Whey Protein Based Edible Food Packaging Films and Coating

Research Signpost 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India

Advances in Agricultural and Food Biotechnology, 2006: 237-261 ISBN: 81-7736-269-0 Editors: Ramón Gerardo Guevara-González and Irineo Torres-Pacheco

11 Whey protein based edible food packaging films and coatings

Regalado, C1., Pérez-Pérez, C2., Lara-Cortés, E1

and García-Almendarez, B1 1DIPA, PROPAC, Facultad de Química, Universidad Autónoma de Querétaro 76000. Qro. Mexico; 2Depto. Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Av. Tecnológico y García Cubas S/N, Celaya, 38010, Gto. Mexico

Abstract Packaging systems are intended to protect the foodfrom its surroundings acting as physical/mechanical, chemical and microbiological barrier to maintain quality, safety, and to prolong the packaged food shelf-life. Food quality and its average shelf-life are decreased when the foodstuff interacts with its environment gaining or losing moisture and aroma, or taking oxygen leading to oxidative rancidity. Additionally, microbial contamination may produce food spoilage, or even food poisoning. In multi-component foods the quality and shelf life are reduced

Correspondence/Reprint request: Dr. Regalado, C, DIPA, PROPAC, Facultad de Química, Universidad Autónoma de Querétaro, 76000. Qro. Mexico. E-mail: [email protected]

Regalado, C. et al. 238

when moisture, aroma or lipids migrate from one food component to another. Food packaging also provides important information to the consumer (nutrition facts, ingredients, expiration date, etc.), and makes the food available for a long period of time.

Introduction A variety of techniques have been developed to maintain the quality and microbial safety of foods, being food packaging one of these methods. Fresh oranges and lemons were wax coated in China in the 12th and 13th centuries, to reduce water loss [46]. The first packaging materials based on cellulose were developed in 1856, and in 1907 phenol-formaldehyde (bakelite) resins were synthesized. This was the starting point of a series of developments and innovations giving birth to a great diversity of packaging materials which nowadays are employed [74]. Packaging systems are intended to protect the food from its surroundings acting as physical/mechanical, chemical and microbiological barrier to maintain quality, safety, and to prolong the packaged food shelf-life [37]. Food quality and its average shelf-life are decreased when the foodstuff interacts with its environment gaining or losing moisture and aroma, or taking oxygen leading to oxidative rancidity. Alternatively, microbial contamination may produce food spoilage, or even food poisoning. In multicomponent foods the quality and shelf life are reduced when moisture, aroma or lipids migrate from one food component to another. Food packaging also provides important information to the consumer (nutrition facts, ingredients, expiration date, etc.), and makes the food available for a long period of time [60]. Initially, food packaging contributed to easy handling of food products to manufacturers, distributors and consumers. However, this has changed due to a growing consumer demand for minimally processed and easy preparation foods, where natural food additives are favored over their synthetic counterparts [12, 84]. Petrochemical based plastics have been widely used because of their availability in large quantities at low cost and favorable mechanical and barrier properties to oxygen, and heat sealability [105]. Nowadays the use of synthetic packaging materials has considerably raised with a concomitant increase in environmental pollution, since they are recalcitrant. Plastic materials may be degraded by naturally occurring microorganisms in the environment, but the process may take about 150 years (low density polyethylene), while paper can be naturally biodegraded in about one year [93]. Inert and non-biodegradable plastic materials account for about 30% by weight of municipal solid wastes, but due to their low density they represent 2/3 of the wastes volume [52]. There has been a growing interest for edible films and coatings in recent years trying to reduce the amount of wastes, capable of protecting the food once the

Food biotechnology 239

primary packaging is open, and because of public concerns about environmental protection [20]. Edible coatings and films can be advantageously used on meat and meat products with the following benefits [39]. Moisture loss reduction during storage of fresh or frozen meats; retention of juices from fresh meat and poultry when packed in plastic trays; oxidation/reduction of lipids and myoglobin; reduction of spoilage and pathogen microorganisms on the surface of coated meats; and restriction of volatile flavor loss and foreign odor pick up. In this chapter it is intended to give an account on the new developments on whey protein edible films. Characteristics of whey proteins, formation of edible films using whey proteins, and the mechanical and physicochemical properties of such films are disclosed. Finally, an account is given on the antimicrobial properties of edible antimicrobial whey protein based films as well as their possible applications in the food industry. Whey proteins Whey is the yellow-green liquid that separates from the curd during manufacture of cheese and casein [96]. In the recent past commercial cheese manufacturers treated whey as sewage or returned it to dairy farms for pig feeding or field spreading. However, since whey generates a biochemical oxygen demand as high as 38,000 mg/L[53], environmental concerns and regulations have become and important issue. Financial costs of disposal have made it profitable to further process whey, especially its protein and lactose for use as food ingredients. Whey protein has been used in confectionery, bakery and ice cream products, infant formula, health foods, and sports bars. Recent investigations aimed to find new uses of whey protein, have made use of the ability of processed whey protein (80 to 90% by weight) to form films and coatings on the surface of food products [7, 108]. Riedel [89], estimated a worldwide annual production of liquid whey of 118 million tons, which is equivalent to about 7 million tons of whey solids. However, in 1995 about 62% of total whey production was used in some application, but the remaining 38% of whey proteins (270,000 tons) were still available as valuable food ingredients. In the USA about 51.2% of the liquid whey produced was used by the food industry [1]. The whey protein is of high quality, since it has all essential amino acids, and a biological value higher than egg or casein proteins [69]. Whey protein has also some functional properties of interest to the food industry, such as solubility, emulsification, foaming, gelation, and viscosity development [2, 110]. Whey proteins represent about 20% of milk proteins, and four proteins represent more than 80% of total protein: β-lactoglobulin (β-LG), α-lactalbumin (α-LA), bovine serum albumin (BSA) and immunoglobulins (Ig) (Table 1).


