Bioresource Technology 100 (2009) 3638–3643                                                               Contents lists a...
E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–3643                             3639   A variety of i...
3640                                     E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–36432.4. Stat...
E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–3643                                         3641     ...
3642                                    E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–3643incubation...
E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–3643                                                  ...
Upcoming SlideShare
Loading in …5
×

Properties of biopolymers produced by transglutaminase treatment of wpi and gelatin

1,530 views
1,475 views

Published on

Byproduct utilization is an important consideration in the development of sustainable processes. Whey
protein isolate (WPI), a byproduct of the cheese industry, and gelatin, a byproduct of the leather industry,
were reacted individually and in blends with microbial transglutaminase (mTGase) at pH 7.5 and 45 C.
When a WPI (10% w/w) solution was treated with mTGase (10 U/g) under reducing conditions, the viscosity
increased four-fold and the storage modulus (G0) from 0 to 300 Pa over 20 h. Similar treatment
of dilute gelatin solutions (0.5–3%) had little effect. Addition of gelatin to 10% WPI caused a synergistic
increase in both viscosity and G0 , with the formation of gels at concentrations greater than 1.5% added
gelatin. These results suggest that new biopolymers, with improved functionality, could be developed
by mTGase treatment of protein blends containing small amounts of gelatin with the less expensive whey
protein.

Published in: Technology, Business
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
1,530
On SlideShare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
38
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Properties of biopolymers produced by transglutaminase treatment of wpi and gelatin

  1. 1. Bioresource Technology 100 (2009) 3638–3643 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortechProperties of biopolymers produced by transglutaminase treatment of wheyprotein isolate and gelatin qEduard Hernàndez-Balada a,b, Maryann M. Taylor a, John G. Phillips a, William N. Marmer a,Eleanor M. Brown a,*a US Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 E Mermaid Lane, Wyndmoor, PA 19038, USAb Department of Chemical Engineering, University of Barcelona, c/ Martí i Franquès 1, 08028 Barcelona, Spaina r t i c l e i n f o a b s t r a c tArticle history: Byproduct utilization is an important consideration in the development of sustainable processes. WheyReceived 12 November 2007 protein isolate (WPI), a byproduct of the cheese industry, and gelatin, a byproduct of the leather industry,Received in revised form 11 June 2008 were reacted individually and in blends with microbial transglutaminase (mTGase) at pH 7.5 and 45 °C.Accepted 18 August 2008 When a WPI (10% w/w) solution was treated with mTGase (10 U/g) under reducing conditions, the vis-Available online 25 March 2009 cosity increased four-fold and the storage modulus (G0 ) from 0 to 300 Pa over 20 h. Similar treatment of dilute gelatin solutions (0.5–3%) had little effect. Addition of gelatin to 10% WPI caused a synergisticKeywords: increase in both viscosity and G0 , with the formation of gels at concentrations greater than 1.5% addedWhey protein isolateGelatin gelatin. These results suggest that new biopolymers, with improved functionality, could be developedMicrobial transglutaminase by mTGase treatment of protein blends containing small amounts of gelatin with the less expensive wheyCrosslink protein.Dithiothreitol Published by Elsevier Ltd.1. Introduction bundle for access by tanning chemicals. The hide may then be split; the upper layer would be tanned to make leather, and the lower Whey and gelatin are both proteinaceous agricultural byprod- layer would provide a rich source for the extraction of type B gel-ucts. Whey is an abundant, inexpensive and readily available atin. Gelatin extracted from limed hides is edible, and is used inbyproduct of the cheese industry. The United States is the world’s sausage casings, as an ingredient in a variety of food products, aslargest whey exporter; latest available figures from the US Dairy well as in pharmaceuticals. Inedible gelatin may be surplus lowerExport Council (USDEC, 2006) show an increase in exportation by grade material from production of edible gelatins or may be from104% between 2001 and 2006. Despite the exports, a considerable an inedible source such as leather waste (Taylor et al., 1994). Theamount of whey is wasted through disposal, and would be avail- highest value