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Effect of sodium alginate and carboxymethyl cellulose edible coating with epigallocatechin gallate on quality and shelf life of fresh pork
International Journal of Biological Macromolecules 141 (2019) 178–184
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Effect of sodium alginate and carboxymethyl cellulose edible coating with epigallocatechin gallate on quality and shelf life of fresh pork Chengcheng Ruan a,b, Yumeng Zhang a,b, Yue Sun a,b, Xueling Gao b, Guoyuan Xiong b, Jin Liang a,b,⁎ a State Key Laboratory of Tea Plant Biology and Utilization/International Joint Laboratory on Tea Chemistry and Health Effects of Ministry of Education, Anhui Agricultural University, 130 West Changjiang Road, Hefei, China b Anhui Engineering Laboratory for Agro-products Processing, College of Tea & Food Science and Technology, Anhui Agricultural University, 130 West Changjiang Road, Hefei, China
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Article history: Received 29 July 2019 Received in revised form 20 August 2019 Accepted 29 August 2019 Available online 30 August 2019 Keywords: Sodium alginate Sodium carboxymethyl cellulose Epigallocatechin gallate Fresh pork Antioxidant Shelf life
a b s t r a c t The active edible coatings were prepared by incorporating epigallocatechin gallate (EGCG) into sodium alginate (SA) and carboxymethyl cellulose (CMC) to investigate the effect of SA-CMC-EGCG coatings on quality and shelf life of fresh pork stored at 4 ± 1 °C for 7 days. The antioxidant effects against lipid oxidation (TBARS), total volatile basic nitrogen (TVB-N) and antimicrobial activity against total viable counts (TVC) were analyzed. Besides, the changes in color parameters and sensory attributes of all pork samples were evaluated. The results showed that fresh pork coated with SA-CMC edible coating with EGCG had a significant inhibitory effect on its microbial growth (P b 0.05), lipid oxidation and TVB-N. SA-CMC-EGCG also increased the L* value and maintained a* value of pork during storage. Besides, the sensory scores of pork samples coated with SA-CMC-EGCG were significantly improved (P b 0.05). Therefore, using SA-CMC-EGCG edible coating could prevent decay and significantly increase the shelf life of fresh pork. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Pork is an important source of dietary nutrition for its rich fat and protein in China [1]. However, fresh pork is easily spoiled by oxidation. Oxidation has been recognized as the main cause of deterioration of fresh pork, which can shorten its shelf life. In addition, oxidation can also change the fat and protein quality in fresh pork, which has a negative impact on the overall acceptance and quality of pork, such as color, odor and tenderness [2]. Besides, pork oxidation can change the compounds in meat, and even form toxic and harmful compounds, such as aldehydes, which can also reduce the nutritional value of pork [3]. In recent years, due to the great potential of antioxidant edible film or coating to meet the demand for fresh meat and prolong its shelf life, more and more attention has been paid to the development and application of antioxidant edible film or coating for fresh meat [4]. Antioxidant edible film or coating formed by biopolymers incorporated with natural antioxidants is an active packaging, which can limit or prevent oxidation and avoid adding active compounds directly to meat. Meanwhile, this edible film or coating can block water vapor, oxygen and carbon dioxide, thus prolonging the shelf life of fresh meat [3,5]. In addition, the antioxidants contained in edible films or coatings can be released slowly and react with free radicals or reactive oxygen species, reducing oxidation and delaying deterioration of meat, fish and poultry products [6,7]. ⁎ Corresponding author. E-mail address: [email protected] (J. Liang).
https://doi.org/10.1016/j.ijbiomac.2019.08.247 0141-8130/© 2019 Elsevier B.V. All rights reserved.