β-Lactoglobulin β-LG represents about 50% of whey protein and act as carrier of retinol from the cow to the young calf. Under physiological conditions is predominantly dimeric, although it undergoes reversible conformational changes known as the Tanford transition at pH 7 [104]. β-LG can dissociate into monomers at pH<3.5 or at elevated temperatures and higher pH. Unfolding of the protein occurs at 72°C (tertiary level) while dimer-monomer dissociation starts at 52°C. The secondary structure showed 54% β-strand, 15% α-helix, 12% β-turns and 19% nonstructured at 15°C [54]. It has been reported eleven genetic variants, with isoforms A and B more prevalent in bovine milk. They differ by two amino acid substitutions: Asp64 and Val118 in isoform A are replaced by Gly and Ala in isoform B, respectively [36]. Native β-LG possesses two disulfide bonds and a solvent inaccessible free sulfhydryl group which upon conformational changes leads to sulfhydryl-disulfide interchange reactions, affecting characteristics such as solubility [101]. The reactivity of the free thiol group increases a pH>6.5 [45]. α-Lactalbumin α-LA is a 123 amino acid globular protein which represents about 20% of total whey protein, and has four intrachain disulfide bridges which confer hydrophobicity and foaming properties to the molecule. It has three genetic variants, the A variant contains a Glu10, while the B variant has Arg substitution at that position. Variant C is not well characterized and has Asn for Asp or a Gln for Glu substitution [36]. The two lobes of the three-dimensional structure can be divided into a acidic lobe rich in β-sheet structure, that contains the Ca2+ domain and a basic lobe rich in α- helical structure [29]. The main function of α LA is to participate in lactose biosynthesis as the regulatory component of the lactose synthase complex [34]. It has the ability to interact with lipid membranes and fatty acids [9], and has 72% sequence identity to human α-LA, which makes it an ideal protein for human infants nutrition [49]. α-LA requires Ca2+ for proper folding and disulfide bond formation, and at pH>5 it can bind to metal ions [29] such as Mn2+, Mg2+. At pH≤4 unfolds and can be digested by pepsin. α-LA and β-LG interact when heating at 75°C, pH 7, forming soluble aggregates whose composition changes with heating time and relative proportions of these proteins [31]. Co-aggregation of α-LA and β-LG may be attributed to the facilitation of intermolecular disulfide bond formation by hydrophobic interactions [50]. When held at temperatures ≥85 °C, α-LA evolves free thiol groups that form intermolecular disulfide bonded aggregates[31, 50]. At 85 °C the surface exposed, highly reactive C6-C120 disulfide bond, drives initiation of


thiol/disulfide interchange, and, subsequently, a neighboring thiol (C111) is the most reactive in forming intermolecular disulfide bonds [64]. The high reactivity on the C-terminus chain may be a result of its known flexibility and the enhanced reactivity of Cys thiols in the proximity of positive charge density [15]. The aggregation of α-LA was enhanced by the presence of calcium, a high degree of purity, excess ionic screening, and heating at 95 °C or above [70].

Table 1. Composition of major proteins in bovine whey, their relative molecular weight (Mr), isoelectric point (pI) and biological functions (adapted from 11, 34, 36, 47, 48).

Whey

Protein

Concentration

(g/L)

Mr

kDa

pI Tda

(°C)

Biological

function

Total 7 -- -- -- -----

β-LG 3 18.6 5.3 71.9 Pro-vitamin A

transfer

α-LA 0.7 14.2 4.8 35

64.3b

Lactose synthesis

Ig 1 150-900 5.5-6.8 Passive immunity

BSA 0.4 66.4 4.7-4.9 72-74 Fatty acid transfer

LFc 0.02-0.35 78.5 9.5 -- Bacteriostatic agent,

iron transport

LPODd 0.01-0.03 77 9.8 -- Antibacterial agent

Lysozyme <0.001 14 10.7 -- Antibacterial agent

Enzymes

(>50)