market for inedible gelatin traditionally was the pho-able for potential new uses. These uses could be for food or non- tographic industry, where the demand for gelatin has declinedfood products. markedly in recent years. The Bloom value, a measure of gel Animal hides, skins and bones, major byproducts of the meat strength of a 6.67% (w/w) gelatin, is often used to predict theindustry, are rich sources of collagen, the structural protein of con- behavior of a gelatin in a particular application. Gelatin suppliersnective tissue. Gelatin, produced by partial hydrolysis of collagen, typically give a nominal Bloom value for the gelatins they market,is a polydisperse mixture of varying sized collagen fragments. Gel- in this case 75 g. High Bloom (> 225 g) gelatins are taken from theatins are classified by type, edibility, and gel strength. Type A gel- highest molecular weight fraction of crude gelatin and are rela-atin is obtained by acid treatment usually of pigskins, while type B tively uniform. High and low molecular weight gelatin fractionsis obtained by alkaline treatment of cattle hides or bones (Rose, are blended to produce low Bloom (<125 g) gelatins, and variation1992). An early step in leather manufacturing is the liming of the between lots may be expected.hide to assist in hair removal and opening up of the collagen fiber Transglutaminase (TGase, EC 2.3.2.13) is a calcium-dependent enzyme that catalyzes an acyl transfer reaction between the c-car- boxyamide of a protein or peptide bound glutamine and a primary amine (Folk and Chung, 1973). An e(c-glutamyl)lysine bond is q Mention of trade names or commercial products in this article is solely for the formed when the primary amine is the e-amino group of a lysinepurpose of providing specific information and does not imply recommendation orendorsement by the US Department of Agriculture. residue. In 1989, Ando et al. used Streptoverticillium mobaraense * Corresponding author. Tel.: +1 215 233 6481; fax: +1 215 233 6795. to produce a calcium-independent form of the enzyme, namely E-mail address: eleanor.brown@ars.usda.gov (E.M. Brown). microbial transglutaminase, mTGase.0960-8524/$ - see front matter Published by Elsevier Ltd.doi:10.1016/j.biortech.2009.02.039
  2. 2. E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–3643 3639 A variety of individual food proteins, including gelatin, casein der mild agitation. Reaction products were then heated at 90 °C forand whey proteins have been polymerized by the formation of 10 min to deactivate the mTGase. Control samples, without mTG-mTGase-mediated intermolecular crosslinks (Sakamoto et al., ase, were subjected to the same thermal treatment.1994; Rodríguez-Nogales, 2006). Biopolymers formed by the enzy- Gelatin and WPI solutions (1–10%) were treated with mTGase atmatic crosslinking of dissimilar proteins have the potential for gen- 10 U/g of protein. The effect of enzyme concentration (0–10 U/g oferating novel products (Motoki and Nio, 1983; Yildirim and protein) was also investigated for gelatin and WPI solutions at 10%.Hettiarachchy, 1997; Oh et al., 2004). The most abundant proteins Finally, the effect of gelatin (0.5–3%) was tested by adding gelatinin whey, b-lactoglobulin and a-lactalbumin, are globular and con- to a 10% WPI sample containing mTGase at either 0 or 2 U/g of totaltain two and four disulfide bonds, respectively (Farrell et al., 2004). protein.The reduction of disulfide bonds in globular proteins, prior to mTG-ase treatment, has been shown (Frgemand et al., 1997) to in- 2.3. Analysescrease access to primary amino groups. Dithiothreitol (DTT) isgenerally the reductant of choice for cleaving disulfide bonds, gi- The gelatin used for this study was characterized by the stan-ven that it is needed in much lower concentration than other dard Bloom test (AOAC Method 948.21, 1990). The strength of areductants to complete the reaction (Grazú et al., 2003). gel formed in a standard 59 mm Bloom jar from a 6.67% (w/w) In this study, biopolymers produced by treating gelatin or whey solution of gelatin at pH 6.5 was measured on a TA.XT2 Textureprotein isolate (WPI) and blends of WPI and gelatin with mTGase Analyzer (Stable Micro Systems, Surrey, UK). Gel strengths ofunder reducing or nonreducing conditions are characterized. In experimental samples were determined by the modified smallblends, WPI, the less expensive of the protein sources, is the pri- sample Bloom test (Wainewright, 1977). Samples were transferredmary component and the focus is on the effect of including small to 39 mm weighing bottles, to a height of 40 mm, cooled to roomamounts of gelatin with the WPI in these biopolymers. temperature and then chilled for 17 h at 10 °C in a constant tem- perature bath. Each sample was then placed under a 0.5 in. diam- eter analytical probe, which then