Green tea is one of the most popular beverages because it contains many active compounds, such as tea polyphenols. Epigallocatechin gallate (EGCG) is the main component of tea polyphenols. It has a relatively high antioxidant capacity because its structure contains multiple aromatic phenol rings [8]. Therefore, EGCG can be considered as a good natural antioxidant. Many researchers have successfully prepared edible coatings containing tea extracts and proved that the prepared coatings have good antioxidant properties [5,9]. The edible film incorporated with EGCG has potential application prospects for reducing oxidation and prolonging shelf life in meat industry. Yang et al. [10] used distiller dried grains protein containing green tea extracts as antioxidant films materials to wrap pork meat, and the prepared film had good antioxidant activity and prolonged the shelf life of fresh pork. However, distiller dried grains protein was difficult to extract and had limited applications, so it was not a suitable coating material for industrial production. Thus, choosing suitable raw materials for preparing edible coating is the key to obtain wide application in pork preservation, especially the selection of renewable, abundant and low cost raw materials. Sodium alginate (SA) as a linear polysaccharide is an attractive edible coating material in food industry due to its good film forming property [5]. In addition, SA has a wide range of sources, low prices and great advantages [11]. It is reported that the edible coatings made of SA have high antimicrobial and oil barrier property [12]. SA-based edible coating has many applications in food industry [13,14]. Hamedi et al. reported SA edible coating containing Ziziphora essential oil could improve the quality of chicken fillet and extend its shelf life during cold storage
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[15]. Carboxymethyl cellulose (CMC) is a biopolymer derived from cellulose. The edible coating prepared by CMC had good barrier property [16,17]. It can be used in meat packaging to block oxygen and reduce oxidation. Muppalla et al. [18] used CMC with clove oil to coat chicken meat and they found meat samples coated by CMC with clove oil displayed a shelf life of 12 days, 8 days longer than control samples during refrigerated storage. Despite the fact that there were some studies on antioxidant films or coatings of SA-CMC [5,19], little is known about the effect of adding EGCG to SA-CMC on practical food, especially meat applications. In previous studies, we successfully prepared antioxidant films with SA-CMC and EGCG and evaluated the composite films properties [5]. Therefore, the purpose of this study was to investigate the effect of SA-CMC edible coating with EGCG on prolonging the shelf life of fresh pork stored at 4 ± 1 °C. The influence of SA-CMC edible coating with different EGCG concentrations on the quality attributes of fresh pork was assessed to determine whether the antioxidant edible coating could extend the shelf life of pork. 2. Materials and methods 2.1. Materials SA was obtained from Qingdao Bright Moon Seaweed Group Co., Ltd. (China). CMC was purchased from Shanghai Shenguang Food Chemicals Co., Ltd. (China). EGCG (98% purity) were offered by Huzhou Rongkai Foliage Extract Co., Ltd. (China). Fresh pork loins were purchased from the local market (Hefei, China). The other chemicals were analytical grade. 2.2. Preparation of SA-CMC edible coating and pork samples The SA-CMC edible coating preparation method has been described in detail in our previous study [5]. Briefly, the EGCG solution with different concentrations (0, 0.8, 1.2 and 1.6 g/100 mL) was mixed with SACMC solution at a volume ratio of 1:1. The resulted edible coating solutions were obtained by stirring for 30 min at 50 °C and degassing. The fresh pork samples were prepared according to previous report [1], with some modifications. The external visible connective tissue and fat of pork loins were removed and about 5.5 kg pork loins were cut into small pieces with an approximate weight of 50 g. Then, pork loins were randomly and equally divided into five groups and each group contained 21 pieces of pork loins: uncoated pork (None), pork with SA-CMC edible coating (SA-CMC), pork with SA-CMC-0.8EGCG edible coating (SA-CMC-0.8EGCG), pork with SA-CMC-1.2EGCG edible coating (SA-CMC-1.2EGCG), pork with SA-CMC-1.6EGCG edible coating (SACMC-1.6EGCG). After that, pork loins were completely immersed into the coating solutions (SA-CMC with or without EGCG) for 5 s. After immersion, the pork loins were dried on grills at room temperature for 15 min, and the process was repeated twice. After coating, each sample was placed in an individual polystyrene tray over-wrapped with a polyethylene plastic film and stored refrigerated at 4 ± 1 °C for a period of 7 days. Weight loss, pH, color, lipid oxidation, protein oxidation, microbial changes and sensory evaluation of pork samples were tested every day to study the effects of different edible coatings on the quality indices over the storage time. 2.3. Weight loss measurement To measure the weight loss during the storage time, pork samples were taken to weight on an automatic electro-balance (Yuyao Jiming Equipment Co. Ltd., China), with a precision of 0.01 g, at each sampling point. Three readings of each pork sample were assessed. For each sample, results were expressed as a percentage of weight loss relative to its