0.03 -- -- -- Health indicators

Proteose-

peptones

≥1 < 12 3.3-3.7 -- Opioid activity

GMPe 0-1.5 8-32 -- -- Regulation of cell

growth and

differentiation

aTd = Thermal denaturation bTd for Ca bound α-LA cLF= lactoferrin dLPOD = lactoperoxidase eGMP = glycomacropeptide


Bovine serum albumin BSA binds insoluble free fatty acids for transportation in the blood and is probable and important source for the production of glutathione in the liver [34]. BSA comprises 583 amino acids arranged in three homologous domains, 6 subdomains, and nine loops, containing a total of 28 helices which are extensively cross linked by 17 disulfide bonds. Cys34 is free and unbound [35]. Concentrated whey protein Whey protein concentrate (WPC) is produced by an industrial fractionation process involving ultrafiltration and diafiltration of pasteurized liquid whey. This is followed by vacuum concentration and spray drying, with a protein concentration ranging from 35% to 80% (e.g. WPC35, WPC80), dry basis [113]. Whey protein isolate (WPI) contains at least 90% (dry basis) protein and can be obtained by ion exchange or by membrane separation processing followed by concentration and drying. Depending on the type and sequence of membrane processing WPI does not preserve the proportions of whey proteins, as it is the case for WPC. The WPI is generally richer in β-LG and α-LA, but proportionally poorer in Ig, lactoferrin (LF), lactoperoxidase (LPOD) and glycomacropeptide (GMP) [30]. The production of 1 kg of WPC80 (80% protein, dry basis) needs 8 kg of whey solids, while the production of WPC90 requires about 9-10 kg of whey solids on the average [90]. Edible films and coatings An edible film is generally defined as a thin layer of edible material formed on a food as a coating or placed on or between food components. This film can be applied over or between foods by immersion, spraying, or panning [112]. When food-grade proteins and other food-grade additives (e.g. plasticizers, surfactants, acid or base, salts, and enzymes) are used and only protein changes due to heating, pH modification, salt addition, enzymatic modification, and water removal occur, the resulting film or coating is edible [60]. This type of materials unlike synthetic polymers, come from natural sources and are biodegradable [43]. A biodegradable material must be degraded completely by microorganisms in a composting process to only natural compounds such as carbon dioxide, water, methane, and biomass [5]. The purpose of edible films is to provide mechanical integrity or handling characteristics of the food, and a selective barrier to oxygen, carbon dioxide, moisture, aroma and lipids [60]. Besides their barrier properties, edible films and coatings may control adhesion, cohesion and durability, and improve the appearance of coated foods [59]. These materials can also act as carriers of active ingredients, such as antioxidants, flavors, fortified nutrients, colorants, antimicrobial agents, or spices [20].


The use of biodegradable polymers in food packaging can reduce the use of non-renewable resources and reduce waste by biological recycling. Natural polymers or those derived from natural monomers (e.g. cellophane, polylactic acid) offer the greatest opportunities, since their biodegradability and environmental compatibility are assured [102]. Edible films and coatings, as well as biodegradable materials offer alternative packaging systems, which may replace some synthetic packaging materials or reduce the use of synthetic materials by partially replacing them. The demand for degradable plastics, including biodegradable, photodegradable, bio-/photodegradable and other degradables has been estimated to reach 1.45 million tons in North America, for the year 2000 [4]. Edible films and coatings can be produced from polysaccharides and their derivatives, from lipid compounds, from proteins of animal or vegetable origin, and also from composites consisting of a blend of the previous materials [55]. A great variety of proteins have been investigated to produce edible films and coatings, such as corn zein, cotton seed, egg white, wheat gluten, soybean, gelatin, fish myofibrillar protein, pea protein, chitosan, collagen, casein, and whey proteins (WPC and WPI) [26, 56, 68, 111].

Edible film and coating formation There are many methods to produce whey protein based edible films, such as heating and irradiation. A process of protein cross linking is necessary to obtain a flexible, easy to handle film. The resulting film properties are affected by the amino acid composition, distribution and polarity; conditions affecting formation of ionic cross linking between amino and carboxyl groups; presence of hydrogen bonding; intramolecular and intermolecular disulfide bonds [40]. However, the formation of whey protein-based films has mainly involved heat denaturation in aqueous solution at 75-100°C, which produces intermolecular disulfide bonds, which might be partly responsible for film structure [106]. Heat treatment promotes water insolubility, which may be beneficial to maintain film and food integrity [85]. Bityrosine bridges between protein chains can be produced by γ-irradiation. This process seems to modify the conformation of proteins, which become more ordered and stable and films can be obtained from the inclusion of these proteins to a matrix such as cellulose [106]. WPI films have also been formed by using enzymatic methods, such as transglutaminase (γ-glutamyltransferase, E.C. 2.3.2.13) as a cross-linking agent [67]. Compression molding of WPI films has also been conducted by using a Carver Press at 0.8-2.2 MPa, at 104-140°C, for up to 2 min. This is the first step toward a continuous extrusion process [100]. Whey protein based films and coatings are generally flavorless, tasteless and flexible materials, water based, and the films varies from transparent to translucent depending on formulation, purity of protein sources and


composition [22]. There are reports [57] were WPI films plasticized with sorbitol (S) or glycerol (G) were slightly sweet and adhesive. On the other hand, the textural impact of glycerol plasticized WPI films could be reduced if film thickness was reduced to about 23 µm, as tested on crackers and melted cheese [65]. Texture perception by consumers of WPI films may also be reduced by making them more soluble, so that they dissolve readily in the mouth during mastication [66].