was driven into the sample to a2. Experimental depth of 4 mm at 1 mm/s. The measured force, using the correction factor (1.389) previously determined for small samples (Taylor2.1. Materials et al., 1994), was expressed in grams. After the determination of gel strength, samples were melted at 60 °C for viscosity mTGase, Activa TG-TI (Ajinomoto USA Inc., Paramus, NJ) with an measurements.active range of pH 4.0–9.0 at 0–70 °C was used without further Viscosity was determined using a Model LV 2000 Rotary Vis-purification. The mTGase activity, in the presence of the malto- cometer (Cannon, State College, PA) equipped with a low Centi-dextrose carrier, under the assay conditions of Folk and Chung poise Adapter and a jacketed sample chamber connected to a(1985) was approximately 100 U/g. Type B gelatin, alkaline ex- refrigerated bath circulator, Model RTE-8 (Neslab, Portsmouth,tracted from bovine skin, was obtained from Sigma (St. Louis, NH). An 18 ml aliquot of each sample was added to the sampleMO), and characterized in this laboratory as 115 g Bloom. WPI, Ala- chamber and equilibrated for at least 10 min. Viscosity was mea-cen 895, containing 93.2% protein (manufacturer’s data), was gen- sured at a spindle speed of 60 rpm, corresponding to a shear rateerously supplied by NZMP (formerly New Zealand Milk Products; of 73.42 sÀ1. After the viscometer had been stabilized for one min-Lemoyne, PA). DTT was obtained from Calbiochem (San Diego, ute, readings were taken at 60 °C for gelatin as recommended byCA). All other chemicals were reagent grade and used as received. Rose (1992) and at 25 °C for WPI and blended biopolymer solutions.2.2. Sample preparation Dynamic oscillatory rheometric measurements were carried out in a controlled stress AR-2000 Rheometer (TA Instruments, New Solutions of gelatin or WPI ranging from 1% to 10% (w/w) were Castle, DE) at room temperature (21 ± 2 °C) after enzyme deactiva-prepared by suspending protein powder in the required weight of tion and equilibration of samples for 1 h. The sample was placeddeionized water. WPI–gelatin solutions (10% WPI with 0.5–3% gel- between parallel 25 mm diameter plates and the gap betweenatin) were prepared by suspending the required amounts of WPI them was set to 2.5 mm. Excess sample was trimmed off and a thinand gelatin powders in deionized water and stirring for about layer of mineral oil applied to the exposed free edges of the sample30 min to ensure a uniform suspension. To test the effect of a to prevent moisture loss. Time sweep measurements were used toreducing environment, a 10% DTT (w/v) solution was prepared study the evolution of the storage modulus G0 as a function of time.and the volume necessary to give a concentration of 10 mg DTT A strain sweep test was performed at a constant frequency ofper g protein was included in the preparation of selected samples. 0.1 Hz to find an oscillation stress value that was within the linearSamples were allowed to swell at room temperature for 4 h, and viscoelastic range. This value, 0.5 Pa, was then used to performthen adjusted to pH 7.5 with 1 N NaOH or 1 N HCl. Samples were time sweep measurements at a constant 0.1 Hz frequency. Storagethen heated at 38 °C for 1 h, cooled to room temperature and modulus G0 was recorded and analyzed over 20 h, at a rate of twostored overnight at 4 °C. points per hour. Typical conditions for mTGase-mediated crosslinking of gelatin Inter-protein crosslinking was evaluated by polyacrylamide gelare pH 6.5 and 50 °C for 4 h (Taylor et al., 2001). For whey proteins, electrophoresis in sodium dodecyl sulfate (SDS–PAGE) (Laemmli,the optimum conditions are pH 7.5 and 40 °C for 8 h (Truong et al., 1970) using precast gradient gels (4–15%). Gels were calibrated2004). To provide a basis for comparison, all reactions in this study using the broad range (BRM) SDS calibration standard (Bio-Rad,were performed at pH 7.5 and 45 °C for 5 h, considering that WPI Hercules, CA) that contains a mixture of nine proteins ranging inwas the major protein component in the mixtures. size from 6.5 to 200 kDa. Samples (approximately 0.5 mg) of lyoph- For crosslinking experiments, protein samples were prepared in ilized protein dissolved in sample buffer (10 mM Tris–HCl at pH 8.010 ml less than the required volume. The appropriate concentra- containing 1 mM EDTA, 25 mg/ml SDS, 50 ll/ml b-mercap-tion of mTGase was then prepared in 10 ml of water and this solu- toethanol and 0.1 ll/ml bromphenol blue) were heated at 40 °Ction added with stirring to gelatin, WPI, or WPI–gelatin solutions to for 4 h. Separation was achieved using a Phast System (Pharmaciagive the desired final protein concentration. Samples were read- Biotech Inc., Piscataway, NJ). Gels were stained with Coomassiejusted to pH 7.5 and incubated for 5 h at 45 °C in a shaker bath un- Blue.
