu et al., 2005; Reddy and Yang, 2011;u et al., 2011). Proteins are preferred for films especially for foodpplications due to their non-toxicity, biodegradability, absorp-ion of gases and excellent water vapor permeability. Edible filmsave been developed from most plant proteins including peanutroteins (Chen et al., 2012). However, protein based films have rel-tively poor mechanical properties (low strength and elongation)nd water stability compared to films made from synthetic poly-ers. Hydrophilicity of plant proteins is mostly responsible for the
oor wet properties of the films (Su et al., 2010). In addition, mostrotein based films use glycerol as a plasticizer. Although glycerol
mproves the elongation of the films, glycerol is very hydrophilicnd will decrease the mechanical properties of the films. Crosslink-
ng, blending with synthetic polymers are some approaches used to
ake the protein films suitable for practical use. We have recentlyhown that films with excellent dry and wet mechanical properties
∗ Corresponding author at: Department of Biological Systems Engineering, 234,ECO Building, East Campus, University of Nebraska-Lincoln, Lincoln,E 68583-0802, United States. Tel.: +1 402 472 5197; fax: +1 402 472 0640.
can be made from wheat gliadin without the need for crosslinkingagents (Reddy and Yang, 2010).
Plant proteins are preferred for medical applications due to theirbiocompatibility and ability to be easily made into various typesof biomaterials (Reddy and Yang, 2011; Jiang et al., 2010). All thethree major plant proteins, wheat gluten, soyproteins and zeinhave been studied for potential medical applications (Reddy andYang, 2011; Jiang et al., 2010). We have recently developed filmsfrom wheat proteins for tissue engineering (Reddy and Yang, 2010).It was found that gliadin was cytotoxic but gliadin free gluteninhad excellent biocompatibility and promoted the attachment andproliferation of osteoblast cells (Reddy et al., 2011). Similarly,zein and soyprotein and their blends with natural and syntheticpolymers have been studied as potential substrates for tissueengineering.
Protein films have also been used for controlled release ofpharmaceutical and nutraceutical agents and drugs for medicalapplications. Films made from zein were loaded with heparinmicrospheres and studied for potential controlled release applica-tions (Wang et al., 2005). It was reported that both zein and heparinloaded zein microsphere films could suppress platelet adhesion,had good anticoagulation properties and biocompatible to human
umbilical veins endothelial cells (HUVEC’s) (Wang et al., 2005).The kinetics of the breakdown of soyprotein films crosslinked withformaldehyde were studied for potential use for drug delivery(Chen et al., 2008). Soyproteins were blended with chitosan and the
hysical properties and biocompatibility were studied (Silva et al.,005). It was reported that incorporating chitosan into soyproteinsecreased the water absorption and degradation of the membranesSilva et al., 2005).
Although not as extensive as soyprotein, wheat gluten and zein,eanut proteins have also been used to develop films, mostlyor food applications (Liu et al., 2004; Jangchud and Chinnan,999a,b). The physical and mechanical properties of films maderom peanut protein extracted from peanut flour were studied (Liut al., 2004). Chemical treatments using aldehydes and physicalreatments using heat were performed to improve the properties ofhe films. It was concluded that peanut protein films could be usefulor packaging and other applications. The effect of drying temper-ture and pH on the properties of peanut protein films was studiedJangchud and Chinnan, 1999a,b). It was found that tensile strengthnd elongation increased but water vapor permeability decreasedith increase in temperature. pH of the film forming solution also
ffected the physico-chemical and permeability properties of thelms (Jangchud and Chinnan, 1999a).