Plasticizers Sometimes it is necessary to add substances to impart a plasticizing effect, which improve mechanical properties of the edible films. The plasticizers are low molecular weight high boiling point molecules that impart flexibility and extensibility of the edible polymeric film (whey proteins in this case) [42]. They facilitate the processing of the edible films by reduction of the intermolecular forces between adjacent polymer chains, resulting in a reduction of the polymer glass transition temperature [91]. The addition of plasticizer results in a decreased activation energy for diffusion of gases and vapors through the film [8], and can decrease elasticity and cohesion [33]. Commonly used plasticizers in film systems are monosaccharides (glucose), disaccharides (sucrose), oligosaccharides, polyols (S, G, mannitol, glycerol derivatives, polyethylene glycols), and certain lipids and derivatives (phospholipids, fatty acids, surfactants) [41, 99]. Lipids or waxes may interfere with polymer chain to chain interaction and/or provide flexible domains within the film leading to reduction of film strength and increase of film flexibility in whey proteins [94]. Water also acts as plasticizer for edible films and coatings, and hydrophilic plasticizers generally attract additional water. Plasticizers such as G and propylene glycol have shown a secondary role, since they enhanced significantly the formation of cross-links within milk proteins (e.g. caseinates, whey proteins) [13, 73]. Films require plasticizers at 10% to 60% on dry basis, depending on the rigidity of the polymer [41]. The influence of an additive, including plasticizers or functional carriers, on edible film properties will depend on its concentration, chemical structure, degree of dispersion and the extent of its interaction with the polymer [83]. One way to decrease the amount of plasticizer is to reduce the polymer molecular weight to reduce intermolecular forces along the polymer chains, increasing polymer chain end groups and polymer free volume [91]. It has been shown [95] that hydrolyzed WPI has greater solubility and emulsifying activity, while allergenicity of WPI is reduced due to the hydrolysis process [81]. Solubility of 5% and 10% degree of hydrolysis (DH) hydrolyzed WPI edible films (30-75 % soluble protein) was found higher than that shown by unhydrolyzed WPI (about 3%) under equal plasticizer (G) concentration. Insoluble films may be used on high-moisture foods, while readily soluble films can find use in water soluble pouches [97].


Lipids such as butterfat, candelilla wax (CLW), carnauba wax (CW), and beeswax (BW), have been the most commonly used hydrophobic agents added to WPI films to decrease water vapor permeability. BW is a mixture of wax esters (35-80%), wax acids, and hydrocarbons (14%) that exhibits viscoelastic behavior [94]. Wax esters contain mainly C26 and C28 carboxylic acids, and C30 and C32 straight-chain primary alcohols [107]. CW, an exudate from leaves of the Brazilian carnauba palm (Copernica cerifera), is composed mainly of wax esters (85%) containing C24 and C28 carboxylic acids. It also has saturated long-chain mono-functional alcohols (C30-C34; 10-15%), with the overall composition resulting in an elastic behavior [10]. CLW is derived from the leaves of a small shrub native to northern Mexico and the southwestern United States, Euphorbia cerifera and Euphorbia antisyphilitica. It consists of mainly hydrocarbons (50%, C29-C33), esters of higher molecular weight (20-29%), free acids (7-9%) and resins (12-14%, mainly triterpenoid esters) [23, 109]. Mechanical properties Mechanical properties are important for edible films and coatings since they provide an indication of durability of films and the ability of the coating to enhance mechanical integrity of foods [98]. Protein based films are viscoelastic materials that possess characteristics of solids and liquids. Interactions between proteins and small molecules including water, plasticizers, lipids and other dispersed additives also contribute to the mechanical behavior of the film [22]. Tensile and yield strength, elastic modulus, and elongation help relate the mechanical properties of edible films to their chemical structures [82]. Whey protein edible films possess excellent resistance to tension and puncture [58]. pH values above the pI of whey proteins did not show an effect on the mechanical properties of edible films prepared with heat denatured or native WPI (Table 2). Since films made from native WPI had lower elastic modulus, tensile strength, and elongation than heat denatured WPI films, they would be less stiff, weaker and less extensible than denatured WPI. This has been attributed to the covalent cross-linking reactions, such as disulfide/sulfhydryl interchange, thiol/thiol oxidation, and intermolecular disulfide bond formation, due to heat denaturation [80, 85]. The microstructure of WPI edible films was found to depend on WPI concentration, the plasticizer used, and the pH. At increased WPI concentration a highly aggregated structure with larger pores was formed, and these pores tended to be more and smaller when S was used as plasticizer instead of G. When the pH increased from 7 to 9, a denser protein structure was formed and the strain at break increased while the oxygen permeability decreased. On the other hand, S migration can affect the film properties making


Table 2. Effect of pH, plasticizer, and antimicrobial agents on mechanical properties of WPI filmsA.