  3. 3. 3640 E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–36432.4. Statistical modeling Because x, y plots of the data points always showed curvature,second order models in %gelatin or %WPI were generated byregression analysis of viscosity and gel strength data for productsfrom the treatment of combinations of gelatin, WPI, and blendswith mTGase and DTT. These models are of the form:Response = a + b*Gelatin + c*Gelatin*Gelatin, where a, b, and c areregression coefficients. Analysis of covariance (ancova) was per-formed on the models to determine whether the coefficients weresignificantly different between treatments. Comparisons betweentreatments of the slopes and second order coefficients were per-formed using orthogonal contrasts (Littell et al., 2002). All testsof significance were performed at the P < 0.05 level.3. Results and discussion3.1. Gel strength The effects on gel strength of increasing concentrations of gela-tin (1–10%) in the presence or absence of mTGase and DTT wereinvestigated. The second order model showed that gel strengthsof all samples significantly increased (P < 0.05) with increasing gel-atin concentration and were greatest at each concentration forsamples containing gelatin alone (Fig. 1a). Because type I collagen,the primary source of gelatin, has no disulfide bonds, the additionof DTT to gelatin was not expected to have a noticeable effect ongel strength, and at concentrations below 6% gelatin, the additionof DTT did not impact the gel strength. At higher concentrationsof gelatin (6–10%), however, gel strengths were significantly(P < 0.05) reduced by up to 15%. One possible explanation is thepresence of type III collagen, which is generally co-located withtype I collagen in connective tissue (van der Rest et al., 1990).The individual chains of type III collagen are disulfide-linked (Bou-dko and Engel, 2004), and separation of these chains may haveinterfered with the partial renaturation that normally accompaniesgelation. Inclusion of mTGase in dilute (1–5%) gelatin solutions,either with or without DTT, resulted in gel strengths significantly(P < 0.05) lower than for gelatin alone (Fig. 1a). The formation ofintramolecular crosslinks, which would inhibit the attainment of Fig. 1. Gel strengths of gels formed from (a) bovine type B gelatin, 1–10% (w/w),collagen-like structure, is favored over intermolecular ones in di- and (b) WPI–gelatin blends, 10% WPI with 0–3% added gelatin. Gelatin gels werelute solutions (Sakamoto et al., 1994). At higher (6–10%) gelatin prepared in the presence or absence of mTGase (10 U/g protein) and DTT (10 mg/g protein), WPI–gelatin gels in the presence or absence of mTGase (2 U/g protein) andconcentrations where mTGase-mediated intermolecular crosslink- DTT (10 mg/g protein). Each data point represents a separate measurement.ing is more favorable, the resulting gel strengths were about 90% of Abbreviations are: G-gelatin, W-WPI, R-reducing conditions (DTT), E-enzymethose for gelatin alone. (mTGase). When the effect of mTGase (1–10 U/g gelatin) was determined,no trend could be observed in the gel strength of a 10% gelatin(data not shown). The range of values was between 188 and Under reducing conditions, a dramatic increase in gel strength225 g, with an average gel strength of 207 ± 12 g, significantly was seen for mTGase-treated WPI–gelatin blends, the significance(P < 0.05) lower than the 254 ± 1 g determined for 10% gelatin of which was confirmed by the second order statistical modelwithout enzyme. In the presence of the reducing agent, DTT, the (P < 0.05). Because reducing conditions and gelatin were essentialactivity of mTGase was enhanced, as reported by Kolodziejska for enhancing the gel strength, it is likely that crosslinking was be-et al. (2006), and gel strengths (263 ± 14 g) were comparable to tween whey proteins and gelatin chains. The reduction of disulfidethose for gelatin alone. bonds in the globular whey proteins by the action of DTT would ex- The formation of mTGase-mediated WPI gels has been previ- pose reactive groups to the action of the mTGase. In the absence ofously reported at WPI concentrations greater than 10% (Frge- DTT, the WPI proteins were not sufficiently unfolded to serve asmand et al., 1997). Under the conditions of these experiments, no effective substrates for mTGase.gel formation was observed for WPI alone at concentrations upto 10%, with or without mTGase, under reducing or nonreducing 3.2. Viscosityconditions. Gels