Physical Properties of Gelidium corneum–Gelatin Blend Films Containing Grapefruit Seed Extract or Green Tea Extract and Its Application in the Packaging of Pork Loins
ABSTRACT: Edible Gelidium corneum–gelatin (GCG) blend films containing grapefruit seed extract (GFSE) or green tea extract (GTE) were manufactured, and the quality of pork loins packed with the film during storage was determined. Tensile strength (TS) and water vapor permeability (WVP) of the films containing GFSE or GTE were better than those of the control. The film's antimicrobial activity against Escherichia coli O157:H7 and Listeria monocytogenes increased with increasing antimicrobial concentration, resulting in a decrease in the populations of bacteria by 0.77 to 2.08 and 0.91 to 3.30 log CFU/g, respectively. Pork loin samples were inoculated with E. coli O157:H7 and L. monocytogenes. The samples packed with the GCG film containing GFSE (0.08%) or GTE (2.80%) had a decrease in the populations of E. coli O157:H7 and L. monocytogenes of 0.69 to 1.11 and 1.05 to 1.14 log CFU/g, respectively, compared to the control after 4 d of storage. The results showed that the quality of pork loins during storage could be improved by packaging them with the GCG film containing GFSE or GTE.
Biodegradable films have been studied to address environmental concerns and suitability for use with natural antimicrobials, and edible films provide barriers to moisture migration, oxygen transfer, lipid oxidation, and volatile flavors (Ko and others 2001; Ku and Song 2007; Rojas-Graü and others 2007). Typically, antimicrobial edible film and coating have been used for inhibition of microbial growth and extending the shelf life of food products (Min and others 2007).
Antimicrobial agents used in the edible films can be weak organic acids, antimicrobial enzymes, bacteriocins, triclosan, grapefruit seed extract (GFSE), green tea extract (GTE), EDTA, essential oils, fungicides, BHT, α-tocopherol, and chitosan (Cha and Chinnan 2004; Kim and others 2006; Jongjareonrak and others 2008). Among the antimicrobials, GFSE and GTE contain polyphenolic compounds with antioxidant activity and bactericidal activity against a wide range of Gram-positive and Gram-negative bacteria (Heggers and others 2002; Reagor and others 2002; Si and others 2006; Xu and others 2007).
Gelidium corneum (GC) is a type of red algae containing agarose. It has been used for processed food, culture media, and pharmaceutical products, because the GC extract contains a high amount of dietary fiber with calcium and iron (Kim and others 1997; Ku and others 2007). In addition, preparation of the GC film and its application in sausage has been reported in a previous study (Ku and others 2008). However, the GC film needs improvement in physical property due to weak tensile strength and poor elongation. Therefore, the GC-gelatin blend film was considered to improve the physical property of the film, because gelatin has excellent film-forming ability and good physical property (Hulda and Carlos 2006; Cao and others 2007).
Pork consumption keeps on increasing, and it suggests the necessity of ensuring microbial safety in pork meat. Pathogenic bacteria such as E. coli and Listeria cause spoilage in pork, and its consumption results in a disease to humans (Lee and others 2004; Rojas and Brewer 2007). In particular, L. monocytogenes causes a food safety problem, since it can grow even at refrigeration temperatures (Trivedi and others 2008). Therefore, the objective of this study was to investigate the feasibility of using a Gelidium corneum–gelatin blend film containing GFSE and GTE to improve microbial safety, maintain the quality, and extend shelf life of pork loins.
Materials and Methods
The Gelidium corneum used in this study was harvested from Jeju Island, Korea. The gelatin and glycerol used in this study were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Mo., U.S.A.). The grapefruit seed extract was obtained from ABC Techno Inc. (Desfan-100, Tokyo, Japan), and the green tea extract was purchased from Amorepacific Co. (Jincheon, Korea).
