Parameter Optimization of Protein Film Production Using Microbial

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Biomacromolecules 2010, 11, 896–903

Parameter Optimization of Protein Film Production Using Microbial Transglutaminase Katja Patzsch, Kristin Riedel, and Markus Pietzsch* Department of Downstream Processing, Institute of Pharmacy, Faculty of Sciences I, Martin Luther University Halle-Wittenberg, 06099 Halle (Saale), Germany Received November 2, 2009; Revised Manuscript Received February 5, 2010

Sodium caseinate films were produced using microbial transglutaminase as a protein cross-linking biocatalyst. Basic parameters for the film production, such as buffer type and concentration, pH, temperature, plasticizer concentration and its influence on transglutaminase activity, mold material for film casting, specimen width, and cutting method, were investigated and compared with standardized methods (DIN EN ISO 527-3). Surprisingly, a previously described sodium phosphate buffer (50 mM, pH 8.0) resulted in crystals after drying the films for 48 h. To avoid this deteriorating effect, the buffer system was optimized and finally a Tris-HCl buffer (20 mM, pH 7.0) was chosen for the production of transparent, smooth films without crystallization. Incubation time and temperature during enzyme treatment had a considerable influence on the mechanical properties of the films.

1. Introduction Naturally occurring biopolymers like polysaccharides or proteins are of growing interest as alternatives to petroleumbased materials. There are numerous reports in the literature on protein based films. Besides their origin from renewable biological resources, protein based materials have been shown to be fully biodegradable.1 Films have been produced from a variety of different proteins, for example, collagen, gelatin, dairy proteins, corn zein, wheat gluten, and soy protein.2-4 Depending on their origin, the protein fractions used are more or less complex mixtures of different macromolecules and are used directly for film production or as blends, for example, with polysaccharides. As for petroleum-based polymer films, good mechanical properties and barrier properties against gases, vapors, and solutes are important for biodegradable films. So far, the application of protein films is limited by their poor water vapor resistance and lower strength when compared to synthetic polymers.5,6 To improve mechanical and surface properties of biodegradable films numerous physical, chemical, or enzymatic treatments have been used. For example, the addition of plasticizers like glycerol or sorbitol is a successful way to obtain a flexible proteinous material by weakening the hydrogen bonding. Cross-linking may be catalyzed by enzymes or by using cross-linking reagents as, for example, formaldehyde.7 So far, enzymatic cross-linking is limited to the application of microbial transglutaminase (protein-glutamine γ-glutamyltransferase, EC 2.3.2.13, MTG), which catalyzes an acyl transfer reaction between a γ-carboxyamide group of a glutamine residue of a protein or peptide and the ε-amino group of a lysine residue. This reaction leads to the formation of covalent ε-(γ-glutamyl-) lysine bonds.8 The effects of MTG treatment on the film properties have been studied for several proteins, for example, gelatin,9 casein and whey protein,10 pea protein,11 soy protein isolates,12 and pectin-soy flour.13 During the past years, a variety of different methods has been established for the preparation of protein films, sample prepara* To whom correspondence should be addressed. Tel.: +49 345 55 25 949. Fax: +49 345 55 27 260. E-mail: markus.pietzsch@ pharmazie.uni-halle.de.

tion and characterization. As solvent for the protein, distilled water,6,13 sodium phosphate buffer (50 mM, pH 8.0;10 0.1 M, pH 6.0),14 or Tris-HCl buffer (0.1 M, pH 8.0)15 have been used. After homogenization and degassing, the film was usually cast into Petri dishes with a diameter of 6013,14 or 140 mm10 consisting of polystyrene or Teflon-coated glass. The subsequent drying (and the simultaneous cross-linking) process was carried out overnight at room temperature (RT)10 for 24 h6 or at 50 °C overnight.13 The reaction catalyzed by MTG occurred either at RT10 or at 50 °C for 15 min6 or for 4 h,9 after which the reaction was stopped by heat deactivation of the enzyme (85 °C, 10 min). To measure the mechanical properties of the protein films, for example, tensile strength (TS) and elongation at break (EB), the dried films were cut into stripes mostly using a sharp razor blade or a scalpel.13,16 Sample dimensions (width × grip separation) were very different: 20 × 20 mm,10 25.4 × 50 mm,6 or 10 × 90 mm.13,14 The aim of the present paper is to compare the different methods with regard to solvent/buffer system, film preparation, and sample dimensions for testing mechanical properties. Furthermore, the influence of glycerol on the activity of MTG was investigated because this has not been reported so far. The effect of different MTG reaction conditions (temperature and time) on the mechanical properties of protein films was also investigated.