did form when small amounts of gelatin (0.5–3%) were included in 10% WPI solutions (Fig. 1b). Gelatin alone The viscosities at 60 °C, of gelatin solutions (1–10%), with andat these concentrations forms only very weak gels, and so long as without DTT and mTGase, were determined (Fig. 2a). Neither DTTthe reaction conditions were not reducing, the gel strength was nor mTGase alone had a significant effect on gelatin viscosity atnot significantly altered whether the gelatin was alone or in a this temperature. Similar to the case for gel strength, the effect of10% WPI solution, with or without mTGase at 2 U/g WPI–gelatin. mTGase under reducing conditions was not significant at gelatin
  4. 4. E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–3643 3641 centrations less than 7%, neither DTT nor mTGase, alone or to- gether, had any significant effect on the viscosity of the solution. Between 7% and 10% WPI, mTGase alone did not affect the viscosity (data not shown), and by inference had little ability to crosslink the proteins. In this concentration range, the viscosity was decreased slightly when disulfide bonds were reduced and the protein con- formation was less well defined. A highly significant effect on the viscosity (P < 0.001) was observed as a result of the treatment that combined the action of DTT and mTGase on a 10% WPI solution; a four-fold increase in viscosity was attained. Viscosities measured at 25 °C for blends of gelatin (0.5–3%) with 10% WPI were always higher than those for gelatin alone (Fig. 2c). When mTGase was included in blends of 10% WPI with gelatin, the increase in observed viscosity was not significant. When both DTT and mTGase were included in the blend, their effect on viscosity became more significant (P < 0.001). In fact, viscosities for blends of 10% WPI with more than 1.5% gelatin could not be measured be- cause the solution formed a gel that did not melt below 60 °C. 3.3. Rheological properties The storage modulus (G0 ) was determined for 10% WPI, blends of 10% WPI with 3% gelatin, and samples treated with mTGase un- der reducing or nonreducing conditions (Fig. 3). A 10% WPI solu- tion, which remained liquid at 20 °C, exhibited a constant value less than 1 for G0 . In contrast, for a 10% WPI solution treated with mTGase under reducing conditions, G0 remained nearly constant and low for the first 10 h and then increased to 300 Pa between 10 and 20 h, suggesting the formation of a stable and permanent gel structure (Comfort and Howell, 2002). Because G0 can be re- lated to the amount of crosslinking, the similar and flat response of the WPI sample in a reducing environment and the WPI sample with mTGase under nonreducing conditions suggest that no signif- icant crosslinking occurred under these conditions. WPI–gelatin blends under reducing conditions without mTGase or with mTGase in a nonreducing environment showed a small increase in G0 be- tween 10 and 20 h, possibly due to noncovalent interactions be- tween gelatin and whey protein molecules. Chen and Dickinson (1999) suggested that electrostatic interactions, hydrogen bonding or hydrophobic interactions that have a physical nature and are temperature-dependent might contribute to the appearance of weak gel-like behavior. A similar study of 3% gelatin samples with or without mTGase (2 U/g gelatin) produced a constant G0 value near zero (data not shown). Blends of 10% WPI with 3% gelatin afterFig. 2. Viscosities of solutions of (a) gelatin, (b) WPI and (c) WPI–gelatin blendsprepared as described for Fig. 1. Viscosities were measured at 60 °C for gelatinsolutions and at 25 °C for WPI or WPI–gelatin blends. Abbreviations are as reportedfor Fig. 1.concentrations in the 1–6% range. Above 7% gelatin, the effect wasa significantly dramatic increase (P < 0.001) in viscosity to valuesgreater than 1800 mPa s, beyond the limit of the viscometer. Theseresults are consistent with the formation of intramolecular cross-links at low gelatin concentrations and both intra- and intermolec-ular crosslinks at higher concentrations (Clark and Courts, 1977; Yi Fig. 3. Time sweep analysis of 10% (w/w) WPI and WPI–gelatin blends. Blendset al., 2006). contain 10% (w/w) WPI and 3% (w/w) gelatin, mTGase was 10 U/g protein for WPI The viscosities at 25 °C of solutions of WPI (1–10%) with and and 2 U/g protein for WPI-gelatin blends, DTT was 10 mg/g protein. Abbreviationswithout DTT and mTGase were determined (Fig. 2b). At WPI con- are as designated for Fig. 1.