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initial weight. The weight loss was calculated as followed: Weight loss ð%Þ ¼ ðW 0 −W Þ=W 0 100 where W0 is the initial weight and W is the final weight at each sampling point. 2.4. Determination of pH The pH of pork samples were determined based on the previous method reported by Zhang et al. [1], with some modification. 10 g of each pork sample was chopped and then homogenized thoroughly with 100 mL distilled water at 6000 rpm for 5 min. The pH of the pork sample solution was measured using a pH meter (OHAUS, USA). The pH test was repeated three times for each sample. 2.5. Color measurement The pork sample colors of L* (lightness/darkness), a* (redness/ greenness) and b* (yellowness/blueness) were measured directly on the sample surface by Minolta colorimeter (CR 400; Minolta, Japan) with a 10° observer angle, D65 illumination and an 8 mm aperture. Besides, white board calibration and background were used. The analysis was performed on three samples from each treatment and five measurements at randomly selected points were recorded per sample. 2.6. Total viable counts (TVC) The total viable counts (TVC) measurements were performed for microbiological analysis, according to the method of Feng et al. [20]. A minced pork sample (25 g) was mixed with 225 mL sterile normal saline in a sterile plastic bag and homogenized for 1–2 min. Serial dilutions (1:10 v/v each time) were made. TVC was determined using spread plate technique on standard plate count agar (PCA) after 48 h incubation at 37 °C. TVC values were expressed as log CFU/g meat. All the experiments were carried out in triplicate. 2.7. Lipid oxidation measurement (TBARS) Lipid oxidation of pork samples was assessed by the 2-thiobarbituric acid (TBA) assay method as described by Zhang et al. [1], with some modifications. In brief, a minced pork sample (5 g) was added to 50 mL of TCA solution (7.5% trichloroacetic acid and 0.1% ethylenediaminetetraacetic acid) and mixed well. The mixture was oscillated on a thermostatic oscillator (Guoyu Instrument Manufacturing Co. Ltd., China) at 50 °C for 30 min. After cooling to room temperature, the mixture was filtered through two Whatman No.1 filter papers and 5 mL of filtrate was mixed with 5 mL 20 mM 2-thiobarbituric acid (TBA) and incubated at 90 °C for 30 min. After that, the mixture was cooled to room temperature with running tap water. The absorbance of the solution was determined at 532 nm with Lambda 35 UV–Vis spectrophotometer (PerkinElmer Ltd., USA). A standard curve was prepared using 1, 1, 3, 3tetramethoxypropane (MDA) and TBARS values were expressed as mg of MDA equivalent per kg of meat. All the experiments were repeated in triplicate. 2.8. Total volatile basic nitrogen (TVB-N) TVB-N was determined according to previous method [4] with some modifications. A minced pork sample (20 g) was added to 100 mL distilled water in a beaker. The beaker was mixed by gently shaking for 30 min to obtain a homogeneous solution. The solution was filtered through a Whatman No.1 filter paper. Afterwards, 5 mL filtrate was alkalinized by adding 5 mL of MgO solution (10 g/L). A Kjeldahl distillation unit was used to carry out the steam distillation for 5 min. The distillate was collected in a receiving flask with 10 mL boric acid
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(20 g/L) and a few drops of methyl red (0.1%) and bromocresol green (0.1%) indicators. The distillate solution was finally titrated with HCl (0.01 mol/L) and TVB-N values were expressed as mg/100 g meat. All the experiments were repeated in triplicate.
2.9. Sensory evaluation The sensory quality of pork sample during storage time was evaluated by 10 trained members from the Anhui Engineering Laboratory for Agro-products Processing (Anhui Agricultural University). According to the method of Mohammadi et al. [4], pork samples with different treatments were independently presented in white polyethylene trays to each panellist and a fresh pork sample was offered to panellists for comparison purposes along with the test samples. Panellists gave scores for sensory characteristics, including color, odor, and overall acceptance using a 5-point descriptive scale [7]. The color value expressed the degree of color darkening in pork samples: 5 = none; 4 = slight; 3 = small; 2 = moderate; and 1 = extreme. The odor value represented the degree of spoilage odor in pork sample: 5 = none; 4 = slight; 3 = small; 2 = moderate; and 1 = extreme. The overall acceptance value indicated the degree to which panellists liked the pork sample as a whole, where 5 = like extremely and 1 = dislike extremely. Pork was considered unacceptable when the score was below 3.
2.10. Statistical analysis All indicators in the experiment were performed in triplicate. The results were presented as means ± standard deviation of three parallel assays. The data were analyzed by IBM SPSS Statistics V17.0 and Origin 9.0. Statistics on a completely randomized design were performed with the analysis of variance (ANOVA) procedure in SPSS software. Duncan's multiple range test (P b 0.05) was used to detect differences among mean values of pork samples' properties in all tests.