2. Materials and Methods 2.1. Materials. Sodium caseinate was obtained from Rovita GmbH (Engelsberg, Germany). Microbial transglutaminase (MTG; TG ActivaWM, 81-135 U/g preparation, enzyme content 1% in maltodextrin) was obtained from Ajinomoto (Hamburg, Germany). MTG amounts are given in U/g protein. Z-Gln-Gly was purchased from Bachem (Weil am Rhein, Germany). Stripes for sensitive measurement of the pH (PEHANON, pH 6.0-8.1 and pH 8.0-9.1) were obtained from Roth (Karlsruhe, Germany). All other chemicals (e.g., plasticizers, buffer salts) were purchased in analytical grade from Fluka-Aldrich (Taufkirchen, Germany) or Roth (Karlsruhe, Germany). Maltodextrin was obtained from Fluka (Taufkirchen, Germany) and had a molecular weight of 1672 g/mol (degree of polymerization ) 10.32 dextrose units). The protein marker solution (Protein Molecular Weight Marker) used for

10.1021/bm901248d  2010 American Chemical Society Published on Web 03/10/2010

Parameter Optimization for Protein Film Production

Figure 1. Mold for film preparation consisting of a PTFE plate that is fixed on top of an aluminum plate (8 mm) to prevent deformation. Into the PTFE plate a pocket was milled; the surface was ground with sandpaper (grit size 1000) and polished with rough tissue paper.

SDS-PAGE was purchased from Fermentas (St. Leon-Rot, Germany). Deionized water was used throughout the experiments. 2.2. Methods. 2.2.1. Film Preparation: Standard Method. The enzymatic cross-linking was carried out using the commercially available ActivaWM preparation as a biocatalyst. Activa consists of 1% (w/w) MTG and 99% (w/w) maltodextrin as filler. Maltodextrin is most probably added to increase the stability of MTG during spray drying and storage. In addition, the handling is simplified since larger amounts can be weighted more accurately. As a control, non-cross-linked films were prepared by using the equal amount of pure maltodextrin. The film-forming solution was prepared according to Oh et al. by mixing 3 g glycerol with 110.4 g solvent.10 A total of 6 g sodium caseinate and 1.5 g maltodextrin (for samples without MTG) were dissolved in the plasticizer-buffer solution. The homogenization at 90 °C was done for 30 min because of a volumetric scale up compared to Oh et al. Thereafter, the solution was cooled to RT and the pH was adjusted to pH 7.0 by adding 1 M NaOH. The solution was equilibrated to a temperature of 50 °C at which the MTG shows optimum activity.17 A solution of MTG (1.5 g TG ActivaWM) in 6 mL of preheated buffer (50 °C) was added to the film-forming solution (for samples without MTG, 6 mL of buffer was used). After centrifugation at 177 g and 40 °C for 1 min to force degassing, the solution was poured immediately into a 200 × 200 mm mold consisting of PTFE fixed on an aluminum plate to prevent bending (Figure 1). This mold was manufactured inhouse using a CNC lathe. The surface was polished using a P 1000 threads per inch wet milling waterproof sandpaper. The casted film was kept in level at RT in a fume hood to facilitate drying, removed from the mold when dry (after 48 h) and equilibrated for 48 h at 23 ( 2 °C and 50 ( 3% relative humidity (RH) using a cabinet over a saturated Ca(NO3)2 solution for humidity control. All heating and cooling steps during film production were done using water baths. 2.2.2. Film Preparation: InVestigation of the Buffer System and pH. Sodium caseinate films were prepared essentially as described above. A quarter of the given amounts were used and the film-forming solution was poured into frames. Because of a high number of films and no intended mechanical testing, the films were prepared in acrylic glass frames with dimensions of 90 × 100 mm and a PTFE foil as base. Sodium phosphate or Tris-HCl buffer with concentrations of 0, 5, 10, 20, 50, and 100 mM were used as well as pH values of 6.0, 7.0, and 8.0 (phosphate buffer) or 7.0 and 8.0 (Tris-HCl buffer). Optical properties of the protein films (transparency, crystallization) and buffer capacity were analyzed visually and microscopically. The buffer capacity was investigated by adding MTG (0.375 g dissolved in 1.5 mL buffer) to the film-forming solution at RT for 16 h and the pH was measured using pH stripes (accuracy 0.3 pH units). The difference between the set pH before and the measured pH after the enzyme reaction was used to evaluate the capacity of the buffer. A film forming solution with 50 mM Tris-HCl buffer and pH values of 6.0, 7.0, 8.0, and 9.0 were prepared to investigate the influence on