  5. 5. 3642 E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–3643incubation with mTGase (2 U/g protein) under reducing conditionsshowed an exponential increase of G0 from approximately 70 Pa toapproximately 5000 Pa within the first 10 h. This remarkable gainsuggests the existence of an initial gelled network that becamemore reticulated and stable with time (Eissa et al., 2004). Similarenhancements in storage modulus were reported for blends of gel-atin with small amounts of chitosan, a larger polymer, and mTGase(Chen et al., 2003).3.4. SDS–PAGE Gelatin, WPI, and blended biopolymers were analyzed bySDS–PAGE. Ten percent gelatin (Fig. 4, lane 2) reflects a polydis-perse mixture of different sized collagen fragments with someaggregated material in the stacking gel at the top, and faintbands visible through the general smear in the separating gel. Fig. 4. SDS–PAGE gels: lane 1, molecular weight markers, ranging in size fromAlthough more distinct bands can be seen in SDS–PAGE patterns 200 kDa at the top to 6.5 kDa at the bottom; lane 2, gelatin 10% (w/w); lane 3, 10%for gelatins isolated from a specific source (Taylor et al., 1994) gelatin after treatment with mTGase (10 U/g) under reducing conditions; lane 4, WPI 10% (w/w); lane 5, 10% WPI after treatment with mTGase (5 U/g); lane 6, 10%or high Bloom (250 g) commercial gelatins (Tosh et al., 2003), WPI after treatment with mTGase (5 U/g) under reducing conditions; lane 7, WPIthe pattern seen here is typical of low Bloom (115 g) commer- 10% (w/w) with gelatin 1.5% (w/w); lane 8, WPI 10% (w/w) with gelatin 1.5% aftercial gelatins (Taylor et al., 2004), which are sold on the basis treatment with mTGase (2 U/g); and lane 9 WPI 10% (w/w) with gelatin 1.5% afterof gel strength, and are blends of gelatins from different prepa- treatment with mTGase (2 U/g) under reducing conditions. This is a composite figure produced from two gels that were run simultaneously.rations. The pattern for 10% gelatin treated with mTGase underreducing conditions (Fig. 4, lane 3) shows only a faint smear,representing small collagen fragments that most likely lack thelysine or glutamine sidechains necessary for mTGase-mediated and rheological data that the inclusion of 1.5% gelatin in 10% WPIcrosslinking. Although samples prepared for lanes 2 and 3 were induces gel formation and enhances physical properties, the bio-of the same weight, the faintness of the pattern in lane 3 and polymers formed range in size from those formed by WPI whenthe lack of material in the stacking gel and the upper range treated with mTGase under reducing conditions to the much lar-of the separating gel suggests the formation of aggregates so ger aggregates that do not penetrate the gel.large they could not penetrate the stacking gel (Taylor et al.,2004). A similar observation (Sharma et al., 2002) was inter- 4. Conclusionspreted to suggest that the protein had become so highly poly-merized that it could not penetrate the stacking gel; they also Both gelatin (ICIS, 2006) and whey (NASS, 2006) are relativelydemonstrated that although the highly polymerized products low value byproducts of the American food industry. While thedid not appear on the gels, their presence could be confirmed actual prices vary over time the ratio is relatively constant atby chromatography. approximately 5:1 (gelatin:whey). The addition of minor The SDS–PAGE patterns for 10% WPI alone (Fig. 4, lane 4) or amounts of relatively low quality gelatin to whey protein im-after incubation with mTGase 5 U/g under nonreducing conditions proves the strength and stability of gels formed by the action(Fig. 4, lane 5) show the major whey proteins, b-lactoglobulin (MW of mTGase in a reducing environment. As a byproduct of theapproximately 18 kDa) and a-lactalbumin (MW approximately meat industry, and a breakdown product of collagen, gelatin14 kDa) as well as faint bands for bovine serum albumin (MW comes in a range of qualities. The higher quality gelatins findapproximately 66 kDa) and lactoferrin (MW approximately a variety of uses in food, pharmaceutical and industrial products86 kDa). Incubation of 10% WPI with mTGase 5 U/g under reducing and are heavily traded. Whey is a lower value byproduct thatconditions (Fig. 4, lane 6) resulted in bands, above both the