One of the major disadvantages and limited use of peanut pro-eins for film applications is the high cost compared to other plantroteins (Liu et al., 2004). All of the previous reports on devel-ping peanut protein films have extracted proteins from peanutour. However, peanut meal ($250 per tonne) is much less expen-ive than peanut flour and contains up to 40% proteins. The lowrice of peanut meal compared to common synthetic thermoplas-ics such as polyethylene and polypropylene ($1000–$1200 peronne) makes peanut meal very attractive to develop thermoplas-ics. In this research, peanut proteins extracted from peanut mealere used to prepare the films. The films were crosslinked with
itric acid and the tensile properties, water vapor permeability andotential of using the peanut protein films for tissue engineeringere studied.
. Experimental
.1. Materials
Peanut meal was supplied by Golden Peanut Company, GA. Cit-ic acid, sodium hypophosphite and glycerol were purchased fromWR International.
.2. Extracting protein from peanut meal
The peanut meal was treated with a 0.5% (w/w) sodium hydrox-de solution at room temperature for 1 h. After the treatment, theeanut protein mixture was centrifuged at 8000 rpm for 10 min.upernatant was collected and acetic acid was slowly added torecipitate the proteins. The precipitated proteins were washed inater and dried at 50 ◦C before using them to prepare the films
.3. Preparing peanut protein films
Various ratios (% w/w) of peanut protein were added into% (w/w) sodium hydroxide solution. The solution was graduallyeated to 90 ◦C in approximately 20 min with vigorous stirring andeld at 90 ◦C for 5 min. The heated solution was cooled down to0 ◦C and the required amount (0–15%) of glycerol was added. Theixture was then poured onto Teflon coated glass plates. Filmsere dried under ambient conditions and peeled from the Teflon
oated glass for further analysis.
.4. Crosslinking with citric acid
Citric acid was used for crosslinking because citric acid is bio-ompatible, can crosslink proteins and provides good improvement
nd Products 39 (2012) 26– 30 27
in dry and wet tensile properties and was recently found to be non-cytotoxic when used to crosslink electrospun zein nanofibers (Jianget al., 2010). The predetermined amount of crosslinking agent (cit-ric acid) along with sodium hypophosphite as the catalyst (50% onweight of citric acid) was added into the alkali solution containingthe peanut protein. Films were prepared as described above. Thedry films were cured at 150 ◦C for 30 min in a hot air oven for thecrosslinking reaction to occur.
2.5. Tensile properties
All samples for dry tensile testing were conditioned at 21 ◦C and65% relative humidity for at least 24 h before testing. Tensile prop-erties of the films were measured on an MTS tensile tester (modelQTEST 10) equipped with a 50 N load cell according to ASTM stan-dard D882. Gauge length was 2 inches and crosshead speed was10 mm/min. Wet strength of the films was determined after condi-tioning the films at 21 ◦C and 90% relative humidity for 24 h. Twentysamples randomly selected from three separately cast films weretested for each condition and the average and ±one standard devi-ation are reported.
2.6. Thermal behavior
Thermal behavior of the peanut protein, films with and withoutcrosslinking was studied using a differential scanning calorimeter(DSC) (Mettler Toledo, Model 822e). Samples were placed in sealedaluminum pans and heated at a rate of 20 ◦C/min from 25 ◦C to200 ◦C under nitrogen atmosphere.
2.7. Morphology
The surface features of the films were observed under a variablepressure scanning electron microscope (VP-SEM) (Hitachi ModelS300N Variable Pressure SEM). Films placed on conductive tapeswere sputter coated with gold palladium and observed in themicroscope at a voltage of 25 kV.
2.8. Cell culture
Peanut protein films crosslinked with 3% citric acid were usedas substrates for cell culture. Mouse fibroblast cells (NIH3T3) wereseeded onto the films at a density of 1 × 105 cells/well in 24-wellplates and cultured in Dulbecco’s modified eagle media (DMEM)at 37 ◦C with humidified 5% CO2 for 5 days. After culture, thecells attaching on the films were rinsed in PBS, fixed with 4 vol.%paraformaldehyde. To observe the spreading of the cells, the F-actin and the cell nuclei were labeled by the fluorescent dyesPhalloidin 633 and Hoechst 33342, respectively. Confocal LaserScanning Microscope (CLSM) was used to observe the cell mor-phology and spreading. The presence of cells on the films was alsoobserved using a scanning electron microscope (SEM). After cul-ture, the cells on the films were dehyhdrated using ethanol and latercritical point dried using carbon dioxide to preserve the morphol-ogy of the cells. The critical point dried films were coated with goldpalladium and observed under the SEM to study the cell growthand morphology.