3. Results and discussion 3.1. Weight loss Weight loss is an important indicator of fresh meat quality, because it can change the color, flavor and texture of fresh meat, affect the taste and sensory quality [21]. Table 1 showed the effects of SA-CMC edible coating containing EGCG on the weight loss of fresh pork samples. SACMC edible coating significantly reduced the weight loss of pork samples during storage. In particular, the weight loss of pork decreased with the increase of EGCG content. The relevant research in recent years showed that the less weight loss by SA-CMC edible coating might be caused by water barrier property of SA-CMC [20]. With the increase of EGCG, the structure of SA-CMC edible coating became denser [5] and water barrier property improved, which reduced the weight loss of pork samples. This was consistent with previous studies [2,22].
3.2. pH value The pH changes of fresh pork samples with SA-CMC-EGCG edible coating during storage were shown in Fig. 1. As shown in Fig. 1, the pH of all samples generally showed an overall upward trend during storage, in line with the previous research [23]. The pH value increased first, then decreased slightly, and then increased gradually with time. This might be due to the fact that there were some aerobic bacteria on the surface of pork samples at the early stage of storage. Under sufficient oxygen and nutrient conditions, the proteins in pork samples were rapidly decomposed and alkaline substances were produced, which increased the pH value of pork [20]. With the increase of storage time, the internal environment of packaging gradually became hypoxia and high carbon dioxide, which inhibited the growth of aerobic bacteria and created a suitable environment for the growth of lactic acid bacteria. The decrease in pH value of pork samples might be due to the accumulation of lactic acid bacteria [24]. At the later stage of storage, proteins in pork samples were degraded into volatile alkaline nitrogen molecules, including ammonia and primary amines, secondary amines and tertiary amines, through microbial activity and meat endogenous proteases (e.g. calpains), resulting in an increase in the pH of pork samples [2,25]. Especially, the pH value of pork samples coated with SA-CMC and SA-CMCEGCG was lower than that of uncoated pork samples. With the increase of EGCG content, the pH value gradually decreased. This might because protein degradation was significantly inhibited by edible coatings, while EGCG as an active antioxidant and antimicrobial component inhibited microbial activity and produced fewer amines [25]. Similar results were reported by Zhang et al. [1] and Siripatrawan et al. [26]. Therefore, the pH value of pork samples coated with SA-CMC-EGCG was more stable than that of uncoated samples. It indicated that the quality of fresh pork samples by using edible coating treatment was maintained during storage. 3.3. Color The changes of color parameters L*, a* and b* were shown in Fig. 2. The L* value increased on the first day, probably due to changes in pork structure, such as protein denaturation and protein conformation, which might increase light dispersion [27]. At the later stage of storage, pork samples were further oxidized by enzymes and microbial reproductive activities, which oxidized myoglobin to brown metmyoglobin (MetMb) and reduced L* value [28]. In particular, uncoated pork samples had the lowest L* value, while those coated with SA-CMC and SACMC-EGCG had higher L* value at the end of storage. This might be because the edible coating could block oxygen, while EGCG as an antioxidant reduced oxidation, so the L* values of SA-CMC and SA-CMC-EGCG pork samples were relatively high. Huang et al. [29] and Cardoso et al. [30] also reported similar results. The a* represents the freshness of pork sample. As shown in Fig. 2b, all pork samples had the highest a* values on the first day and then decreased during storage. This was mainly because most of the surface myoglobin would bind with oxygen on the first day to form the red color pigment of oxymyoglobin and a* values increased [25]. In
Table 1 Effect of SA-CMC based coating solutions with EGCG at different concentration on weight loss of pork samples during 4 ± 1 °C storage. Sample
Storage time (d) 1
None SA-CMC SA-CMC-0.8EGCG SA-CMC-1.2EGCG SA-CMC-1.6EGCG
2 a
0.87 ± 0.58 0.53 ± 0.09a 0.52 ± 0.09a 0.51 ± 0.17a 0.51 ± 0.20a
3 a
8.27 ± 0.61 2.81 ± 0.67b 2.50 ± 1.88b 1.70 ± 1.00b 1.32 ± 0.92b
4 a
10.92 ± 0.83 4.55 ± 1.25b 3.27 ± 0.82b,c 2.64 ± 1.10b,c 2.00 ± 1.17c
5 a
15.05 ± 0.93 7.57 ± 0.70b 6.27 ± 1.55b,c 4.05 ± 1.08c,d 3.19 ± 0.90d
6 a
24.32 ± 0.58 12.12 ± 0.75b 7.93 ± 1.56c 5.45 ± 0.82d 4.41 ± 0.98d
7 a
36.98 ± 1.45 16.06 ± 1.30b 9.79 ± 0.90c 7.29 ± 1.22c,d 6.05 ± 0.83d
All values are presented as mean ± standard deviation. Data in the same line with different superscript letters are significantly different (P b 0.05).
