Determination of Shelf Life Model of Pork Cutlet and Pork Lard during Accelerated Storage Conditions

One type of processed meat products was selected and purchased from market in Korea. Pork cutlet (M) manufactured by one companies was selected. The pork lard sample was obtained from one company (L). Each sample was transported to the laboratory at the temperature of distribution, and initial quality levels were analyzed immediately.

The cutlet meat was stored at -18℃, -6℃, and -1℃ (denoted M1-18, M1-6, and M1-1). Temperature conditions (-18℃) of cutlet meat was necessary to maintain the product qualities listed on the package based on legal standards (MFDS, 2017). The remaining temperature conditions (-6℃ and -1℃) reflected noncompliance with storage temperature conditions by either manufacturers or consumers. The prescribed storage condition of pork lard is usually room temperature, but pork lard is often stored outside of warehouses. Therefore, in this study, pork lard was stored at 10℃, 25℃, 35℃, and 45℃ (denoted L1-10, L1-25, L1-35, and L1-45), temperature conditions akin to real distribution conditions. The pork cutlet and pork lard samples were stored for 204 days, and experiments were performed once per week during that time. All analyses were performed in triplicate.

Quality indicators were selected according to the characteristics of each of the samples. Pork cutlet was analyzed using microbiological analysis methods (total aerobic counts, TAC; coli form counts), physico-chemical methods (thiobarbituric acid reactive substances; thiobarbituric acid reactive substances (TBARS) assay, volatile basic nitrogen (VBN), and pH), and sensory evaluation. Pork lard was analyzed using physico-chemical methods (TBARS assay; acid value, AV; peroxide value, POV) and sensory evaluation. All analyses were carried out in triplicate for each formulation.

Each 10 g sample was diluted (1:10) in distilled water and homogenized using a stomacher (Stomacher^® 400 Circulator, Seward, Ltd., UK) for 2 min at room temperature. Serial dilutions (1:10) from the homogenized microbial extracts (0.1 mL) were plated separately on each plate and spread thoroughly. TAC was determined using plate count agar (Difco, USA) incubated at 37±1℃ for 48 h. Coliform group counts were enumerated using deoxycholate lactose agar (Difco, USA) and were incubated at 37±1℃ for 24 h. All analyses were performed in triplicate, and results were expressed as logarithm colony-forming units per milliliter (Log CFU/mL).

TBARS assay was performed in triplicate using the modified method proposed by Witte et al. (1970) to assess lipid oxidation. On each sampling day, 10 g minced sample was homogenized with 25 mL 20% (w/v) aqueous trichloroacetic acid in 2 M phosphoric acid at room temperature. The homogenization was performed at 14,000×g for 1 min with a homogenizer (AM-7, Nihonseiki Kaisha Ltd., Tokyo, Japan). After homogenization and adjustment to 20 mL using distilled water, the supernatant was filtered through a Whatman paper (No. 1). After filtering, a 5 mL sample was reacted with 5 mL 2-TBA (0. 005 M in distilled water) in a test tube and stored for 15 h at room temperature in darkness. The TBA complex was measured at 530 nm using a spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK). The results were calculated using the following equation and reported in milligrams malonaldehyde (MDA) per kilogram: all determinations were performed in triplicate.

VBN concentration is usually used to represent the level of corruption in meat. VBN was measured in the experimental sample in triplicate according to the Conway micropipette diffusion method (Pearson, 1976). Each 5 g sample was combined with 15 mL distilled water and homogenized with a homogenizer (AM-7, Nihonseiki Kaisha Ltd., Tokyo, Japan) at 10,000×g for 1 min. The supernatant was filtered using a Whatman paper (No. 1), and the filtrate was placed in a conical tube and adjusted to a final volume of 50 mL with distilled water. A 1 mL volume of filtered sample solution and 1 mL saturated potassium carbonate were placed in the outer section of a Conway micro diffusion cell (Sibata Ltd., Tokyo, Japan). Boric acid (0.01 N) was carefully transferred into the inner section, and the lid was immediately closed and the solution mixed carefully. The cell was incubated at 37℃ for 2 h, and then the solution in the inner section was titrated with 0.02 N sulfuric acid until the green solution turned pink. The concentration of VBN was calculated using the following equation: all determinations were performed in triplicate.