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sodium caseinate cross-linking. Samples were taken from the filmforming solution after 30 min, diluted 1:100 with SDS-sample buffer, heated at 99 °C for 5 min and analyzed by SDS-PAGE as described below. 2.2.3. Film Preparation: Variation of Cross-Linking Time and Temperature. Sodium caseinate films were prepared according to the standard method (see above) using Tris-HCl buffer (20 mM, pH 7.0). To investigate the influence of the enzyme reaction parameters on the mechanical properties of protein films, three different methods were investigated. Method 1: According to the method published by Oh et al., the MTG was added to the film-forming solution at 50 °C and the film was dried at RT.10 Method 2: MTG was added to the film-forming solution at 50 °C and the film was dried overnight at 50 °C as described by Di Pierro et al.14 Method 3: According to Chambi and Grosso, MTG was added to the film-forming solution at 50 °C and was kept for 15 min at 50 °C. The enzyme was inactivated by heating the solution at 90 °C for 10 min. The film was dried at RT.6 For method 2, in a separate experiment samples were withdrawn at regular time intervals and analyzed by SDS-PAGE and for MTG activity using the standard assay. For SDS-PAGE, the sample was diluted 1:100 with SDS-sample buffer and heated at 99 °C for 5 min. 2.2.4. Measurement of Mechanical Properties: Standard Protocol. The mechanical properties such as tensile strength and elongation at break of the sodium caseinate films were measured using a Zwick universal testing machine (model BDO-FB0.5TH, Zwick GmbH&Co. KG, Ulm, Germany) according to the standard procedure of DIN EN ISO 527-3. The films were cut into specimens of 15 × 150 mm and mounted between chuck jaws. The initial distance between the top and bottom jaws was 100 mm. The test speed was set to 50 mm/min. The specimens were stretched until broken. Tensile strength (N/mm2) was calculated as peak load [N]/initial cross-sectional area (mm2). Elongation at break (%) was expressed as percentage of [final gap (mm)/initial gap (mm)] × 100. Initial gap was the start distance between the two jaws and final gap was the length at which the film was broken. Usually, five test pieces were analyzed and the average was calculated. The standard deviation was usually in the range of 2-10%. Thickness of protein films prepared according to the standard method was determined by a micrometer screw to be 220 ( 30 µm. 2.2.5. Variation of Specimen Cutting and Sample Dimensions. To investigate the influence of the sample dimensions on the mechanical properties, specimens with 10 or 15 mm width and 60, 80, or 150 mm length were used. The initial distance between the top and bottom jaws was set to 32, 50, or 100 mm. Specimen were cut by a commercial cutter knife, scissors or a self-designed double-bladed roller knife to analyze the influence of the sample preparation. The cutting edge was investigated by a light microscope. The mechanical properties of the films were measured using the standard protocol. 2.2.6. Determination of Transglutaminase ActiVity: Standard Hydroxamate Assay. The activity of MTG was assayed according to the colorimetric hydroxamate procedure (MTG standard assay).18 Usually, the enzyme sample volume was 10 µL and the volume of the substrate solution 140 µL (130 + 10 µL additives). A calibration curve was measured using commercially available L-glutamic acid γ-monohydroxamate in the range from 0 to 5 mM. One unit of MTG is defined as the formation of 1 µmol L-glutamic acid γ-monohydroxamate per min at 37 °C and pH 6.0. The absorption was measured using a microtiter plate reader (Fluostar Galaxy, BMG Labtech GmbH, Offenburg, Germany). 2.2.7. Determination of Transglutaminase ActiVity in the Presence of Glycerol. Glycerol in the absence of MTG was added to the substrate solution of the standard activity test (10 µL) in different concentrations 0-3.33% (w/w) to analyze the influence of glycerol on the assay itself. MTG was added to analyze the influence of glycerol on the activity of MTG. In a second experiment, the MTG was stored in a glycerol buffer solution with concentrations of 0-50% (w/w) for 3.5 h at RT to analyze the influence of glycerol on the stability of MTG. A solution without glycerol was used as blank.

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Patzsch et al. Table 1. Optical Properties of Sodium Caseinate Films (without MTG) and Buffer Capacity (pH-Shift During MTG Reaction) Dependent on Concentration and pH of Tris-HCl Buffer

Figure 2. Light microscope image (magnification 134×) of a sodium caseinate film prepared using 50 mM sodium phosphate buffer, pH 8.0, showing severe crystallization.