sepa- can be recovered from the waste stream of the cheese industry.rating gel and the stacking gel, showing formation of high molecu- The use of mTGase to catalyze the formation of crosslinks in andlar weight polymers in addition to the typical WPI bands. An earlier between protein molecules is well established, as is the efficacystudy (Frgemand et al., 1997) produced similar results. of its usefulness with either gelatin or WPI. When a small The addition of 1.5% gelatin to 10% WPI had little effect on the amount of gelatin was added to WPI, before mTGase treatmentgel pattern (Fig. 4, lane 7). In an earlier study, when small under reducing conditions, a dramatic rise in viscosity, higheramounts of WPI were added to 10% gelatin samples (Taylor gel strengths, and the appearance of high molecular weightet al., 2006), the whey component was clearly visible in the gel bands due to inter-protein crosslinking in SDS–PAGE gel patternspatterns, and its participation in the formation of crosslinked bio- than for either gelatin or WPI treated separately were observed.polymers could easily be monitored. Here, the lack of defined These results suggest that the reducing environment partiallymolecular weight bands in patterns for gelatin and the low con- unfolds the whey proteins, increasing access to glutamine andcentration of gelatin in the blends contribute to its invisibility. lysine side chains, and that the gelatin chains crosslink the wheyIncubation of the WPI–gelatin blend with mTGase under nonre- proteins to form a network. The improvement in physical prop-ducing conditions resulted in the appearance of a high molecular erties over either protein component, given the same treatment,weight band, possibly representing crosslinked gelatin fragments, suggests the possibility of greater utilization and new productsbut had little effect on the WPI pattern (Fig. 4, lane 8). The gel from these byproducts.pattern (Fig. 4, lane 9) for a similar sample incubated underreducing conditions shows a decrease in intensity of the WPI Acknowledgementsbands, an increase in high molecular weight aggregates and a de-crease in total protein. The pattern is consistent with the forma- The authors would like to thank Lorelie Bumanlag, Paul Pierlott,tion of WPI–gelatin biopolymers in addition to aggregates of the Michael Tunick and Carlos Carvalho for their technical support inindividual biopolymers. Although it is clear from the gel strengths this work.
  6. 6. E. Hernàndez-Balada et al. / Bioresource Technology 100 (2009) 3638–3643 3643References NASS, National Agricultural Statistics Service, 2006, Dairy product prices, June 30, 2006. <http://usda.mannlib.cornell.edu/MannUsda/viewDocumentInfo.do? documentID=1450>.Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., Tanaka, H., Oh, J.H., Wang, B., Field, P.D., Aglan, H.A., 2004. Characteristics of edible films made Motoki, M., 1989. Purification and characteristics of a novel transglutaminase from dairy proteins and zein hydrolysate cross-linked with transglutaminase. derived from microorganisms. Agric. Biol. Chem. 53, 2613–2617. Int. J. Food Sci. Tech. 39, 287–294.Association of Official Analytical Chemists, 1990. Jelly strength of gelatine, final Rodríguez-Nogales, J.M., 2006. Effect of preheat treatment on the transglutaminase- action (948.21). In: Helrich, K. (Ed.), Official Methods of Analysis of the catalyzed cross-linking of goat milk proteins. Process Biochem. 41, 430–437. Association of Official Analytical Chemists, 15th ed. AOAC, Arlington, VA, p. 929. Rose, P.I., 1992. Inedible gelatin and glue. In: Pearson, A.M., Dutson, T.R. (Eds.),Boudko, S.P., Engel, J., 2004. Structure formation in the C terminus of type III Inedible Meat By-Products, Advances in Meat Research. Elsevier Applied collagen guides disulfide cross-linking. J. Mol. Biol. 335, 1289–1297. Science, New York, pp. 217–263.Chen, J., Dickinson, E., 1999. Interfacial ageing effect on the rheology of a heat-set Sakamoto, H., Kumazawa, Y., Motoki, M., 1994. Strength of protein gels prepared protein emulsion gel. Food Hydrocoll. 