3. Results and discussion
3.1. Morphology of the films
Peanut protein films were homogenous and had had a smoothsurface with very few particles even at a magnification of 400× asseen from Fig. 1. Heating the peanut proteins in alkali solution at
28 N. Reddy et al. / Industrial Crops and Products 39 (2012) 26– 30
Fig. 1. SEM image of the surface of the peanut protein films shows a smooth andhomogenous surface.
Table 1Effect of concentration of glycerol on the tensile properties of peanut protein filmsprepared with a protein concentration of 10%.
igh temperatures was able to dissolve the proteins and provide aniform film. The films were reddish in color but were transparent.
.2. Effect of increasing ratio of glycerol
Increasing the concentration of glycerol considerably reducedhe tensile strength and modulus but substantially increased thereaking elongation as seen from Table 1. Without glycerol, thelms had high tensile strength (12.3 MPa) but were brittle due tohe low breaking elongation (3.9%). Adding 3% glycerol reducedhe strength by about 3.5 times and modulus by about 4 timesut increased the elongation by 156%. Further increase in glyc-rol concentration reduced the strength and modulus but increasedhe breaking elongation. Glycerol concentrations of 5 and 7.5%rovided similar tensile properties. Films with glycerol concentra-ions above 7.5% further increased the elongation but considerablyecreased the breaking strength and modulus. Therefore, a glyc-rol concentration of 7.5% was chosen to optimize the other filmorming conditions.
Previous researchers have also used plasticizers such as glyc-rin, sorbitol or polyethylene glycol to develop films from peanutroteins. Glycerine (0.67, 1.17 and 1.67 g/g of protein) was used
o obtain films with tensile strength ranging from 4.1 to 5.1 MPand elongation ranging from 105–164% at 25 ◦C and 50% rela-ive humidity (Jangchud and Chinnan, 1999a,b). On a study of the
able 3ffect of increasing citric acid concentration on the tensile properties of peanut protein fi
effect of physical and chemical treatments on properties of peanutprotein films, it was reported that heat curing was most effectivein improving the strength, water resistance and made the filmsless permeable to water vapor and oxygen (Liu et al., 2004). How-ever, the strength of the films developed in this research was lowat 0.55 MPa (Liu et al., 2004). Heat curing at 90 ◦C improved thestrength of the films to 1.27 MPa with an elongation of 98%. Chem-ical crosslinking with formaldehyde, glutaraldehyde, succinic andacetic anhydride was also found to improve the properties of thefilms. Glutaraldehyde crosslinked peanut protein films providedthe highest strength (1.98 MPa) and good elongation (117%) usingprotein to glycerol ratio of 90/100 (Liu et al., 2004). The effect of pHand temperature of the peanut protein solution on the propertiesof the films were studied (Jangchud and Chinnan, 1999a,b). It wasfound that increasing temperature from 70 to 90 ◦C increased thetensile strength and elongation at all three pH’s studied (pH 6, 7.5and 9.0). Increasing pH from 6 to 7.5 improved the tensile propertiesbut pH 9 decreased the tensile strength at high temperatures with aprotein to glycerin ratio of 3:5 (Jangchud and Chinnan, 1999a,b). Asseen from the above reports, the strength of films obtained in thisresearch is several folds higher than those reported earlier. Sim-ilarly, much lower amounts of glycerol was used in this researchcompared to 90–167% glycerol on weight of the proteins used inprevious researches.