2.2.8. SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE). SDS polyacrylamide gel electrophoresis was performed according to the method of Laemmli19 using a Mighty Small apparatus from Hoefer (Amersham Biosciences, Freiburg, Germany). The gels consisted of a resolving (12.5% acrylamide) covered by a stacking (4.5% acrylamide) gel and were stained with Coomassie blue. Usually, 10 µL of the respective sample (containing approximately 1 mg protein per mL) and 5 µL of the molecular weight marker were added to the SDS-PAGE gel. After destaining, the gels were dried between two cellophane foils fixed in a frame.

3. Results and Discussion 3.1. Optimization of the Buffer System. To study the crosslinking efficiency of MTG, sodium caseinate films were prepared according to Oh et al. using 50 mM sodium phosphate buffer, pH 8.0 as the solvent.10 After 48 h of equilibration in the cabinet at 23 ( 2 °C and 50 ( 3% RH, unexpected crystallization was observed in the protein films (see Figure 2 for a microscope image). The crystals caused inhomogeneities in the films and the measurement of mechanical properties was difficult because samples broke preferentially at the crystals. To prevent crystallization, buffer type, concentration, and pH were varied as described in Materials and Methods. Pure distilled water was investigated as a reference. Crystals were only observed at 50 and 100 mM sodium phosphate buffer of both pH 7.0 and 8.0. The crystallization was observed 96 h after film casting. This effect was not described by Oh et al., who dried their films in Teflon-coated glass Petri dishes (140 mm) overnight and equilibrated them at 23 °C and 50% RH for 24 h. Obviously, this period was too short for the formation of crystals. Because of possible but unwanted crystallization, a sodium phosphate buffer is not suitable for the preparation of protein films. With Tris-HCl buffer as the solvent (over the full range of concentration and pH), no crystallization was found but the sodium caseinate films showed different optical properties (Table 1, left). At high concentrations of Tris-HCl at both pH 7.0 and 8.0, opaque films were observed, which were also partially uneven. As expected, when using pure water, the setting of the pH turned out to be very difficult (even though caseinate could itself act as a buffer). Very careful addition of NaOH and extensive mixing was necessary to prevent precipitation. The capacity of Tris-HCl buffer at various concentrations and pH values is shown in Table 1 (right). At pH 7.0, a maximum

optical properties (films without MTG cross-linking)

pH shift (pH units; films with MTG cross-linking)

Tris-HCl buffer (mM)

pH 7.0

pH 8.0

pH 7.0

pH 8.0

0 5 10 20 50 100

transparent, plane transparent, plane transparent, plane transparent, plane opaque, plane opaque, uneven

transparent, plane transparent, plane transparent, plane transparent, plane transparent, plane opaque, plane

–0.4 –0.4 –0.3 –0.2 –0.2 –0.1

–0.8 –0.7 –0.5 –0.5 –0.3 –0.2

deviation of 0.4 pH units was observed at low Tris-HCl concentrations. The highest deviation of 0.8 pH units was detected for pH 8.0. At pH 8.0, a buffer concentration of g50 mM was necessary to stabilize the pH during the MTG reaction. For the preparation of transparent films at a maximum buffer capacity, a 20 mM Tris-HCl buffer, pH 7.0, or a 50 mM TrisHCl buffer, pH 8.0, is suitable. To select between pH 7.0 and 8.0, samples were taken during the film preparation procedure and analyzed by SDS-PAGE (Figure 3). To investigate the possible pH window at which the MTG is active, experiments were also performed at pH 6.0 and 9.0. Lane 1 shows casein without enzymatic treatment as a reference for non-cross-linked protein. The three protein bands (30-35 kDa) represent R-, β-, and κ-casein, respectively. At pH 6.0, 7.0, and 8.0 (lanes 3-5) the non-cross-linked casein was completely converted to protein polymers. Two main bands were observed: The first band (B1) was located between the stacking and the resolving gel and the second one (B2) was observed on top of the stacking gel, indicating proteins which were not able to enter the polyacrylamide gel at all due to their high molecular weight. The second (high MW) band was mainly observed at pH 6.0 and 7.0. At pH 8.0, large molecules were observed as well; however, the first band dominates. At pH 9.0 (lane 6), the high MW band was completely missing and some non-cross-linked casein remained. In conclusion, a 20 mM Tris-HCl buffer of pH 7.0 was chosen as the optimum buffer for further film production and optimization experiments. Films prepared using this buffer were transparent, planar, did not contain any crystals, and the pH remained almost constant during the enzymatic cross-linking reaction.