13, 363–369. with microbial transglutaminase as related to reaction conditions. J. Food Sci.Chen, T., Embree, H.D., Brown, E.M., Taylor, M.M., Payne, F.P., 2003. Enzyme- 59, 866–871. catalyzed gel formation of gelatin and chitosan: potential for in situ Sharma, R., Zakora, M., Qvist, K.B., 2002. Susceptibility of an industrial applications. Biomaterials 24, 2831–2841. a-lactalbumin concentrate to cross-linking by microbial transglutaminase. Int.Clark, R.C., Courts, A., 1977. The chemical reactivity of gelatin. In: Ward, A.G., Courts, Dairy J. 12, 1005–1012. A. (Eds.), The Science and Technology of Gelatin. Academic Press, New York, pp. Taylor, M.M., Diefendorf, E.J., Marmer, W.N., Brown, E.M., 1994. Effect of various 209–247. alkalinity-inducing agents on chemical and physical properties of proteinComfort, S., Howell, N.K., 2002. Gelation properties of soya and whey protein isolate products isolated from chromium-containing leather waste. J. Am. Leather mixtures. Food Hydrocoll. 16, 661–672. Chem. Assoc. 89, 221–228.Eissa, A.S., Bisram, S., Khan, S.A., 2004. Polymerization and gelation of whey protein Taylor, M.M., Cabeza, L.F., Marmer, W.N., Brown, E.M., 2001. Enzymatic modification isolates at low pH using transglutaminase enzyme. J. Agric. Food Chem. 52, of hydrolysis products from collagen using a microbial transglutaminase I. 4456–4464. Physical properties. J. Am. Leather Chem. Assoc. 96, 319–332.Frgemand, M., Otte, J., Qvist, K.B., 1997. Enzymatic cross-linking of whey proteins Taylor, M.M., Marmer, W.N., Brown, E.M., 2004. Molecular weight distribution and by Ca2+-independent microbial transglutaminase from Streptomyces lydicus. functional properties of enzymatically modified commercial and experimental Food Hydrocoll. 11, 19–25. gelatins. J. Am. Leather Chem. Assoc. 99, 129–141.Farrell Jr., H.M., Jiménez-Flores, R., Bleck, G.T., Brown, E.M., Butler, J.E., Creamer, L.K., Taylor, M.M., Marmer, W.N., Brown, E.M., 2006. Preparation and characterization of Hicks, C.L., Hollar, C.M., Ng-Kwai-Hang, K.F., Swaisgood, H.E., 2004. biopolymers derived from enzymatically modified gelatin and whey. J. Am. Nomenclature of the proteins of cows’ milk-sixth revision. J. Dairy Sci. 87, Leather Chem. Assoc. 101, 235–248. 1641–1674. Tosh, S.M., Marangoni, A.G., Hallett, F.R., Britt, I.J., 2003. Aging dynamics in gelatinFolk, J.E., Chung, S.I., 1973. Molecular and catalytic properties of transglutaminases. gel microstructure. Food Hydrocoll. 17, 503–513. Adv. Enzymol. Relat. Areas Mol. Biol. 38, 109–191. Truong, V.D., Clare, D.A., Catignani, G.L., Swaisgood, H.E., 2004. Cross-linking andFolk, J.E., Chung, S.I., 1985. Transglutaminases. Method. Enzymol. 113, 358–375. rheological changes of whey proteins treated with microbial transglutaminase.Grazú, V., Ovsejevi, K., Cuadra, K., Betancor, L., Manta, C., Batista-Viera, F., 2003. J. Agric. Food Chem. 52, 1170–1176. Solid-phase reducing agents as alternative for reducing disulfide bonds in US Dairy Export Council’s, 2006. Annual Report at <http://usdec.org> (accessed proteins. Appl. Biochem. Biotech. 110, 23–32. 31.08.07.).ICIS News, Chemical Industry News, Chemical Prices and Chemical Suppliers from van der Rest, M., Dublet, B., Champliaud, M.-F., 1990. Fibril-associated collagens. ICIS, 2006 at <http://www.icis.com/Articles/2006/08/28/2015785/Chemical- Biomaterials 11, 28–31. Prices-F-J.html>. Wainewright, F.W., 1977. Physical tests for gelatin and gelatin products. In: Ward,Kolodziejska, I., Piotrowska, B., Bulge, M., Tylingo, R., 2006. Effect of G.A., Courts, A. (Eds.), The Science and Technology of Gelatin. Academic Press, transglutaminase and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide on New York, pp. 507–534. the solubility of fish gelatin-chitosan films. Carbohydr. Polym. 65, 404–409. Yi, J.B., Kim, Y.T., Bae, H.J., Whiteside, W.S., Park, H.J., 2006. Influence ofLaemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the transglutaminase-induced cross-linking on properties of fish gelatin films. J. head of bacteriophage T4. Nature 227, 680–685. Food Sci. 71, E376–E383.Littell, R.C., Stroup, W.W., Freund, R.J., 2002. SAS for Linear Models, fourth ed. SAS Yildirim, M., Hettiarachchy, N.S., 1997. Biopolymers produced by cross-linking Institute Inc., Cary, NC. soybean 11S globulin with whey proteins using transglutaminase. J. Food Sci.Motoki, M., Nio, N., 1983. Crosslinking between different food proteins by 62, 270–274. transglutaminase. J. Food Sci. 48, 561–566.

×