3.3. Effect of protein concentration
Increasing the concentration of proteins in the solution contin-ually increased the tensile properties as seen from Table 2. Thetensile strength increased from 2.7 to 5.1 MPa, breaking elongationincreased from 14 to 77% and modulus showed a relatively mod-est increase from 88 to 113 MPa when the concentration of theproteins was increased from 8 to 14%. Previous reports had usedpeanut protein concentrations from 3 to 5% to prepare the films(Jangchud and Chinnan, 1999a,b; Liu et al., 2004). Increasing pro-tein concentration increased the interaction between the proteinsleading to increased tensile properties. However, it was difficultto completely dissolve protein concentrations above 14% in the 1%sodium hydroxide solution even after heating at 90 ◦C. Since thetensile properties at 12 and 14% were similar, we chose 12% proteinconcentration to optimize the crosslinker concentration.
Crosslinking improved the tensile strength and modulus butdecreased the breaking elongation as seen from Table 3. However,
lms prepared with a protein concentration of 12% and 7.5% glycerol.
ncreasing concentration of the crosslinker, citric acid above 1%id not have any considerable influence on the dry tensile prop-rties. The highest tensile strength and modulus were obtained at
citric acid concentration of 1%. However, the breaking elongationf the films decreased by about 43% after crosslinking with citriccid. Crosslinking improved the intermolecular crosslinks in theroteins and creates strong linkages leading to an increase in thetrength and modulus. However, crosslinking limits the flexibilitynd therefore the movement of the protein molecules during ten-ile testing leading to a decrease in breaking elongation. Increasingoncentration of citric acid above 1% over-crosslinked the pro-eins and we therefore observed a decrease in tensile properties.revious reports on crosslinking peanut protein films with differ-nt crosslinking agents have also reported an increase in breakingtrength and modulus and decrease in breaking elongation afterrosslinking (Liu et al., 2004). However, a much lower concentra-ion (1%) of citric acid was sufficient to improve the dry and wetensile properties of the peanut protein films.
.5. Tensile properties at high humidity
Protein films, especially those containing hydrophilic plasticiz-rs, absorb appreciable amounts of moisture and lose considerabletrength and modulus making the films unsuitable for most appli-ations. Crosslinking with citric acid improved the wet tensileroperties of the peanut protein films as seen from Table 4. Withoutrosslinking, the tensile strength of the films was just 5% of the drytrength. Elongation and modulus of the uncrosslinked films alsoecreased considerably at high humidity. Films crosslinked with% citric acid did not show any substantial improvement in wetensile properties compared to the properties of the uncrosslinkedlms. Increasing citric acid concentration to 3% improved theet strength of the films by nearly 3 times and elongation andodulus were almost doubled compared to the properties of the
ncrosslinked samples. Although the dry strength of the citric acidrosslinked films was similar to that of the uncrosslinked samples,he wet strength of the crosslinked samples was higher than thatf the non-crosslinked samples. This could be due to the differencen swelling between the crosslinked and non-crosslinked samples.he uncrosslinked samples swell considerably and have poor inter-ction between the molecules leading to easier movement (sliding)f the molecules during tensile testing and therefore poor strength.rosslinking interconnects the molecules, provides better resis-ance to swelling and therefore the wet tensile properties of therosslinked samples are better than that of the non-crosslinkedamples. Previous studies on developing peanut protein films haveot reported the wet stability. The films developed in this researchere stable in water and could be used for various applications inater or at high humidities.
.6. Water vapor permeability (WVP)
Crosslinking slightly decreased the WVP but there was no sig-ificant difference in WVP between the samples crosslinked witharious concentrations of citric acid as seen from Table 3.
Fig. 2. DSC thermograms of the peanut protein powder and films before and aftercrosslinking with 3% citric acid.