Figure 3. SDS-PAGE showing the cross-linking of sodium caseinate by MTG dependent on the pH. Samples of 100 µL were taken from the film-forming solution, diluted 1:100 with SDS-sample buffer and heated at 99 °C for 5 min. A total of 10 µL of the sample and 5 µL of the molecular weight marker were added to the SDS-PAGE gel (50 mM Tris-HCl buffer; R, non-cross-linked sodium caseinate as reference; M, molecular weight marker).


Figure 4. Relative activity of MTG, (A) depending on glycerol concentration present in the activity test and (B) after storage at RT for 3.5 h at different glycerol concentrations. The MTG activity of the solution was determined immediately after sampling by using the standard hydroxamate assay. Data points represent the mean value of four replicates and standard deviations are shown as error bars.

3.2. Effect of Glycerol on the Activity of MTG. Glycerol is often used as a plasticizer in protein film production.6,9,10,14 However, so far it was not reported if glycerol itself does have an influence on the activity of MTG. Therefore, the influence of different concentrations of glycerol present in the standard hydroxamate assay was investigated as described in Materials and Methods. The maximum concentration investigated (3.33%) corresponds to the amount of glycerol introduced to the assay solution through the sample taken from the stability test at 50% glycerol. The latter concentration is approximately 20 times the starting glycerol concentration in the film-forming solutions. The glycerol concentration is increasing during the drying process of the films to a final concentration of 28%. Therefore, in this experiment, the range of glycerol concentrations to which the MTG is exposed was covered. As can be seen from Figure 4A, there was no significant change in activity up to a glycerol concentration of 3.33% (w/w) present in the standard assay. Figure 4B shows the effect of glycerol on the activity of MTG during storage for 3.5 h at RT. The relative activity of MTG increased with rising glycerol concentration. At 50% glycerol, the relative activity was nearly 30% higher than for the control without glycerol. This effect could be explained by the function of glycerol to act as a stabilizer to enzymes as it was shown for aldehyde dehydrogenase.20 To sum up, there is a positive effect of glycerol on the stability of the MTG under film-forming conditions. 3.3. Influence of Sample Preparation and Sample Dimensions on the Mechanical Properties of Protein Films. In the literature, a variety of different methods is used for the preparation of specimen and testing of protein films (whether cross-linked or not), and a standardized method is missing. Sample width and initial grip separation differ as well as the cutting procedure. The only method commonly used by all authors of recent papers is the preincubation of films at 23 ( 2 °C and constant humidity of 50 ( 3% RH according to DIN EN ISO 291 prior to measurement.21 The period of preincubation varies from 2410 to 72 h.6 In the present paper, 48 h were selected, which is sufficient for thin protein films (e250 µm) and between the instructions of ASTM D 882-02 (g40 h) and DIN EN ISO 291 (88 h). Because these parameters may influence the mechanical properties some of the published methods were compared and a standard protocol was developed. So far, protein films were produced mainly in Petri dishes and test specimens with a maximum length of 50-100 mm


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could be prepared. Therefore, different initial grip separations of 2010 and 50 mm6 as well as various sample widths were used for mechanical testing, which do not reflect the European standard DIN EN ISO 527-3. Therefore, the influence of changing sample dimensions to the mechanical properties was investigated. In the present study, all films were casted in a 200 × 200 mm frame and test pieces with lengths g150 mm were produced by cutting. With regard to standardization, an initial grip separation of 100 mm according to DIN EN ISO 527-3 was used.22 Three different initial grip separations and two sample widths were compared. The testing machine used in the present study was limited to a minimal distance between the chuck jaws of 32 mm. The results are shown in Figure 5. A small but significant influence of the initial grip separation on tensile strength was detected for 15 mm specimens. Tensile strength decreased by 9.5% from 2.46 N/mm2 (32 mm initial grip separation) to 2.25 N/mm2 (100 mm initial grip separation). This effect is caused by the larger sample area when using 100 mm separation. The risk of inhomogeneities and possible points of fracture being present is therefore increased. At 10 mm width, an initial grip separation of 100 mm resulted in a TS of 2.44 N/mm2, which was higher than with 32 mm initial grip separation (2.31 N/mm2). The reason for this effect is unclear but could be explained by the relatively high standard deviation. For the elongation, the measured values were within the range of error and therefore not significant. To obtain comparable results, for protein film preparation the standard according to DIN EN ISO 527-3 should also be used. For using a specimen type 2 (stripe of g150 mm length and 10-25 mm width), the standard stipulates an initial grip separation of 100 ( 5 mm. Because of the lower standard deviation, in this study a sample width of 15 mm was chosen. Another difference in literature is the way of cutting the specimen out of the protein film. Cutting by a cutter knife and scissors were compared with a new tool. The new tool, a doublebladed roller knife shown in Figure 6B consists of two parallel knives, which allows a uniform sample width and an easy and fast handling. As can be seen from Figure 6A, the cutting method does have an influence on the tensile strength and elongation at break and also on the respective standard deviation. Highest standard deviations were found for the cutter knife, whereas the scissors and the roller knife showed comparable low standard deviations. As can be seen from the microscope images (Figure 6C-E), the cutting edges slightly differ for the three tools. The use of a cutter knife resulted in ripped edges reflecting defects, which could result in preferred rupture at these positions. The relatively high standard deviations might be caused by these defects. With the scissors and the double-bladed roller knife, sharp edges could be produced, with the scissors producing the sharpest cut. Compared to the double-bladed roller knife, the scissors cut needs some more steps (marking cutting lines, two cuts). As a result, the double-bladed roller knife is a useful cutting tool because of the sharp cutting edges, reproducible mechanical properties, and easy preparation of the specimens. 3.4. Influence of the MTG Cross-Linking Conditions on the Mechanical Properties. In the literature, different reaction conditions for enzyme catalyzed cross-linking have been applied: (1) MTG addition at 50 °C and film drying at RT;10 (2) MTG treatment and film drying at 50 °C;14 and (3) MTG treatment at 50 °C for 15 min, inactivation for 10 min at 90 °C and film drying at RT.6 So far, the effect of the cross-linking temperature was only investigated for soy protein isolate films.23 The authors