Preparation of film-forming solution
The Gelidium corneum was washed to remove foreign substances and bleached using 5% chlorine dioxide at 60 °C for 90 min, followed by treatment with 8.5 g/h ozone gas (Ku and others 2007). The bleached and dried samples were cut, ground, and prepared using a 200-mesh sieve. To make the GCG film, 0.75%Gelidium corneum powder and 5% gelatin were dissolved in distilled water and mixed with 2.5% glycerol. The film-forming solutions were then conditioned in a water bath at 90 °C for 30 min, and then cooled at room temperature. Subsequently, varying amounts (0%, 0.02%, 0.04%, 0.08%, 0.10%) of GFSE and GTE (0%, 0.5%, 1.4%, 2.8%, 4.2%), which were based on their antimicrobial activity, were incorporated into each film-forming solution (100 mL).
Film casting and drying
The film-forming solutions were strained through cheesecloth and cast on flat, teflon-coated glass plates (24 × 30 cm). The film forming solution was uniformly spread on the plates by controlling the speed of pouring while leveling the glass plates up. A uniform film thickness was maintained by casting the same amount (80 mL) of film-forming solution on each plate for the GCG film containing GFSE. On the other hand, the GCG film containing GTE had different amounts (80, 75, 65, 60, 55 mL) of film-forming solution for uniform thickness, depending on the concentration of GTE (0%, 0.5%, 1.4%, 2.8%, 4.2%). The plates were then dried at 25 °C for 24 h. The dried films were peeled intact from the casting surface. Specimens were cut to measure water vapor permeability (2 × 2 cm) and tensile strength (2.54 × 10 cm).
Determination of film thickness
The film specimens were conditioned in an environmental chamber at 25 °C and at 50% relative humidity (RH) for 2 d. The film thickness was measured using a micrometer (Mitutoyo, Model Nr 2046-08, Tokyo, Japan) at 5 random positions, and the mean value was determined.
Measurement of tensile strength and elongation
The film's tensile strength (TS) and elongation at break (E) were determined using the Instron Universal Testing Machine (Model 4484, Instron Co., Canton, Mass., U.S.A.) according to the ASTM Standard Method D882-91 (ASTM 1993). The film specimens were conditioned in an environmental chamber at 25 °C and 50% RH for 2 d. An initial grip distance of 5 cm and crosshead speed of 50 cm/min were used. The TS was calculated by dividing the maximum load by the initial cross-sectional area of a specimen, and the elongation was expressed as a percentage of the change in the initial gauge length of a specimen at the point of sample failure. Five replicates of each film were tested.
Measurement of water vapor permeability
The water vapor permeability (WVP) of the edible film was determined according to the modified ASTM E 96-95 method (ASTM 1983) at 25 °C and 50% RH using a polymethylacrylate cup. The cup was filled to 1 cm with distilled water and covered with a film specimen that had been previously conditioned in an environmental chamber at 25 °C and 50% RH for 2 d. The weight loss of the cups with time was measured. A linear regression analysis was performed to calculate the slope. The WVP (ng m/m2s Pa) values were then calculated using the following formula:
where the water vapor transmission rate (WVTR, g/m2s) was calculated by dividing the slope by the open area of the cup. L is the mean thickness (m), and △p is the corrected partial vapor pressure difference (Pa) across the film specimen. The corrected partial vapor pressure was determined by maintaining in an environmental chamber at 25 °C and 50% RH.
E. coli O157:H7 (NCTC12079) and L. monocytogenes (KCTC 3710) cultures were grown at 37 °C for 24 h in 50-mL Corning tubes containing 25 mL of Luria-Bertani broth (LB, Difco Co., Detroit, Mich., U.S.A.) and Listeria enrichment broth (Oxoid, Basingstoke, U.K.), respectively.