Each 5 g sample was combined with 15 mL distilled water and then homogenized with a homogenizer (AM-7, Nihonseiki Kaisha Ltd., Tokyo, Japan) at 10,000×g for 1 min. The supernatant was filtered using a Whatman paper (No. 1), and then the filtrate was adjusted to a final volume of 50 mL with distilled water. Measurements were taken with a digital pH meter (AM-7, Nihonseiki Kaisha Ltd., Tokyo, Japan). All determinations were performed in triplicate.

AV measurements were performed in triplicate to assess lipid oxidation using a standard titration method according to the methods described in the Food Code of Korea (MFDS, 2015). Samples were melted at 60℃ before the experiments. Each 5 g sample was placed in an Erlenmeyer flask and mixed thoroughly with 100 mL neutralized ethanol-ether (1:2) mixture. The mixture was titrated with a solution of 0.05 M ethanolic potassium hydroxide as the standard reagent to phenolphthalein. AV was calculated using the following equation: all determinations were performed in triplicate.

Analyses of lipid oxidation were conducted by assessing following the official methods described in the Food Code of Korea (MFDS, 2015). Each 5 g sample was dissolved slightly by heating with 25 mL acetic acid-chloroform (3:2) mixture. This solution was homogenized slightly with 1 mL saturated potassium iodide solution and then placed in darkness for 10 min, after which it was homogenized thoroughly with 30 mL distilled water. The mixture was titrated with 0.01 N sodium thiosulfate using 1 mL starch solution as an indicator. Measurements were corrected with the blank test and carried out in at least triplicate.

Samples were prepared for sensory evaluation, which was carried out using 12 consumer-type panels on each day of sampling. Each panel independently evaluated the samples for appearance, flavor, and texture according to a hedonic rating method described (9=excellent, 5=acceptable, and 1=vile).

The availability of so many processed products with long shelf lives adds difficulty to shelf-life estimations. Decreasing experimental costs and reducing the time spent, accelerated shelf life tests are often used to overcome this problem (Labuza and Schmidl, 1985; Nelson and Labuza, 1994). Accelerated shelf life tests using various kinetic models are useful for assessing the effects of temperature changes on product quality (Jedermann et al., 2009). Data obtained with accelerated shelf life tests was calculated through reaction order, Arrhenius equation, and Q₁₀ value. These kinetic parameters have been described in previous studies (Kong et al., 2007; Wang et al., 2004).

The data obtained from each experiment was adjusted according to the formula below. The zero-order reaction model, one of the best known models for establishing shelf life, was used to represent a linear evolution:

The data obtained from each experiment was adjusted according to the formula below. The first-order reaction model, another well-known model for establishing shelf life, was used to represent an exponential evolution of the parameter:

The Arrhenius equation is usually used to describe the temperature dependence of a reaction rate on isothermal conditions. The formula is as follows:

Q₁₀ value is a frequently used parameter to describe the temperature dependence of a reaction rate. It can be estimated via the quality changes at increases of 10℃. Q₁₀ value is calculated as follows:

It can be transformed via the Arrhenius equation:

All tests were conducted at least three times for each experimental condition, and mean values are reported. Analysis of variance was performed using SAS software (SAS ver. 9.3, SAS Institute Inc., Cary, NC, USA, 2010). Duncan’s multiple range test (p<0.05) was used to determine the differences between the means.