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Figure 5. Mechanical properties (A, tensile strength; B, elongation at break) of sodium caseinate films dependent on specimen width and initial grip separation at 32, 50, and 100 mm (6 g sodium caseinate, 3 g glycerol, and 1.5 g maltodextrin in 110.4 mL of 20 mM Tris-HCl buffer, pH 7.0, dried at RT for 48 h; cross-head speed was 50 mm/s). Data columns represent the mean value of five replicates and standard deviations are shown as error bars.

showed that an increase in temperature from 18 to 50 °C resulted in a decrease of TS and EB. As a reason, the faster loss of MTG activity at higher temperatures was discussed. In the present study, the three methods mentioned above were compared with respect to cross-linking time and temperature under standard conditions (20 mM Tris-HCl buffer, pH 7.0) for the preparation of caseinate films. The results are shown in Figure 7. Temperature and time of MTG addition and film drying had an influence on the tensile properties. The films produced with method 1 showed the highest mechanical properties with a TS of 4 N/mm2 and an EB of 184%. After drying the films at 50

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°C according to method 2, an equal TS of 4 N/mm2 was measured. However, the EB dropped to 124%. Method 3 resulted in films with a TS of 2.46 N/mm2 and an EB of 154%. Compared with methods 1 and 2, the TS was reduced by 50% and the EB value was between the other two values. These results can be explained by a varying degree of crosslinking of caseinate. An influence of the MTG concentration on TS and EB of caseinate films has been reported by Bruno et al.24 They found an increase in TS for MTG concentrations from 0 to 2 mg/g (MTG/caseinate). The EB showed a maximum for an MTG concentration of 1 mg/g (MTG/caseinate). To verify this explanation, for method 2 the progress of cross-linking was investigated by SDS-PAGE as described in Materials and Methods. As can be seen from Figure 8A, after 15 min, a cross-linking reaction could be detected. However, non-cross-linked casein was still detectable and polymers were able to enter the stacking gel (compare Figure 3, protein band 1). After this time, according to method 3, the reaction was stopped. The crosslinking did not result in high MW polymers of protein band 2. Consequently, the low mechanical strength observed for this method is a result of incomplete cross-linking. With increased incubation time, the non-cross-linked casein bands disappeared almost completely and more polymers were formed that could not enter the stacking gel because of their high molecular weight. After 120 min, the film was completely gelatinized and no more samples could be taken. To examine if the reduced EB in method (2) is a result of decreased MTG activity as suggested by Jiang et al.23 for soy protein isolate, samples from the film casting solution were analyzed for MTG activity. As can be seen from Figure 8B, for caseinate there is no loss in MTG activity during 90 min of reaction time at 50 °C. MTG is significantly stabilized by the high concentration of caseinate. The reduced EB is most probably a result of too many cross-links as suggested also by Bruno et al. for MTG concentrations >1 mg/g (MTG/caseinate).24 Under optimum conditions (method 1), the films with MTG treatment showed an increase in TS of 66% compared to noncross-linked sodium caseinate films (Figure 6A) resulting in 4 N/mm2. Also, EB was improved by 40% to a value of 183%. This result is in partial contrast to the results of Oh et al. who reported on decreased TS in MTG cross-linked casein films.10 However, the reason for this is unclear and can not be discussed since information about the MTG concentration used by Oh et al. is missing. The enzyme-catalyzed reaction is producing distinct isopeptide bonds between the side chain amide residue of glutamine and the ε-amino group of lysine. By this “vulcanization” reaction, polymers of large molecular weight are produced. Most probably, there is a three-dimensional network formed; however, detailed investigations about the quantity and cross-linking degree are lacking so far. Because the cross-linking biocatalyst itself is a macromolecule of nearly 38.000 g/mol, one should await that there is an increasing diffusion limitation with increasing degree of cross-linking. The commercial preparation of transglutaminase contains large amounts of maltodextrin (99%) as filler and stabilizer. The maltodextrin can not easily be removed from the preparation without loosing the enzymatic activity. Only recently, a genetically optimized variant of the transglutaminase was screened, which shows one single amino acid exchange and a remarkably increased thermostability compared to the wild-type (commercial) enzyme.25 At present, the production of this biocatalyst