Film type pH Elastic

modulus

(MPa)

Tensile

strength

(Mpa)

Elongation

at break

(%)

Reference

5% WPI, 2.1% G

6

7

8

195a

199a

170a

6.5a

6.9a

6.0a

4.3a

4.1a

5.4a

85

5% WPI (native),

2.1% G

4

5

6

7

8

102b

104b

88b

100b

105b

2.2a

2.9a

2.8a

3.1a

3.2a

3c

7b

9b

7b

10b

85

10% WSM, 23% S

W:SC 1:1

W:SC 1:0.6

W:SC 1:0.43

10

10

10

---

---

---

14.5±4.5a

7.9±1.4b

4.7±1.3b

64.5±26.8a

87.8±13.5b

128.0±18.8c

25

5% WPI, 2.1% GB 5.2 --- 6.5a 12.4a 18

5% WPI, 2.1% G,

1% PABAB

5.2 --- 19.5b 40.7b 18

5% WPI, 2% G,

0% SA

0.5% SA

0.75% SA

1% SA

1.5% SA

0.5% PABA

0.75% PABA

1% PABA

1.5% PABA

5.2

(adjust

ed with

lactic

acid)

---

---

---

---

---

---

---

---

---

5.88±1.38a

4.85±2.70b

4.87±0.54b

3.75±0.18c

3.05±0.45d

5.38±0.97a

5.23±2.84a

4.36±0.18a

5.31±0.14a

6.37±3.28a

20±14.6b

26.58±3.24c

67.78±6.40e

73.01±2.07e

18.32±5.58b

20.85±8.47b

34.73±6.27c

34.98±10.6d

16

10% WPI, 5% G

VE: 0 %

VE 0.1 %

VE 0.2 %

7.41a

7.17b

7.26b

---

4.55a

4.45ab

4.05b

12.14a

29.42b

52.40c

72


Table 2. continued

10% WPI

WPI:G 1:1

WPI:G 1.5:1

WPI:G 2:1

WPI:G 2.5:1

WPI:G 3:1

WPI:BW:G 1:1:1

WPI:BW:G1.5:1.5:1

WPI:BW:G 2:2:1

WPI:BW:G 2.5:2-5:1

WPI:BW:G 3:3:1

WPI:CW:G 1:1:1

WPI:CW:G 1.5:1.5:1

WPI:CW:G 2:2:1

WPI:CW:G 2.5:2.5:1

WPI:CW:G 3:3:1

---

41±4

188±26

273±38

450±57

---

39±12

142±46

178±63

390±27

430±59

124±29

338±40

---

---

---

2.9±0.4

6.0±0.4

8.4±1.1

11.2±0.6

---

1.2±0.1

2.8±0.2

3.6±0.5

6.3±1.1

6.1±0.2

3.1±0.1

4.8±1.0

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

---

103

10% WPI, WPI:G 1:1, LPOS:

LPO:GO:Glu:KSCN:HP

1:0.35:108.7:1.09:2.17

LPOS: 0 %

LPOS: 0.7 %

---

---

20.21±2.26

22.57±2.57

1.90±0.12

2.09±0.16

126.8±3.9

129.5±3.4

77


LPO:GO:Glu:KSCN:HP

1:0.35:108.7:1.09:2.17

LPOS: 0 %

LPOS: 0.6 %

LPOS: 1.2 %

LPOS: 3 %

LPOS: 5 %

---

25.8±2.9a

23.57±2.0a

23.21±2.5a

17.16±2.0b

14.47±2.3b

2.31±0.32a

2.09±0.10a

2.09±0.06a

1.09±0.04b

0.96±0.07c

140.3±21.6a

129.50±10a

136.60±12a

119.5±6.7a

91.99±16b

78

ANumbers with different letters in the same column, same authors, are significantly different (p<0.05), BTests conducted before any cooking or smoking treatment on the sausage casings. Abbreviations: BW= beeswax; CW=carnauba wax; G= glycerol, GO= glucose oxidase, Glu= glucose, HP= hydrogen peroxide, KSCN= potassium thiocyanate, LPO= lactoperoxidase, LPOS= lactoperoxidase system, PABA= p-aminobenzoic acids, SA= sorbic acid, SC= sodium caseinate, VE= α-tocopheryl acetate, W= whey powder, WPI= whey protein isolate, WSM= mixture of whey and sodium caseinate.