Previously reported research has indicated that measures of microbial growth are the main components of estimations of shelf life for meat and processed meat products (Georgantelis et al., 2007; Heo et al., 2008; Roller et al., 2002; Zhao et al., 1994). Therefore, two microbiological experiments were carried out in this study, including total aerobic bacteria (TAC) in the ground meat kind of pork cutlet. The initial TAC value of pork cutlet (M) was 2.87±0.03 Log CFU/mL. Samples were stored at each experimental temperature over 204 days, and experiments were carried out on each weekly sampling day. The TAC value of pork cutlet -18℃ (M1-18) was 7.75±0.05 Log CFU/mL at day 204 that of pork cutlet -6℃ (M1-6) was 8.09±0.05 Log CFU/mL at day 204, and that of pork cutlet -1℃ (M1-1) was 6.31±0.03 Log CFU/mL at day 78. These values in ground meat samples differed significantly during the storage period according to temperature conditions (p<0.05), a result consistent with those of a study by Kim et al. (2008), who reported that TAC values of ground meat patties increase significantly during storage periods. This tendency that increase of TAC value during storage periods is consistent with that reported by a previous study (Heo et al., 2008). The TAC values of the ground meat samples are shown in Table 1 and 2. In case of coliform group, all sample were not detected.

TBARS assay is a widely using method for measuring contents of MDA generated from hydroperoxides. Previous studies have reported that MDA content is closely related both to the amount of lipids present during storage period and off flavor (Tarladgis et al., 1960; Teets et al., 2008). In this study, lipid oxidation was determined according to the method described by Witte et al. (1970) and detected at 530 nm. The TBARS value of pork cutlet meat and pork lard are presented in Table 1, 2, 3, and 4. First, in case of cutlet meat, the TBARS value for M1 was 0.88±0.00 MDA mg/kg at day 0, and thereafter, samples were stored at different temperatures (-18℃, -6℃, and -1℃). Each sample was tested on every weekly sampling day over 204 days. The TBARS value of M1-18 was 4.18±0.01 MDA mg/kg at day 204, that of M1-6 was 7.19±0.05 MDA mg/kg at day 204, and that of M1-1 was 5.42±0.06 MDA mg/kg at day 78. Second, the initial TBARS value of the pork lard samples was 0.09±MDA mg/kg. This value was also measured in samples stored under each temperature condition. The TBARS value for L1-10 was 0.35±0.01 MDA mg/kg at day 204, that of L1-25 was 1.10±0.05 MDA mg/kg at day 204, that of L1-35 was 0.76±0.01 MDA mg/kg at day 78, and that of L1-45 was 1.22±0.05 MDA mg/kg at day 78. Significant difference was found between days 0 and 204 (p<0.05). Lipid oxidation showed a tendency to increase over the storage period as well; the higher the storage temperature, the higher the lipid oxidation. This result was similar with study of Lee et al. (2004).

VBN value has also been used as a good indicator of bacterial growth and protein deterioration and decomposition (Fanco et al., 2002; Kang et al., 2002; Vinci and Antonelli, 2002). One study (Field and Chang, 1969) has reported that enzymes and microorganisms in food decompose protein into peptides and amino acids. Therefore, VBN value was selected as an indicator and measurements were shown in Table 1 and 2. Samples pork cutlet (M) showed significant differences during the storage period (p<0.05). The initial value for pork cutlet (M) was 8.68±0.02 mg% and samples were kept at each experimental temperature during the 204 day study period, and then VBN values were measured once each week. VBN values of M1-18, M1-6, and M1-1 were 15.00±0.02 mg% at day 204, 16.66±0.30 at day 204, and 11.77±0.45 mg% at day 78, respectively. The results confirmed that VBN value is an appropriate proper measure for processed meat products.

pH is a widely used estimate of the quality of food, including meat products (Jeremiah et al., 1991). Several studies have reported that microorganisms degrade protein and produce organic sulfides and amines that increase pH during storage (Brown et al., 1998; Devine et al., 1993; Gregory, 2005; Hambrecht et al., 2003). Therefore, pH was selected as an indicator of freshness and pH measurements for the samples appear in Table 1 and 2. The pH of the M1 was measured over 204 days on every weekly sampling day. The initial pH value of was 6.45±0.04; these values decreased significantly during the storage period (p<0.05). The pH values of the individual samples (M1-18, M1-6, and M1-1) were 6.12±0.00 at day 204, 5.37±0.33 at day 204, and 4.73±0.16 at day 78, respectively.