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Figure 6. Investigations on specimen preparation from sodium caseinate films. (A) Mechanical properties (6 g sodium caseinate, 3 g glycerol, and 1.5 g maltodextrin in 110.4 mL of 20 mM Tris-HCl buffer, pH 7.0, dried at RT for 48 h; cross-head speed was 50 mm/s, sample width was 15 mm, and initial grip separation was 100 mm). Data columns represent the mean value of five replicates and standard deviations are shown as error bars. (B) Engineering drawing of double-bladed roller knife. (C-E) Light microscope images (magnification 33.5×) of test specimens prepared by (C) cutter knife; (D) scissors; and (E) double-bladed roller knife.

is optimized to provide sufficient amounts for the preparation of cross-linked protein films without maltodextrin.

4. Conclusions

Figure 7. Mechanical properties of sodium caseinate films dependent on MTG reaction conditions. Sodium caseinate (6 g) and glycerol (3 g) in 110.4 mL of 20 mM Tris-HCl buffer, pH 7.0, addition of 1.5 g TG ActivaWM in 6 mL of 20 mM Tris-HCl buffer, pH 7.0, times and temperatures of MTG incubation and film drying are given in the graph; cross-head speed was 50 mm/s, sample width was 15 mm, and initial grip separation was 100 mm. Data columns represent the mean value of five replicates and standard deviations are shown as error bars.

In the present study different parameters influencing the properties of sodium caseinate films cross-linked by microbial transglutaminase were (re)investigated. Unwanted crystallization of sodium phosphate originating from a previously described buffer was detected. However, this effect could be used if opaque films are requested. For the production of transparent and smooth films, the buffer had to be changed. As a plasticizer for sodium caseinate films, glycerol is commonly used. It was shown for the first time that glycerol does not negatively affect the activity of microbial transglutaminase. By producing 200 × 200 mm sodium caseinate films, it was possible to measure mechanical properties according to DIN EN ISO 527-3 with an initial grip separation of 100 mm and a sample width of 15 mm. Both parameters influenced the mechanical properties slightly and a comparison with other methods used in the literature should be carefully considered. The enzyme reaction conditions like time and temperature during enzyme treatment and film drying, respectively, had a remarkable influence on the mechanical properties of the films. Drying at 50 °C for 24 h resulted in brittle, strong films with a tensile strength of 4 N/mm2

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N/mm2 and elongation at break of 184% (increase of 51 and 63%, respectively, in comparison to non-cross-linked films). Standardized methods as DIN EN ISO 527-3 should be used for specimen preparation and characterization to produce comparable results. In Figure 9, the mechanical properties of enzymatically crosslinked caseinate films prepared in the present study are compared with commercial polymer films. Values for petroleum-based films (LDPE, HDPE, PP) and films based on other renewable resources (Mater-Bi (starch based) and Ecoflex (biologically degradable copolyester) are taken from the literature.26,27 The tensile strength of cross-linked caseinate films was found to be 2.5-8 times lower than the one of commercial films. However, the elongation at break of the protein films was comparable with HDPE and PP. The differences can be explained by the manufacturing process. Whereas the commercial films were produced by thermoplastic processing, the caseinate films were produced by casting. At present, experiments on the extruderbased thermoplastic manufacturing of protein films with enzymatic cross-linking are carried out.

