Chapter 7
Films and Coatings from Commodity Agroproteins 1
1
1
Nicholas Parris , Leland C. Dickey , Peggy M . Tomasula , David R. Coffin , and Peter J. Vergano 1
2
1
Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, P A 19038 Department of Packaging Science, 225 Poole Agricultural Center, Clemson University, Clemson, SC 29634-8370
2
Edible films and coatings from agricultural proteins have been investigated as an alternative to synthetic wrapping material for food protection and preservation. Milk protein films are hydrophilic, which limits their utility for long-term storage. Films prepared from casein precipitatedfromskim milk with CO were found to be more hydrophobic and exhibited better water vapor barrier properties than calcium caseinate films. Unplasticized corn zein films, cross-linked with 20% polymeric dialdehyde starch, exhibited the best water vapor barrier properties. Zein proteins isolated from dry milled corn were less denatured and could be recovered more economically than commercial zein preparations from corn gluten. Paper coated with zein isolate (80-85% protein and 15-20% oil) exhibited good grease resistance and water vapor barrier properties. 2
Agriculturally derived alternatives to the polyolefin packaging materials currently used by the food industry provide opportunities to strengthen the agricultural economy and reduce importation of petroleum and petroleum products. Interest in the preparation of biopolymer films and coatings from agricultural proteins has recently been renewed. Such films alone or in combination with polysaccharides, lipids etc. have the potential to control mass transfer of gases and thus extend food Mention of brand or firm name does not constitute an endorsement by the U.S. Department of Agriculture above others of a similar nature not mentioned. 118
© 2001 American Chemical Society
119 shelf life. Comprehensive review articles have been published on the use of edible films and coatings for food preservation (1,2,3,4)· Polymeric films are inexpensive to use when applied to food as wrapping. Films made from soluble agricultural proteins, although more expensive may be applied to food by dipping or spraying, thus eliminat ing packaging waste and manufacturing costs. Edible films, however, are sensitive to relative humidity and are less likely to replace synthetic packaging materials for prolonged storage of food (2). In addition, films prepared from water soluble proteins such as gelatin, casein, serum albumin, and egg albumin are poor water vapor barriers. Films and coatings of soy and grain (wheat and corn) proteins are better water vapor barriers. In this study we have reported isolation techniques and compositional factors affecting the physical properties of films and coatings made from milk and corn protein. The research was directed to the preparation of biodegradable biopolymer films and coatings from renewable resources which could potentially replace synthetic materials.
Milk Protein Films (Nonfat D r y M i l k ) N D M The principal proteins in cow's milk are caseins (~30g/L) which exhibit amphipathic character and assemble to form micelles, and whey proteins (~4.5g/L) which are globular and easily denatured. N D M powder prepared from skim milk has become increasingly important as a food ingredient because of its ease of handling, storage, and nutritional value. To meet some N D M product specifications on protein denaturation, skim milk is heated before spray-drying. For example, high pre-heat treatment (85 °C for 30 min) of N D M used as an additive in baking is essential to improve the extensibility and water absorption of dough (5). A whey-casein complex has been shown chromatographically to form in N D M preheated to 74 and 85 °C for 30 min and is responsible, in part, for the functional properties of the product (6). Maynes and Krotchta (7) found that formation of N D M films was impeded due to the high concentration of lactose (~50g/L) in the reconstituted milk which crystallized during film drying. To overcome this problem, lactose was enzymatically hydrolyzed, complexed with potassium sorbate or removed by ultrafiltration (UF). They obtained homogeneous films from N D M after the lactose had been hydrolyzed with β-D-galactoside galactohydrolase and then heated at 100°C for 30 min. The monomers, galactose and glucose, are more soluble and do not crystallize during film formation. Solutions of N D M containing 10% potassium sorbate and ultrafiltered milk with 25% glycerol formed films after similar heat treatment. The presence of potassium sorbate interferes with the crystalline environment of lactose and thus inhibits crystal growth. UF-total milk protein (TMP) films had the lowest permeability followed by potassium sorbate complexed-NDM and lastly, N D M with lactose hydrolyzed to monomers (Table I). The relative humidity (RH), at the inner surface of the film which was corrected for stagnant air effects, was between 65 and
120 Table I. Water Vapor Permeability (WVP) of N D M Edible Films Film"
Thickness (mm)
RH(%f
Permeability (g-mm/m -h-kPa) 2
N D M , hydrolyzed lactose
0.12
72
3.76
N D M : Κ Sorbate (9:1)
0.11
74
3.30
0.07
65
2.93
c
UF-TMP :Glycerol(4:l) fl
Heated at 100°C for 30 min. ^Relative humidity (RH) at the inner surface of the film. T M P = Total milk protein (ultra filtered rehydrated NDM). SOURCE: Adapted from ref (7).