Antimicrobial activity of the films against pathogenic bacteria
The antimicrobial activity of the GCG film containing GFSE or GTE was determined using the method of Ku and others (2008). E. coli O157:H7 and L. monocytogenes were incubated at 37 °C in LB broth and Listeria enrichment broth, respectively, until 107 to 109 CFU/mL. Fifteen microliters of each bacterial suspension were placed on each film disc (0.02 g). The film discs were then incubated at room temperature for 60 min. After the incubation, each disc was placed in 0.98 mL of 0.1% peptone water and homogenized for 3 min, and the solution was then diluted with 0.1% peptone water. E. coli O157:H7 counts were determined by plating appropriately diluted samples onto Chromogenic E. coli coliform medium (EC, Oxoid), and plates were incubated at 37 °C for 24 h. For L. monocytogenes, the diluted samples were plated onto Listeria selective agar (Oxoid), and plates were incubated at 37 °C for 48 h. Each microbial count was the mean of 3 determinations, and the microbial count was expressed as a log colony forming unit (CFU)/g.
Inoculation of pork loins and packaging
The pork loins were cut into 10-g slices. E. coli O157:H7 and L. monocytogenes were incubated at 37 °C in LB broth and Listeria enrichment broth, respectively, until 106 CFU/mL. E. coli O157:H7 and L. monocytogenes cultures (0.5 mL) were then spread on the pork loin surfaces, respectively, with a sterile glass rod and allowed to drain for 10 min. The initial inoculation levels of the E. coli O157:H7 and L. monocytogenes in the pork loin samples were 5.16 and 4.80 log CFU/g, respectively. The inoculated pork loins were packed in direct contact by GCG film containing GFSE (0.08%) or GTE (2.80%). Pork loins packed in direct contact by GCG film without GFSE or GTE were used as controls. All pork loin samples were stored at 4 °C for 10 d using a sterile polystyrene tray according to the film type.
Antimicrobial activity of coating against pathogenic bacteria
Five grams of each pork loin sample were taken and placed in 45 mL of 0.1% peptone water. The sample was then homogenized for 3 min in a sterile stomacher bag using a Stomacher (MIX 2, AES Laboratoire, Combourg, France), filtered through sterile cheesecloth, and diluted with peptone water to determine its microbial count. To determine the microbial count, serial dilutions were performed and plated on selective agar plates to count bacteria, as described in the previous method section.
Measurement of lipid oxidation
The degree of lipid oxidation of the pork loins was determined using the method of Ahn and others (1998). Five grams of each sample were homogenized in 15 mL of distilled water using a blender for 1 min. A 1-mL sample solution was transferred into a disposable test tube, and a 2-mL 2-thiobarbituric acid/trichloroacetic acid (TBA/TCA) solution was added. The mixture was then vortexed and boiled in a water bath for 15 min. The sample was cooled at room temperature for 10 min and centrifuged for 15 min at 2000 ×g. The absorbance of the resulting supernatant solution was measured at 531 nm, and the thiobarbituric acid-reactvie substance (TBARS) value was determined and expressed as mg malondialdehyde per kg sample (mg MDA/kg).
Analysis of variance (ANOVA) and Duncan's multiple range tests with significance at P < 0.05 were performed using the SAS (2001) program. Duncan's test was used to check the differences between pairs of groups, and all experiments were repeated 3 times.
Results and Discussion
Physical properties of the film
The physical properties of the GCG film were improved over those of the GC film reported in a previous study (Ku and others 2008) by blending the GC extract with gelatin. The TS of the GCG film increased to 6.23 MPa, compared to 2.53 MPa for the GC film, and the percent elongation also increased to 63.86%, compared to 28.96% for the GC film (Table 1). The reason for the difference may be attributed to the gelatin molecules, which have good film forming ability and a more organized network. It has been reported that a konjac glucomannan and gelatin blend film improved the tensile strength and water vapor permeability of the film (Li and others 2006).