it stiffer and less stretchable with time [3]. S may crystallize on the surface of plasticized WPI films making them opaque with a slightly undesirable appearance. The first signs of S crystallization can take up to four months, depending on the amounts of plasticizer used. However, in our laboratory we have made WPI films plasticized with non-crystallizable S (7-12% by weight), which have been kept clear for about 8 months (unpublished data). The smaller size of the G molecule permits more influence on the film properties than the S molecule. Thus, higher amounts of S than G are needed to obtain similar tensile properties [71]. When WPC80 was plasticized with increasing concentrations of G, the resulting film showed increased water solubility, but decreased mechanical resistance, Young modulus and glass transition temperature. Enhancement of mechanical properties, water insolubility and increased glass transition temperature were achieved by incorporation of formaldehyde as cross linking agent [38]. Addition of α-tocopherol (VE) to WPI films greatly improved the elongation at break values, but an addition of 0.2% of VE significantly reduced the tensile strength compared to the control film with no VE addition [72] (Table 2). The tensile properties of WPI containing up to 0.06 g of lactoperoxidase system (LPOS)/g film (dry basis) were not significantly affected by the incorporation of LPOS. However, significant reductions in elastic modulus and tensile strength were observed at ≥0.15 g LPOS/g film [78] (Table 2). Water vapor permeability Moisture transfer is often the most important factor leading to changes in the food quality during distribution and storage. Critical levels of water activity (Aw) of food products must be maintained for optimal quality and acceptable safety [55]. Whey protein films generally provide poor moisture barrier. S has the ability to bind less water than glycerol and can provide a better barrier against water vapor at low and intermediate relative humidity (RH). However, G and S migrate during the lifetime of the WPI films, which could be desirable when the film is used to carry antimicrobial agents or antioxidants that will migrate into the packed food and prolong its shelf life [3]. When WPI concentration increases, there is also an increase in water vapor permeability (WVP) but a reduced oxygen permeability (OP) [3]. Films thickness may not be an important issue here, since thin films of WPI (20 mm) have shown similar water barrier properties than thicker films (80 µm) [65]. Edible WPI films may be produced from hydrolyzed and heat denatured (5% and 10% DH), non-hydrolyzed and heat denatured, and non-hydrolyzed native whey protein. These treatments have resulted in films with no


significant differences in WVP at similar plasticizer content. However, an increase in G content of the hydrolyzed WPI led to increased WVP, indicating that hydrolysis may be an alternative to minimize permeability of films while producing the required film flexibility [85, 97]. WPI films made by compression-molding incorporating glycerol as plasticizer were flexible, and partially soluble when temperature was kept at 104°C, but insoluble at 140°C. Glycerol content, molding pressure and temperature had little effect on water vapor permeability values [100]. Incorporation of lipids can improve the moisture barrier properties producing protein/lipid emulsion films with increased hydrophobicity [32]. The WVP rate increases with higher water sorption because water acts as plasticizer and increases the free volume of the polymer. Water sorption isotherms can assess the equilibrium moisture content of WPI edible films at a given relative humidity to estimate their barrier properties [56]. Equilibrium moisture content of WPI films was largely influenced by the amount of plasticizer [28]. Oxygen permeability Factors which affect permeability properties of edible films are: microstructure, plasticizer, density, orientation, cross-linking, and molecular weight of the polymer chains, nature of the permeant, etc. [74]. WPI films, like other protein made films have excellent oxygen, carbon dioxide, aroma, and lipid barrier properties, particularly at low RH [103]. Hydrolyzed WPI films showed OP values similar to WPI, but with more flexibility, at the same G content. Sothornvit and Krochta [98], concluded that hydrolyzed WPI films and coatings can be used to protect foods from oxygen while reducing plasticizer incorporation and allergenicity of the whey protein. OP of some WPI edible films is reported in Table 3, together with that of different synthetic polymers as a means of comparison. Incorporation of the lactoperoxidase system (LPOS; 40 mg/g film, dry basis) to a WPI film did not significantly affect OP, which was true even for additions of up to 59 mg LPOS/g of film [79]. However, OP was significantly reduced at concentrations ≥0.15 g/g film, dry basis [78]. Active packaging Active packaging has been defined as a system in which the product, the package and the environment interact in a positive way to extend shelf life or to achieve some characteristics that cannot be obtained otherwise [75]. Active packaging can be achieved by incorporation of actively functional ingredients into the packaging system. Some of these active ingredients can be antimicrobial agents (organic acids, bacteriocins, chelating agents, proteins like lysozyme,


plant and herb essential oil and extracts, lactoferrin, lactoperoxidase system and bacteriocins), antioxidants, nutraceuticals, flavors, pigments, moisture control agents, nutrients, etc. (Table 4). A more thorough description of the antimicrobial agents’ characteristics, spectra, and uses in other edible films is given elsewhere [19]. Antimicrobial Table 3. Oxygen permeability of different WPI based edible films. Numbers with different letters are significantly different (p<0.05).