39.82 ± 1.36a 18.05 ± 1.45b 11.69 ± 0.70c 9.32 ± 0.92c,d 7.72 ± 1.33d
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Fig. 1. Effect of SA-CMC based coating solutions with EGCG at different concentration on pH values of pork samples during 4 ± 1 °C storage.
addition, the a* value of uncoated pork samples on the first day was the highest because uncoated pork was directly combined with more oxygen. After the first day, the a* values of all pork samples continued to decline, although a* values of SA-CMC-EGCG pork samples decreased more slowly than uncoated and SA-CMC samples. The decrease of a* might be due to the formation of MetMb, which was related to the oxidation of oxymyoglobin or deoxymyoglobin [31]. In our previous studies, SA-CMC-EGCG edible film showed good antioxidant activity [5], which might lead to its effective delay of pork myoglobin oxidation and discoloration. In some previous studies, the b* value was not discussed or showed no significant change [22,30]. However, our results showed that the b* value increased gradually during the storage shown in Fig. 2c. This might be due to the formation of hydrogen sulfide (H2S), which was produced by microorganisms and enzymes that degraded proteins, thus binding to hemoglobin to form yellow complexes [32]. Especially compared with SA-CMC and SA-CMC-EGCG pork samples, the b* value of uncoated pork samples increased more. The b* value of SA-CMC and SA-CMC-EGCG pork samples increased slowly because edible coating could block oxygen and EGCG could reduce oxidation as an antioxidant. Therefore, adding EGCG in SA-CMC edible coating had better color protection effect on fresh pork. 3.4. Total viable counts In order to study the inhibitory effect of edible coating on the growth of microorganisms in pork samples, the total viable count (TVC) was determined. Fig. 3 showed the TVC changes of uncoated and coated pork samples. From 0 to 7 days, it increased gradually in all groups, especially in the uncoated pork samples. The initial microorganism counts in the pork samples were around 3.48 log CFU/g, which was a relatively low, similar with the previous report [1]. However, on the seventh day, TVC reached at 9.79, 10.77, 8.92, 8.17 and 6.13 log CFU/g for uncoated, SA-CMC, SA-CMC-0.8EGCG, SA-CMC-1.2EGCG and SA-CMC-1.6EGCG pork samples, respectively. At the end of storage, the TVC value of SACMC pork samples was higher than that of uncoated pork samples. This might attribute to the good barrier property of SA-CMC edible coating. The uncoated pork samples lost more weight than that of SA-CMC coated, thus losing more water and nutrients, making microorganisms difficult to survive in the last period [20,33]. Fresh pork with TVC b6 log CFU/g is acceptable to consumers [4]. Our experiments showed that the TVC value of uncoated pork samples exceeded 6 log CFU/g on the third day. SA-CMC and SA-CMC-0.8 EGCG exceeded the threshold on the fourth day. However, SA-CMC-1.2 EGCG and SA-CMC-1.6 EGCG
Fig. 2. Effect of SA-CMC based coating solutions with EGCG at different concentration on changes in color parameters of pork samples during 4 ± 1 °C storage. a: L* for lightness; b: a* for redness; c: b* for yellowness.
exceeded the threshold on day 5 and 7, respectively. These results indicated that SA-CMC edible coating with EGCG could significantly inhibit the microbial growth and extend the shelf life of pork. The enhanced
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feel unpleasant flavor [34]. This indicated that consumers could detect severe oxidation and decay in uncoated and SA-CMC pork samples on the third day. However, with the increase of EGCG, the TBARS values of pork samples at 4, 5 and 7 days were N0.6 mg MDA/kg meat respectively, indicating that the SA-CMC edible coating with EGCG could prolong the shelf life of fresh pork for 1–3 days. Compared with the SA-CMC pork samples, the TBARS value during storage was lower when EGCG was added to the SA-CMC edible coating. This might be attributed to the antioxidant property of EGCG. The incorporation of EGCG into SACMC edible coating made the structure of SA-CMC edible coating dense and improved oxygen barrier property, further reducing the exposure of pork samples to oxygen, thus retarding lipid oxidation. Similar report showed that pork meat wrapped with protein films containing tea extracts decreased the TBARS value and had the slowest lipid oxidation during storage [10]. These results indicated that the addition of EGCG in SA-CMC edible coating had a strong inhibitory effect on lipid oxidation of fresh pork during storage. 3.6. Total volatile basic nitrogen (TVB-N) Fig. 3. Effect of SA-CMC based coating solutions with EGCG at different concentration on TVC values of pork samples during 4 ± 1 °C storage.