74%. These values were considerably lower than those obtained for synthetic films (compare Table I and Table IV).
Casein Casein can be isolated from milk through the action of lactic acid bacteria during cheese making or by acidification of skim milk by an acid such as HC1 or H S 0 . A c i d precipitation of casein has also been carried out by dissolution of C 0 in skim milk (8). The use of C 0 as a precipitant is attractive because it eliminates the formation of ionic byproducts from mineral acids. Typically casein was precipitated by injecting C 0 into skim milk at -5000 kPa and 38 °C. The precipitate was washed with distilled water to remove whey proteins, lactose and unbound minerals. The calcium content of casein precipitated in this way was higher than that precipitated using mineral acids. Films from C0 -casein were found to be more hydrophobic and only 7% of the material dissolved in water compared to 90% of the calcium caseinate films (Table 2). Increased solubility was observed in both cases for films containing glycerol. The increased solubility for the films containing G L Y appears to be due to the presence of the plasticizer, because both film have the same protein content. The solubility of plasticized C0 -casein films is comparable to that of plasticized zein films made from corn zein, a prolamine, which is soluble in aqueous alcohol mixtures. Water vapor permeability (WVP) values for C0 -casein films are lower than those for the calcium caseinate films (Table 3). The lower R H for the calcium caseinate films, at comparable thickness to the C0 -casein films, is attributed to greater absorption of water by the protein resulting in swelling of the film. Both types of films showed an increase in W V P values with increasing film thickness which is characteristic of hydrophilic films due to swelling. Such differences in the properties of the two types of films are indicative of structural dissimilarities. 2
4
2
2
2
2
2
2
2
121 Table Π . Water Solubility of CQ.-Casein Films Protein
film
Material Dissolved in Water (g/lOOml)
Reference
7.1
Reference (20)
COj-casein-30% G L Y
16.8
Reference (20)
Calcium caseinate
90.0
Reference (20)
100.0
Reference (20)
14.2
Reference (14)
C0 -casein 2
Calcium-caseinate-30% G L Y Zein-30% G L Y
Table ΙΠ. WVP of Casein Edible Films Film"
Thickness (mm)
RH(9o)
Film Swelling Permeability* (g-mm/m -h-kPa) 2
C 0 casein 2
C a caseinate
0.112
85.8
No
2.22a
0.163
87.7
No
2.58b
0.184
87.9
No
3.21c
0.277
89.7
No
3.80d
0.171
86.5
Yes
3.18c
0.222
85.5
Yes
4.45e
"30% Glycerol. *Means in the same column with no letter in common are significant at Ρ < 0.05 using the
Bonferroni LSD multiple-comparison method. SOURCE: Adapted from ref (20).
Whey Protein Whey proteins can be isolated after precipitation of casein from skim-milk. Whey films have been prepared by heating an 8-12% solution of whey protein isolate (WPI) to temperatures from 75 to 100°C (9). The effects of heat treatment, protein concentration, and p H on plasticized films were examined. The best film formation occurred at neutral p H for a 10% solution heated at 90°C for 30 min. Heat was essential to form intermolecular covalent bonds and intact films (9). The pure whey protein films, however, were too brittle and required plasticizer, which significantly increased their W V P values (Table IV). A t comparable concentrations and relative humidity conditions, sorbitol plasticized whey protein films had lower W V P values than glycerol plasticized
122 films. Despite many attempts by researchers to make hydrophilic films more hydrophobic, the W V P values for synthetic films are still orders of magnitude lower. Comparisons of oxygen permeability, however, showed that hydrophilic milk protein films are less permeable than synthetic packaging materials (4). Covalent crosslinking of whey protein films with transglutaminase results in very strong films. One should be aware that the effectiveness of this crosslinked coating depends on the acidity and the proteolytic activity of the surface to be coated (10).