Table 1—. Mechanical properties of the Gelidium corneum–gelatin blend film containing grapefruit seed extract or green tea extract.
|Grapefruit seed extract||0 || 98.87 ± 7.94a|| 6.23 ± 0.61d||63.86 ± 5.23a||4.64 ± 0.29a|
|0.02|| 96.87 ± 4.91a|| 6.70 ± 1.01d||53.62 ± 3.60b||3.72 ± 0.73b|
|0.04|| 92.80 ± 2.97a|| 7.78 ± 0.87c||49.07 ± 3.78c||3.70 ± 0.67b|
|0.08||100.00 ± 4.08a|| 8.97 ± 0.57b||41.99 ± 1.81d||3.27 ± 0.17b|
|0.10|| 98.20 ± 5.46a||11.33 ± 0.36a||42.88 ± 0.11d||3.21 ± 0.20b|
|Green tea extract||0 || 98.87 ± 7.94c|| 6.23 ± 0.61d||63.86 ± 5.23a||4.64 ± 0.29a|
|0.5 || 97.80 ± 5.50c||12.92 ± 0.01b||46.05 ± 3.01b||3.87 ± 0.91a|
|1.4 || 102.27 ± 5.91bc||11.84 ± 0.92c||25.74 ± 3.94c||3.79 ± 0.73a|
|2.8 || 108.13 ± 3.63ba||12.77 ± 0.33b||18.12 ± 3.24d||3.64 ± 0.07a|
|4.2 ||112.40 ± 4.15a||13.95 ± 0.83a|| 6.64 ± 2.22e||3.60 ± 0.07a|
Incorporation of GFSE or GTE into the GCG film affected the mechanical properties of the film. The TS of the GCG film increased with increasing GFSE or GTE concentrations with the exception of 0.5% GTE (Table 1). The TS of the GCG films increased to 11.33 and 13.95 MPa, with the addition of 0.1% GFSE and 4.2% GTE, respectively, compared to 6.23 MPa for the control. These results are in good agreement with those of Sivarooban and others (2008), where they reported that the TS of soy protein film increased with the addition of 1% grape seed extract (GSE) because of hydrogen bonding between protein molecules and GSE polyphenols, and intermolecular cross-linking of the polyphenols. In addition, the TS of the GCG film containing GFSE or GTE was better than the 1.54 MPa TS of the bilayer film of zein and medium-chain triglyceride (MCT) oil (Weller and others 1998) and better than the 0.44 MPa of the soy protein isolate film containing green tea extract (Si and others 2006).
By contrast, the percent elongation of the GCG film decreased with increasing GFSE or GTE concentrations (Table 1). Regarding a decrease in percent elongation, a similar result was observed in the study of the round scad muscle protein-based films incorporating palm oil and chitosan (Prodpran and others 2007). Water vapor permeability (WVP) is an important factor in the selection of food packaging materials because it affects the shelf life of food. The WVP of the GCG film decreased with increasing GFSE or GTE concentrations (Table 1). The WVP of the film incorporated with 0.1% GFSE or 4.2% GTE were 3.21 and 3.60 ng m/m2sPa, respectively, compared to 4.64 ng m/m2sPa for the control. This difference can be attributed to the formation of a compact network by the addition of GFSE or GTE.
Antimicrobial activity of the GCG film against pathogenic bacteria
The antimicrobial activities of the films containing GFSE or GTE against E. coli O157:H7 and L. monocytogenes are presented in Table 2. Increasing GFSE or GTE concentration in the GCG film increased the inhibition of bacterial growth. The GCG film containing 0.1% GFSE decreased the populations of E. coli O157:H7 and L. monocytogenes by 2.08 and 3.30 log CFU/g, compared to that of the control. For the GCG film containing 4.2% GTE, the bacterial populations were reduced by 0.77 and 0.91 log CFU/g, respectively. Kim and others (2006) reported that soy protein isolate film containing 4% GTE had antimicrobial activity against Staphylococcus aureus and Streptococcus mutans. Ha and others (2001) also reported that LDPE film coated with 1.0% GFSE had a positive antimicrobial activity against E. coli, S. aureus, and Bacillus cereus. Along with the reports, our results indicate that GTE or GFSE can be used as an antimicrobial agent in the films. In this study, the GFSE and GTE concentration used in the GCG film for pork loin packaging was determined to be 0.08% and 2.80%, respectively, based on the physical properties and the antimicrobial activity.