Lipid oxidation is a main factor of quality deterioration in pork. As an index of lipid oxidation, the TBARS values of pork samples were measured. As shown in Fig. 4, it increased continuously during storage. Compared with uncoated pork samples, the TBARS values of pork samples coated with SA-CMC or SA-CMC-EGCG decreased significantly during storage (P b 0.05). The TBARS value of uncoated pork samples increased rapidly during storage, reaching 0.63 ± 0.02 mg MDA/kg meat on the third day and 1.94 ± 0.03 mg MDA/kg meat on the seventh day. While the TBARS value of SA-CMC pork samples increased relatively slowly, reaching 0.61 ± 0.01 mg MDA/kg meat on the third day and 1.71 ± 0.06 mg MDA/kg meat on the seventh day. It has been reported that when TBARS value exceeds 0.6 mg MDA/kg meat, customers could
TVB-N is the main product of protein decomposition by spoilage bacteria in pork. Protein provides abundant nutrition for microbial growth [35], which is an index of pork quality evaluation. Fig. 5 showed the changes of TVB-N values of all pork samples with storage time. The initial TVB-N value of fresh pork was 4.36 mg/100 g, and its value increased with the prolongation of storage time. It was speculated that this upward trend might be due to the growth and reproduction of microorganisms, which degraded protein in pork samples and destroyed the integrity of muscle cell structure [36]. Therefore, a large number of endogenous enzymes was released from broken cells, which accelerated protein degradation and the release of amino acids [37]. It was noteworthy that during storage, the TVB-N values of uncoated pork samples were significantly higher than those of SA-CMC and SA-CMC-EGCG pork samples. This might be because the oxygen barrier of SA-CMC and SA-CMC-EGCG pork samples had been improved, which delayed the oxidation of pork samples and reduced the protein decomposition caused by microorganisms. When EGCG was present, the TVB-N value of SA-CMC-EGCG pork samples was lower during storage. This effect was related to the concentration of EGCG, especially the highest concentration (SA-CMC-1.6 EGCG) could more effectively delay the oxidation process of pork samples. The result was consistent with previous study [4], which showed that the concentration of okra mucilage in CMC edible coating would influence the TVB-N values of chicken breast
Fig. 4. Effect of SA-CMC based coating solutions with EGCG at different concentration on TBARS values of pork samples during 4 ± 1 °C storage.
Fig. 5. Effect of SA-CMC based coating solutions with EGCG at different concentration on TVB-N values of pork samples during 4 ± 1 °C storage.
antimicrobial activity might be attributed to EGCG, which reduced negative charges and led to cell rupture or stomatal formation, leading to microbial death [25]. Similar reports also showed that tea polyphenol incorporated into chitosan coating had lower microbial counts compared to that without coating [23]. 3.5. Lipid oxidation
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Table 2 Effect of SA-CMC based coating solutions with EGCG at different concentration on sensory attributes of pork samples during 4 ± 1 °C storage. Parameter
Color scores
Odor scores
Overall acceptance
Sample
None SA-CMC SA-CMC-0.8EGCG SA-CMC-1.2EGCG SA-CMC-1.6EGCG None SA-CMC SA-CMC-0.8EGCG SA-CMC-1.2EGCG SA-CMC-1.6EGCG None SA-CMC SA-CMC-0.8EGCG SA-CMC-1.2EGCG SA-CMC-1.6EGCG
Storage time (d) 1
2
3
4
5
6
7
4.83 ± 0.24a 4.53 ± 0.17a 4.63 ± 0.19a 4.60 ± 0.08a 4.67 ± 0.12a 4.43 ± 0.33a 4.57 ± 0.12a 4.70 ± 0.16a 4.73 ± 0.12a 4.80 ± 0.08a 4.17 ± 0.12b 4.60 ± 0.08a 4.70 ± 0.16a 4.77 ± 0.12a 4.83 ± 0.09a
4.73 ± 0.21a 4.43 ± 0.33a 4.33 ± 0.29a 4.40 ± 0.22a 4.63 ± 0.17a 3.43 ± 0.33b 3.80 ± 0.16b 4.47 ± 0.12a 4.60 ± 0.08a 4.63 ± 0.12a 3.17 ± 0.24c 3.83 ± 0.17b 4.33 ± 0.12a 4.60 ± 0.14a 4.73 ± 0.17a
2.50 ± 0.41b 2.63 ± 0.54b 3.80 ± 0.24a 4.17 ± 0.29a 4.40 ± 0.14a 2.17 ± 0.24b 2.50 ± 0.36b 3.67 ± 0.12a 4.07 ± 0.12a 4.10 ± 0.16a 2.23 ± 0.17b 2.57 ± 0.42b 3.67 ± 0.26a 4.00 ± 0.16a 4.07 ± 0.19a
2.33 ± 0.47b 2.40 ± 0.22b 2.83 ± 0.29b 3.80 ± 0.22a 4.10 ± 0.08a 1.43 ± 0.33c 2.33 ± 0.12b 2.83 ± 0.29b 3.43 ± 0.17a 3.77 ± 0.21a 1.23 ± 0.21d 2.00 ± 0.16c 2.27 ± 0.21c 3.27 ± 0.21b 3.73 ± 0.17a