Table I V . W V P of Whey Protein Edible Films Film
Thickness (mm)
RH(%)
Permeability (g-mm/m -h-kPa) 2
Hydrophilic:
a
WPI, 50% glycerol
15
0.12
59
6.44
WPI, 37.5% glycerol
0.12
65
4.99
WPI, 50% sorbitol
0.14
75
3.53
WPI, 37.5% sorbitol
0.13
79
2.58
0.0078
99.8
0.0022
99JS
0.0016
Synthetic:
0
polyethylene
poly(vinylidene chloride) 0.0084 SOURCE: Adapted from ref (9)andref (21). (w/w)-
b
Cora Protein Films Zein Isolation and film-forming properties of corn zein was investigated in order to identify a more inexpensive and more hydrophobic film or coating. Classification of corn proteins into albumins, globulins, prolamines and glutelins traditionally has been based on solubility (11). Newer techniques of protein identification have shown that zein, a mixture of prolamine rich proteins, comprises approximately 50% of the protein in the corn kernel (12). The most interesting property of zein for use as an industrial protein is its ability to form a tough, glossy, and grease-proof coating. It can be cured with various aldehydes to form an essentially insoluble product. Zein has been commercially isolated from corn gluten meal (60% protein) at 60 °C using 88% isopropanol containing 0.25 wt% N a O H (75). Less than half of the protein in the meal, however, is recovered at low temperatures, after the supernate is decanted. The principal proteins in
123 commercial zein are the α-zeins which are a complex group of closely related prolamines with molecular weights of 19 and 22 kDa. Their molecular weights can readily be estimated by sodium dodecyl sulfate capillary electrophoresis (SDS-CE) by comparing their migration time to a calibration plot of molecular weight standard proteins. Zein proteins gel, or polymerize, upon storage in aqueous alcohol solutions at concentrations greater than 20% protein as shown in the capillary electropherogram of zein (Figure la). These proteins associate through disulfide linkages, since only the monomer plus a small amount of dimer was present after reduction with 2-mercaptoethanol (Figure lb).
Commercial Zein Films Zein films have been prepared by heating 10% solutions of zein and plasticizer at 60°C for 10 min. The solutions were then dried in a vacuum oven adjusted to 10 inches mercury, at approximately 50 °C (14). Cross-linked films were prepared by adding the cross-linking agent to the mixture which was then heated at 70°C for 30min in a sealed container and dried as above. Zein films containing no plasticizer had the lowest W V P values (Table 5). O f these, films cross-linked with 20% polymeric dialdehyde starch (PDS) exhibited the best water barrier properties. Films prepared in acetone had lower W V P values than those prepared in ethanol. Incorporation of plasticizer into zein films significantly reduced water vapor barrier properties resulting in an almost doubling in W V P values (Table 5). In addition, W V P values, and the corresponding variation between zein films with the same plasticizer composition, decreased with increasing poly(propylene glycol) (PPG) concentrations (Figure 2). This variation could be attributed to preferential separation of G L Y over P P G from the film. Films containing a G L Y : P P G ratio of 1:3 exhibited elongation values almost fifty times greater than G L Y plasticized film. Incorporation of cross-linking agents into zein films resulted in approximately a 2-3-fold increase in tensile strength values (14).
Table V. Water Vapor Permeability of Zein Films Film Type
Casting Solvent
Plasticizer
Permeability (g-mm/ m -h- kPa) 2
zein
ethanol
none
0.620c
zein
acetone
none
0.577c
zein
ethanol
zein/PDS (20%)
ethanol
none
zein/PDS (20%)
ethanol
G L Y : P P G (30%)
The ratio of mixed plasticizers was 1:3. SOURCE: Adapted from ref(i^).
GLY:PPG(30%)
a
a
1.060ab 0.533c 1.150a
124
.050 .039 .028 .017 .006 -.005 0.0
2.1
.050
4.2 6.3 Time (min)
8.4
21kDa
.039 ^
24kDa
.028 .017 H 45kDa
.006 — \
L
-.005 00
2.1
4.2 6.3 Time (min)
8.4
Figure 1. Sodium dodecyl sulfate capillary electrophoresis of zein: unreduced (a) and reduced (b).
125
1.6 +
0.6 -I GLY
1
3:1
1
1:1
i
1:3
1 PPG
GLY:PPG Figure 2. Effect ofPPG concentration on zeinfilmwater vapor permeability (WVP). SOURCE: Adapted from ref(14)
126 Zein Isolate New markets for zein are severely limited because of its cost. Presently commercial zein sells for about $ 10/lb which is too much to compete with markets presently held by other raw materials such as edible shellac at $2-3/lb. Dickey et al., (15) estimated the cost of ethanol extraction of zein/oil mixtures from ground dent corn to be significantly cheaper than commercial purified zein. Dent maize, milled to a median size of 2mm was extracted with 70% ethanol as described in Figure 3. The extract was separated from the corn by centrifugation and diluted to 40% ethanol by vacuum evaporation. The precipitated product was scraped from the bottom and walls of the evaporation vessel and dried in a lyophilizer. The typical analysis for the dried product was 80-85% protein; 15-20% oil;