Film Type Relative

HumidityA

Permeability

[(cm3 µm)/(m2 day kPa)]

Reference

Unhydrolyzed wheyA

WPI:G 3:1 -- 0.8:1

Hydrolyzed wheyA

5.5% DH WPI:G 3:1 – 1-8:1

10% DH WPI:G 3:1 – 1-8:1

50 %

50 %

50 %

41.3 – 333.1

42.2 – 111.9

98


LPO:GO:Glu:KSCN:HP

1:0.35:108.7:1.09:2.17

LPOS: 0 %

LPOS: 0.7% (40 mg/g film)

50 %

50 %

219±14.1

221.5±16.0

77


LPO:GO:Glu:KSCN:HP

1:0.35:108.7:1.09:2.17

LPOS: 0 %

LPOS: 0.6 %

LPOS: 1.2 %

LPOS: 3 %

LPOS: 5 %

50 %

50 %

50 %

50 %

50 %

270±35.49a

240±15.00a

231.7±10.00a

140.96±7.59b

103.76±4.63c

78

LDPE

HDPE

Polyester

EVOH (70% VOH)

EVOH (70% VOH)

50 %

50 %

50 %

0 %

95 %

1870

427

15.6

0.1

12

98

AAll tests done at 23°C Abbreviations: EVOH (70% VOH)= ethylene vinyl alcohol polymer (70% vinyl alcohol), HDPE= High- density polyethylene, LDPE= low-density polyethylene.


Table 4. Application of antimicrobial compounds incorporated in whey protein edible films and coatings.

Antimicrobial Agent Tested Microorganism Substrate/Food

Application

Reference

Lysozyme Brochothrix

thermosphacta

Culture media 44

Sorbic acid, p-amino

benzoic acid

Escherichia coli

O157:H7, Listeria

monocytogenes,

Salmonella typhimurium

Culture media 16

Nisin Listeria monocytogenes Phosphate buffer 58

p-amino benzoic acid Listeria monocytogenes Hot dogs 18

Nisin

(Film made with whey

protein concentrate-70)

Listeria monocytogenes,

Staphyclococcus aureus

Culture media 88

Lysozyme Listeria monocytogenes Culture media

Cold-smoked

salmon

76

Lactoperoxidase system Listeria monocytogenes Smoked salmon 77

Lactoferrin, lysozyme,

lactoperoxidase system

Salmonella enterica

Escherichia coli O157:H7

Culture media 78

Lactoferrin, lactoferrin

hydrolizate,

lactoperoxidase system

Penicillium commune Culture media 79

BLISa from Pediococcus

parvulus

Listeria innocua Culture media 87

aBLIS= Bacteriocin-like inhibitory substance packaging materials may be classified into two types: those containing antimicrobial agents that migrate to the surface of the packaging material and thus can contact the food, and those that are effective against food surface microbiological growth without migration of the active agent to the food. Here we will discuss only the antimicrobial agents incorporated in edible whey protein films. Several factors should be considered in developing antimicrobial films: spectrum of antagonic microorganisms, effect of the antimicrobial agents on the mechanical and physical properties of the films and coatings,


antimicrobial mechanism, migration into the food and toxicological issues, and effect on food product composition [14]. In most solid and semisolid foods microbial growth might start from the surface mainly due to post-processing steps and handling [86]. The use of antimicrobial agents on edible films can be efficient on the microbial growth rate reduction extending lag phase or inactivating the microorganisms. Thus, shelf life can be extended, while food quality and safety is maintained [92]. Antimicrobial agents incorporated into the films can limit or prevent microbial contamination by reducing the growth rate and maximum growth population and/or extending the lag-phase of the target microorganisms or by inactivating microorganisms by contact. Antimicrobial agents may be incorporated in the packaging materials initially and migrate into the food through diffusion and partitioning [44]. Molecular weight, ionic charge, and solubility of different antimicrobial agents, affect the diffusion rate into the polymers [27]. Food safety is an important issue worldwide. Many technologies have been developed to preserve foods based on changes on temperature, water activity reduction, pH control, irradiation, etc. However, all these methods can also be applied in combination with a variety of antimicrobial agents. There is not a single antimicrobial agent that can control all spoilage and pathogenic microorganisms. Instead a more general approach, the hurdle technology, has been applied using a combination of different antimicrobial agents. A variety of antimicrobial agents has been used for food preservation and may be added to the edible film or coating [76]. Active packaging films containing antimicrobial agents work by slow migration of the agent from the packaging material to the surface of the food; thus, acting against contaminant microorganisms [24, 86]. The charge density and cavity size of the three-dimensional protein network of the whey protein film can be adjusted by altering the pH, and the volume ratio of the whey protein and the plasticizer. Thus, the diffusion rate of the incorporated antimicrobial agent can be controlled. The design of protein-based antimicrobial films may be affected by many factors that should take into account the biochemical characteristics of the food (pH, Aw, composition), since they provide different environmental conditions for both, the contaminant microorganisms and the effectiveness of the antimicrobial agents [44]. Antimicrobial agents incorporated Plate counting tests to evaluate the effect of WPI films incorporated with lysozyme (LZ), lactoperoxidase systems (LPOS), lactoferrin (LF) and lactoferrin hydrolizate (LFH) against Gram-positive microorganism (L. monocytogens), and Gram-negative microorganisms (Salmonella enterica, and E. coli O157:H7) have been reported [76, 77, 78]. Antimicrobial tests have