1.57 ± 0.17c 1.87 ± 0.29c 1.93 ± 0.33c 2.87 ± 0.12b 3.47 ± 0.21a \ 1.73 ± 0.17c 2.00 ± 0.16c 2.87 ± 0.21b 3.37 ± 0.19a \ 1.03 ± 0.12d 1.77 ± 0.21c 2.80 ± 0.16b 3.37 ± 0.21a
\ 1.47 ± 0.21c 1.67 ± 0.29c 2.37 ± 0.33b 3.03 ± 0.17a \ 1.03 ± 0.12c 1.40 ± 0.14c 2.27 ± 0.21b 3.03 ± 0.21a \ \ 1.23 ± 0.17c 1.97 ± 0.12b 2.43 ± 0.17a
\ \ 1.10 ± 0.29b,c 1.57 ± 0.33b 2.57 ± 0.25a \ \ \ 1.13 ± 0.09b 2.50 ± 0.22a \ \ 1.03 ± 0.17b 1.07 ± 0.09b 2.00 ± 0.16a
“\” means the score is below 1. All values are presented as mean ± standard deviation. Data in the same line with different superscript letters are significantly different (P b 0.05).
meat and high concentration decreased the TVB-N values more efficiently. It has been reported that the upper limit of pork deterioration TVB-N value is 20 mg/100 g [38]. As shown in Fig. 5, the TVB-N value of uncoated and SA-CMC pork samples exceeded 20 mg/100 g on the third day. However, the TVB-N values of the other three pork samples exceeded 20 mg/100 g on the fourth, fifth and seventh days, respectively. Therefore, the SA-CMC edible coating added with EGCG could effectively prolong the shelf life of fresh pork.
solutions significantly inhibited the growth of microorganism and lipid oxidation of fresh pork during storage at 4 ± 1 °C. Besides, SACMC-EGCG edible coating could reduce the weight loss of pork samples and have protective effect on color of pork samples. The sensory evaluation results also showed that the treatment of SA-CMC-EGCG active coating solution had no adverse effects on the sensory attributes of pork samples, and improved the color, odor and overall acceptance of pork samples. Therefore, SA-CMC-EGCG edible coatings could effectively prolong the shelf life of fresh pork.
3.7. Sensory evaluation Acknowledgements Changes in all sensory properties (color, odor and overall acceptability) of pork samples were shown in Table 2. The color, odor and overall acceptance scores of pork samples, whether coated or not, showed a similar downward trend during storage. On the third day, the color scores of uncoated and SA-CMC pork samples were significantly lower than those of pork samples coated with SA-CMC-EGCG solution (P b 0.05), and reached unacceptable scores. However, the other three pork samples coated with SA-CMC-EGCG solution showed a color scores of b3 points and became unacceptable on the fourth, fifth and seventh days, respectively. This could be due to the antioxidant and antimicrobial properties of EGCG and the inhibitory effect of SA-CMC edible coating on microbial spoilage and lipid oxidation during pork storage. The unacceptable odors were mainly caused by lipid oxidation and microbial spoilage. On the first day, there was no significant difference in odor score between uncoated pork and coated pork samples. However, from the second day, the odor scores of uncoated and SA-CMC pork samples were significantly lower than those of other pork samples. In particular, at the end of storage period, SA-CMC-1.6EGCG pork samples had the highest scores and became unacceptable on the last day of storage. Furthermore, the overall acceptance scores of uncoated and SACMC pork samples were significantly (P b 0.05) lower than those of other three pork samples from the second day of storage and became unacceptable on the third day. With the increase of EGCG, SA-CMCEGCG pork samples reached unacceptable overall acceptance scores (below 3) on the fourth, fifth and sixth day, respectively. It showed that the SA-CMC edible coating with EGCG could prolong the shelf life of fresh pork by 1–3 days, compared with the pork without coating. These results were consistent with the values of pH, TVB-N, TBARS and TVC of pork samples. 4. Conclusion An active edible coating solution for pork preservation was developed by mixing EGCG with SA-CMC. SA-CMC-EGCG edible coating