3.7. Thermal behavior
The thermal behavior of the peanut protein powder and filmsbefore and after crosslinking are shown in Fig. 2. As seen from thefigure, the peanut protein powder has a melting peak at 163 ◦C witha melting enthalpy of 45 J/g. The uncrosslinked peanut protein filmshad a slightly lower melting peak at 157 ◦C but a much higher melt-ing enthalpy of 80 J/g. Alkali treatment during the preparation offilms hydrolyzed the proteins. The hydrolyzed proteins had lowermelting temperatures and better thermoplasticity as seen fromthe broad melting peak. Crosslinking stabilizes the proteins andslightly increases the melting temperature to 161 ◦C but providedsimilar melting enthalpy (75 J/g) compared to the uncrosslinkedfilms. It can be seen that the melting peak for the crosslinkedfilms is similar to that of the peanut protein powder. Some ofthe lower molecular weight proteins that melt at low temperatureand produce a broad melting peak for the uncrosslinked films hadbeen crosslinked and melt at higher temperatures. We thereforedo not see a broad melting peak for the 3% citric acid crosslinkedfilms.
3.8. Biocompatibility
A confocal laser scanning microscope (CLSM) image of the fibro-blast cells on the peanut protein films five days after seeding isshown in Fig. 3. There were few cells on the peanut protein filmsand the cells had poor morphology as seen from the CLSM image.The F-actin did not spread and the typical morphology of a healthycell was not seen indicating poor biocompatibility of the peanutprotein films. The SEM image of the films with cells is shown asFig. 4. The SEM image also shows that few cells have attached andthe cells do not show spreading or development of the F-actin fila-ments and lack the morphology of healthy cells. Plant proteins suchas zein, wheat gluten and glutenin have shown to be biocompatibleand suitable for tissue engineering and controlled release (Reddyet al., 2011; Jiang et al., 2010). However, plant proteins such asgliadin from wheat gluten have been reported to be cytotoxic andexhibited poor cell growth compared to glutenin and gluten (Elliet al., 2003). The reasons for the poor biocompatibility of the peanutprotein films are not obvious at this time but are probably due tothe amino acid composition as in the case of gliadin. Reports have
functions of the cells (epithelial, vascular and endothelial), increasepermeability and invoke an immune response (Finkelman, 2010).The ability of peanut allergens to invoke immune responses couldbe the reason for the poor biocompatibility of the fibroblasts.
30 N. Reddy et al. / Industrial Crops and Products 39 (2012) 26– 30
Fig. 3. CLSM image of cells on peanut protein films depicting the stained nuclei (blue), F-ain this figure legend, the reader is referred to the web version of the article.)
Fs
4
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ig. 4. SEM image of fibroblast cells on peanut protein films five days after seedinghow poor spreading and morphology indicating poor biocompatibility.
. Conclusions
This research demonstrated that peanut proteins can be madento films with good dry and wet strengths after crosslinking withitric acid. At the optimized conditions, peanut protein films had
strength of 4.6 MPa, breaking elongation of 66% and modulusf 103 MPa. Crosslinking with citric acid improved the dry andet tensile strength but decreased the breaking elongation. A low
rosslinker concentration of 1% was sufficient to increase the ten-ile strength from 4.6 to 6.1 MPa. Uncrosslinked films had lowet strength of 0.2 MPa but films crosslinked with 3% CA had aet tensile strength of 0.7 MPa. Crosslinking also improved the
hermal resistance and increased the melting temperature of theeanut proteins. Peanut protein films had poor cell attachment
nd are not suitable for medical applications. However, good drynd wet tensile strengths and water vapor permeability wouldake the peanut proteins films useful for food packaging and other
pplications.
ctin (red) and their overlapped image. (For interpretation of the references to color
Acknowledgements
The authors wish to thank Georgia Peanut Commission, Agri-cultural Research Division at the University of Nebraska-Lincoln,Multi-State Project S 1026 and USDA HATCH act for financialsupport of this work. The sponsors do not endorse the ideas repre-sented in this paper.
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