also been conducted on fungi (Penicillium commune) by antimicrobial WPI edible films incorporating LF, LFH, and LPOS [79]. Only the WPI-LPOS films showed antimicrobial effect against the Gram negative microorganism. WPI-LF, WPI-LFH, WPI-LPOS were all effective against P. commune. WPI incorporated with LZ and LPOS were both effective to control L. monocytogenes in both culture media and smoked salmon. Furthermore these WPI films were also effective to control aerobic microorganisms such as yeasts and molds. The plate counting method has the advantage of providing a quantitative measure of the antimicrobial activity exerted by the WPI films. The same authors also evaluated the ability of these films to inhibit all microorganisms tested by using two methods: the disc-covering test (inoculum placed first), and disc surface-spreading test (inoculum placed after). The disc tests simulate wrapping of foods and the results may resemble what happens when films get in contact with contaminated food surfaces [6]. WPI films adjusted to low pH value (5.2), and incorporated with sorbic (SA), and p-amino benzoic (PABA) safe organic acids were effective to control L. monocytogenes, E. coli O157:H7, and S. thyphimurium in culture media. However, only WPI-PABA low pH film was effective toward L. monocytogenes on surface inoculated hot dogs [16, 18]. Food applications The oxygen-barrier properties of whey protein coatings have potential to increase the shelf life of foods such as roasted peanuts by reducing lipid oxidation rate. However, there is incomplete peanut surface coverage and cracking and flaking of the coating, which must last during storage and transportation. Lin and Krochta [63], have incorporated 0.15% (w/w) sorbitan laureate (Span 20) (three times its critical micelle concentration) into the coating forming solution, based on WPI, and increased peanut coverage to 95%. Oxidation-prone foods like snack peanuts used in candy bars tend to limit the shelf life of the confectionery. Coating of the nuts can improve the quality of the candy bar and extend its shelf life [62]. A good grease barrier can be obtained with paperboard coated with WPI and glycerol (G) as plasticizer, but migration of G into the paperboard produced cracking of the coating during extended testing [21]. A formulation incorporating WPC80 instead of WPI, plasticized with sucrose, was used to coat paperboard. The substantially more economical coating imparted excellent grease resistance, which was retained after ambient storage conditions and extended accelerated testing times (16 h). The plasticizer used here (sucrose) was assumed not to migrate into the paperboard [63]. These coatings may be used on packing products like fast food and pet food. Another application of WPI coatings may be to provide a lining of plastics such as polypropylene, to improve their oxygen-barrier properties [51].


Gloss coatings on chocolate-panned confectionery products is an application where an alternative glaze coating different from shellac (confectioners glaze) is being sought for, because of tight EPA restrictions on this substance. Whey protein-based (WPI) coatings plasticized with G, polyethylene glycol, propylene glycol, and sucrose has been developed for chocolate applications. WPI-sucrose coatings provided the highest and most stable gloss; it is expected that gloss coatings on other confectionery (panned and non-panned) products may be potentially viable [61]. Current research on antimicrobial packaging and coatings is directed toward the surface protection of cheese and meat products. This will reduce the amount of antimicrobial compounds used, since there will be no need to compensate for the amount that moves into the food product. The gradual release of an antimicrobial agent from a packaging film to the food surface may have an advantage over dipping and spraying. In these processes antimicrobial activity may be rapidly lost due to the inactivation of the antimicrobial agent by food components or dilution below active concentration [6]. WPI casings containing p-aminobenzoic acid (PABA) were more effective than WPI casings containing sorbic acid (SA) for inhibiting L. monocytogenes growth on hot dogs. However, when WPI-SA and WPI-PABA casings were used on Bologna and summer sausages, L. monocytogenes, E. coli, and Salmonella typhimurium were reduced by 3.4 to 4.1, 3.1 to 3.6, and 3.1 to 4.1 logs, respectively. The different pH values of these meat products may have influenced the effectiveness of the antimicrobial agents incorporated on the casings [17, 18]. Extended microbial stability of smoked salmon was achieved using WPI-lysozyme coating treatment by inhibition of L. monocytogenes and aerobic microorganisms including yeasts and molds [76]. WPI and lactoperoxidase films and coatings effectively reduced the number of this pathogen, and inhibited cell growth when it was inoculated before and after the application of films and coatings on smoked salmon. Aerobic microorganisms, and yeasts, and molds were also inhibited [77]. Antimicrobial food packaging using edible films should be considered as a part of the hurdle technology that in addition to other processes could reduce the risk of pathogen contamination and extend the shelf-life of perishable food products. References 1. ADPI. 2002. American Dairy Products Institute. Whey products utilization and

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