Table 2—. Antimicrobial activity of the Gelidium corneum–gelatin blend film against major pathogenic bacteria.
|Grapefruit seed extract||0||7.38 ± 0.13a||7.22 ± 0.09a|
|0.02||7.04 ± 0.47ba||6.21 ± 0.14b|
|0.04||6.55 ± 0.23bc||5.74 ± 0.08c|
|0.08||6.36 ± 0.14c||4.74 ± 0.03d|
|0.10||5.30 ± 0.24d||3.92 ± 0.13e|
|Green tea extract||0||8.69 ± 0.13a||6.96 ± 0.12a|
|0.5||8.32 ± 0.03b||6.50 ± 0.07b|
|1.4||8.15 ± 0.06c||6.32 ± 0.10cb|
|2.8||8.10 ± 0.06c||6.12 ± 0.24c|
|4.2||7.92 ± 0.05d||6.05 ± 0.13c|
Microbiological analysis of pork loins during storage
We determined the microbial growth of pathogenic bacteria in pork loins packed with GCG film containing GFSE or GTE during storage. The initial populations of E. coli O157:H7 and L. monocytogenes on inoculated pork loins were 5.16 and 4.80 log CFU/g, respectively (Table 3). The populations of the E. coli O157:H7 in the pork loins increased during storage for 2 d after inoculation and then decreased thereafter through day 10. Our results are in accordance with those of Nissen and others (2001), where the populations of E. coli O157:H7 in beef increased for 7 d and then decreased thereafter. The growth pattern can be explained by the growth conditions of the bacteria and the availability of nutrients for microbial growth during storage. The populations of the pathogenic bacteria in the pork loins packed with the GCG film containing GFSE or GTE significantly decreased during storage, compared to the control. In particular, after 4 d of storage, the populations of E. coli O157:H7 in the pork loins packed with the GCG film containing GFSE or GTE had decreased 1.10 and 0.80 log CFU/g more than the control, respectively (Table 3). Ha and others (2001) reported that the populations of aerobic bacteria and coliform bacteria in ground beef were reduced by packaging with LDPE film containing 0.5% GFSE. Our results are in good agreement with the report, in terms of antimicrobial activity of GFSE in the film. In addition, the population of the bacteria in the control was 5.47 log CFU/g after 10 d of storage, whereas the populations of the bacteria in the pork loins packed with the GCG film containing GFSE or GTE were 4.88 and 4.47 log CFU/g, respectively.
Table 3—. Changes in the population (log CFU/g) of E. coli O157:H7 and L. monocytogenes inoculated on the pork loin packed with the Gelidium corneum–gelatin (GCG) blend film containing grapefruit seed extract (GFSE) or green tea extract (GTE) during storage.
| ||GCG||5.16 ± 0.13a||6.17 ± 0.07a||6.14 ± 0.17a||6.03 ± 0.01a||5.47 ± 0.12a|
|E. coli O157:H7|| GCG + GFSE||5.16 ± 0.13a||5.33 ± 0.14b||5.04 ± 0.23b||4.97 ± 0.06c||4.88 ± 0.10c|
|GCG + GTE||5.16 ± 0.13a||5.42 ± 0.07b||5.34 ± 0.09b||5.20 ± 0.18b||4.47 ± 0.13b|
|GCG||4.80 ± 0.08a||4.86 ± 0.03a||5.03 ± 0.02a||4.81 ± 0.10a||4.75 ± 0.21a|
|L. monocytogenes|| GCG + GFSE||4.80 ± 0.08a||4.15 ± 0.16b||3.89 ± 0.04c||3.85 ± 0.03b||3.42 ± 0.12c|
|GCG + GTE||4.80 ± 0.08a||4.27 ± 0.05b||3.98 ± 0.05b||3.89 ± 0.04b||3.77 ± 0.15b|
For L. monocytogenes in the pork loins, the populations of the bacteria in the control slightly increased up to the 4th day and then decreased through day 10, whereas the pork loins packed with the GCG film containing GFSE or GTE continually decreased from inoculation through day 10 (Table 3). In particular, the populations of L. monocytogenes in the control increased to 5.03 log CFU/g after 4 d, whereas the populations of L. monocytogenes in the pork loins packed with the GCG film containing GFSE or GTE reached only 3.89 and 3.98 log CFU/g, respectively. In addition, after 10 d of storage, the population of L. monocytogenes in the control was 4.75 log CFU/g, whereas the population of the bacteria in the pork loins packed with the GCG film containing GFSE or GTE was 3.42 and 3.77 log CFU/g, respectively.