Figure 8. (A) SDS-PAGE showing the cross-linking of sodium caseinate by MTG within the film-forming solution at 50 °C depending on incubation time. Samples of 100 µL were taken from the filmforming solution, diluted 1:100 with SDS-sample buffer and heated at 99 °C for 5 min. A total of 10 µL of the sample and 5 µL of the molecular weight marker were added to the SDS-PAGE gel. (B) Relative activity of MTG depending on incubation time in a sodium caseinate film solution at 50 °C. The MTG activity of the solution was determined immediately after sampling by using the standard hydroxamate assay.

Figure 9. Mechanical properties of cross-linked sodium caseinate films in comparison to commercial films based on oil and other renewable resources. Values for oil-based films26 and for Mater-Bi and Ecoflex,27 Cas (Na-caseinate), LDPE (low density polyethylene), HDPE (high density polyethylene), PP (polypropylene), Ma-Bi (MaterBi), Eco (Ecoflex), and MTG (microbial transglutaminase).

and elongation at break of 124%. By using a defined enzyme reaction (15 min at 50 °C, inactivation at 90 °C for 10 min), sodium caseinate was only partly cross-linked and just a slight increase in elongation at break was investigated in comparison to non-cross-linked films. In contrast, films produced at room temperature for 48 h showed the highest tensile strength of 4

Acknowledgment. This research was supported by the “Fachagentur fu¨r Nachwachsende Rohstoffe” (FNR, BMELV, Germany, Project Code 22013306). The authors are very thankful to Mr. Frank Ullmann for the manufacturing of the PTFE molds and the double bladed-roller knife and his patience during optimization of the respective design. The authors wish also to acknowledge help by Dr. G. Hause with images of the cut protein films by light microscopy.

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Parameter Optimization for Protein Film Production (14) Di Pierro, P.; Chico, B.; Villalonga, R.; Mariniello, L.; Damiao, A. E.; Masi, P.; Porta, R. Chitosan-whey protein edible films produced in the absence or presence of transglutaminase: analysis of their mechanical and barrier properties. Biomacromolecules 2006, 7 (3), 744– 749. (15) Motoki, M.; Aso, H.; Seguro, K.; Nio, N. Rs1-Casein film prepared using transglutaminase. Agric. Biol. Chem. 1987, 51 (4), 993–996. (16) Schou, M.; Longares, A.; Montesinos-Herrero, C.; Monahan, F. J.; O’Riordan, D.; O’Sullivan, M. Properties of edible sodium caseinate films and their application as food wrapping. LWTsFood Sci. Technol. 2005, 38 (6), 605–610. (17) Lu, S.; Zhou, N.; Tian, Y.; Li, H.; Chen, J. Purification and properties of transglutaminase from StreptoVerticillium mobaraense. J. Food Biochem. 2003, 27 (2), 109–125. (18) Folk, J. E.; Cole, P. W. Mechanism of action of guinea pig liver transglutaminase. I. Purification and properties of the enzyme: identification of a functional cysteine essential for activity. J. Biol. Chem. 1966, 241 (23), 5518–5525. (19) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227 (259), 680–685. (20) Bradbury, S. L.; Jakoby, W. B. Glycerol as an enzyme-stabilizing agent: Effects on aldehyde dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 1972, 69 (9), 2373–2376.


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(21) DIN EN ISO 291, Kunststoffe - Normalklimate fu¨r Konditionierung und Pru¨fung. 1997. (22) DIN EN ISO 527-3, Kunststoffe - Bestimmung der Zugeigenschaften Pru¨fbedingungen fu¨r Folien und Tafeln. 1995. (23) Jiang, Y.; Tang, C.-H.; Wen, Q.-B.; Li, L.; Yang, X.-Q. Effect of processing parameters on the properties of transglutaminase-treated soy protein isolate films. InnoVatiVe Food Sci. Emerging Technol. 2007, 8, 218–225. (24) Bruno, M.; Giancone, T.; Torrieri, E.; Masi, P.; Moresi, M. Engineering properties of edible transglutaminase cross-linked caseinate-based films. Food Bioprocess Technol. 2008, 1, 393–404. (25) Marx, C. K.; Hertel, T. C.; Pietzsch, M. Random mutagenesis of a recombinant microbial transglutaminase for the generation of thermostable and heat sensitive variants. J. Biotechnol. 2008, 136, 156– 162. (26) Crawford, R. J. Plastics Engineering, 3rd ed.; Butterworth-Heinemann: Oxford, 1998; p 22. (27) Otto, S.; Borg, H.; Schnabel, R.; Anton, W.; Jank, M. Biologisch abbaubare Abfallbeutel zur Bioabfallsammlung. Mu¨ll Abfall 2000, 11, 660–666.

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Parameter Optimization of Protein Film Production Using Microbial

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