Lipid oxidation, one of the most important determinants of shelf life, occurs during food production and storage (Castro et al., 2007; Baggio and Bragagnolo, 2006). POV is one of the most widely used methods to evaluate lipid oxidation in processed meat products and represents the content of the primary oxides that form during oxidation. AV is also a popular method with which to measure lipid oxidation. AV is generally associated with lipase activity originating from microorganisms. The quality changes in AV and POV for pork lard under the experimental storage conditions are shown in Table 3 and 4. We observed that the values rapidly increased in all samples. Total samples were divided in accordance with the temperature conditions, and those samples were assessed over 204 days. The initial AV was 0.02±0.01 mg KOH/g, and initial POV was 2.91±0.50 meq/kg. AVs and POV for pork lard samples increased markedly during overall storage. For the temperature conditions of L1-10, L1-25, L1-35, and L1-45, AV values were 0.22 mg±0.01, 0.92±0.02, 0.54±0.02, and 1.48±0.01 mg KOH/g, respectively, at day 204. Respective POV was 12.04±0.10, 57.39±0.24, 41.35±0.20, and 115.10±0.15 meq/kg. The results showed that AV and POV gradually increased without exception under all temperature conditions during overall storage. However, measured POV and AVs of samples stored at higher temperature were higher than those of samples stored at lower temperatures. The results of this study are similar to those of López-Duarte and Vidal-Quintanar (2009). In their research, although POV values were studied in various samples, POV were increased significantly during the storage period. In particular, POV increased significantly with temperature.

Sensory evaluation is a useful tool with which to judge the quality of food. Numerous studies have used sensory evaluation as an indicator of food quality (Ambrosiadis et al., 2004; Bovolenta et al., 2008; Lu et al., 2011). Changes in the sensory evaluation of pork cutlet and pork lard are presented in Table 1, 2, 3, and 4. Sensory analysis was performed using a 9-point hedonic scale. The evaluation criteria were defined such that the initial quality level was 9 points, and the quality limit was 5 points. The relationship between storage period and sensory quality was analyzed using a linear regression equation. Sensory assessments decreased significantly during the experimental period and decreased more rapidly at higher storage temperature (p<0.05).

Cutlet meat product with frozen distribution was selected and kept at -18℃, -6℃, and -1℃ for 204 days. Pork lard was also selected and kept at 10℃, 25℃, 35℃, and 45℃ for 204 days. Accelerated shelf life tests are used because these selected samples have a long shelf-life, assessing their characteristics over time requires comparatively higher costs and more time. Quality indicators were selected according to the characteristics of processed meat products. The selected microbiological indicators were TAC and coli form count, and the selected physicochemical indicators were TBARS, VBN, pH, AV, POV, and Sensory evaluation. Experimental data for pork cutlet and pork lard were analyzed using zero- and first-order reactions, Arrhenius equation, and Q₁₀. Initially, quality changes displayed linear regression according to the zero- and first-order equations, and then reaction order was selected based on higher r². Activation energy and Q₁₀ were calculated using the constant k applied to the Arrhenius equation. Other quality limits were calculated and analyzed using correlated linear regression of sensory evaluation with experimental data.

In this result of estimated shelf-life analysis, the quality limits of cutlet, M1-18, M1-6, and M1-1 were reached at 12.36–13.91, 1.99–3.67 and 1.48–1.97 months (Table 5). And in case of pork lard, L1-10, L1-25, L1-35 and L1-45 were reached 10.17–36.34, 5.76–7.48, 3.21–4.07 and 1.57–2.94 months, respectively (Table 6).