Theivendran and others (2006) reported that the populations of L. monocytogenes in turkey frankfurters coated with soy protein film containing GTE (1%) and nisin (10,000 IU/g) were reduced by 2.80 log CFU/g after 28 d of storage at 10 °C, which was better than our results in terms of reduction. Regarding the growth inhibition pattern of L. monocytogenes by antimicrobials during storage, our results are comparable with those of Ye and others (2008), where the population of L. monocytogenes in ham steaks packed with chitosan-coated plastic film containing 0.01 g sodium lactate decreased during storage at 4 °C, compared to the control. Cagri and others (2002) also reported that whey protein film containing p-aminobenzoic acid and sorbic acid for packaging of sausages retarded the growth of L. monocytogenes, E. coli O157:H7, and S. typhimurium. Along with these reports, our results strongly suggest that GTE or GFSE can be used in the packaging films like other antimicrobials.
Antimicrobial effect of GFSE has been known to inhibit the activity of bacterial enzymes and to weaken the bacterial cell wall and cell membranes (Cho and others 2004; Park and Kim 2006). GTE also has been reported to have antimicrobial activity, because its polyphenolic compounds, such as catechin, disrupt cell division in bacteria and affect DNA and RNA metabolism (Kumudavally and others 2008). Therefore, GFSE or GTE in the GCG film inhibited the growth of the pathogenic bacteria in pork loins.
Lipid oxidation in pork loins during storage
The thiobarbituric acid-reactive substance (TBARS) value represents the degree of lipid oxidation of foods. Lipid oxidation is an important factor in the oxidative deterioration of pork. The TBARS values of the pork loin samples increased during storage (Table 4). The pork loins packed with the GCG film containing GFSE or GTE had a very limited reduction in TBARS value during storage compared to the control (Table 4). However, in contrast to weak initial antioxidant activity of the film, the pork loins packed with the GCG film containing GFSE or GTE showed a decrease in the TBARS value of 0.38 and 0.59 mg MDA/kg more after 10 d of storage, respectively, compared to the control. Our results are comparable with those of Ha and others (2001), where ground beef packed with an LDPE film containing 0.5% and 1.0% GFSE had a slower increase in the TBA value during storage compared to the control. Our results show that lipid oxidation in pork loins during storage may be retarded by packing them with the GCG film containing GFSE or GTE. However, it should be noted that there was a limited reduction in TBARS value during storage except at the 10th d of storage.
Table 4—. Change in TBARS value (mg MDA/kg sample) of pork loin packed with the Gelidium corneum–gelatin (GCG) blend film containing grapefruit seed extract (GFSE) or green tea extract (GTE) during storage.
|GCG||0.81 ± 0.11a||1.28 ± 0.01a||1.42 ± 0.11a||1.75 ± 0.09a|| 2.46 ± 0.20a|
|GCG + GFSE||0.81 ± 0.11a||1.18 ± 0.10a||1.23 ± 0.06a||1.70 ± 0.14a||2.08 ± 0.1b|
|GCG + GTE||0.81 ± 0.11a||1.19 ± 0.09a||1.34 ± 0.12a||1.58 ± 0.25a||1.87 ± 0.0b|
Blending of gelatin with Gelidium cormeum extract and the incorporation of GFSE or GTE into the film forming solution improved the physical property as well as the antimicrobial activity of the film. Application of the GCG film to pork loins was successful in inhibiting microbial growth during storage. This is an interesting new result regarding development of edible film. Thus, the results suggest that the GCG film containing GFSE or GTE can be used in edible packaging for food products.