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Effect of sodium alginate and carboxymethyl cellulose edible coating with epigallocatechin gallate on quality and shelf life of fresh pork Chengcheng Ruan a,b, Yumeng Zhang a,b, Yue Sun a,b, Xueling Gao b, Guoyuan Xiong b, Jin Liang a,b,⁎ a State Key Laboratory of Tea Plant Biology and Utilization/International Joint Laboratory on Tea Chemistry and Health Effects of Ministry of Education, Anhui Agricultural University, 130 West Changjiang Road, Hefei, China b Anhui Engineering Laboratory for Agro-products Processing, College of Tea & Food Science and Technology, Anhui Agricultural University, 130 West Changjiang Road, Hefei, China
a r t i c l e
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Article history: Received 29 July 2019 Received in revised form 20 August 2019 Accepted 29 August 2019 Available online 30 August 2019 Keywords: Sodium alginate Sodium carboxymethyl cellulose Epigallocatechin gallate Fresh pork Antioxidant Shelf life
a b s t r a c t The active edible coatings were prepared by incorporating epigallocatechin gallate (EGCG) into sodium alginate (SA) and carboxymethyl cellulose (CMC) to investigate the effect of SA-CMC-EGCG coatings on quality and shelf life of fresh pork stored at 4 ± 1 °C for 7 days. The antioxidant effects against lipid oxidation (TBARS), total volatile basic nitrogen (TVB-N) and antimicrobial activity against total viable counts (TVC) were analyzed. Besides, the changes in color parameters and sensory attributes of all pork samples were evaluated. The results showed that fresh pork coated with SA-CMC edible coating with EGCG had a significant inhibitory effect on its microbial growth (P b 0.05), lipid oxidation and TVB-N. SA-CMC-EGCG also increased the L* value and maintained a* value of pork during storage. Besides, the sensory scores of pork samples coated with SA-CMC-EGCG were significantly improved (P b 0.05). Therefore, using SA-CMC-EGCG edible coating could prevent decay and significantly increase the shelf life of fresh pork. © 2019 Elsevier B.V. All rights reserved.
1. Introduction Pork is an important source of dietary nutrition for its rich fat and protein in China [1]. However, fresh pork is easily spoiled by oxidation. Oxidation has been recognized as the main cause of deterioration of fresh pork, which can shorten its shelf life. In addition, oxidation can also change the fat and protein quality in fresh pork, which has a negative impact on the overall acceptance and quality of pork, such as color, odor and tenderness [2]. Besides, pork oxidation can change the compounds in meat, and even form toxic and harmful compounds, such as aldehydes, which can also reduce the nutritional value of pork [3]. In recent years, due to the great potential of antioxidant edible film or coating to meet the demand for fresh meat and prolong its shelf life, more and more attention has been paid to the development and application of antioxidant edible film or coating for fresh meat [4]. Antioxidant edible film or coating formed by biopolymers incorporated with natural antioxidants is an active packaging, which can limit or prevent oxidation and avoid adding active compounds directly to meat. Meanwhile, this edible film or coating can block water vapor, oxygen and carbon dioxide, thus prolonging the shelf life of fresh meat [3,5]. In addition, the antioxidants contained in edible films or coatings can be released slowly and react with free radicals or reactive oxygen species, reducing oxidation and delaying deterioration of meat, fish and poultry products [6,7]. ⁎ Corresponding author. E-mail address: [email protected] (J. Liang).
https://doi.org/10.1016/j.ijbiomac.2019.08.247 0141-8130/© 2019 Elsevier B.V. All rights reserved.
Green tea is one of the most popular beverages because it contains many active compounds, such as tea polyphenols. Epigallocatechin gallate (EGCG) is the main component of tea polyphenols. It has a relatively high antioxidant capacity because its structure contains multiple aromatic phenol rings [8]. Therefore, EGCG can be considered as a good natural antioxidant. Many researchers have successfully prepared edible coatings containing tea extracts and proved that the prepared coatings have good antioxidant properties [5,9]. The edible film incorporated with EGCG has potential application prospects for reducing oxidation and prolonging shelf life in meat industry. Yang et al. [10] used distiller dried grains protein containing green tea extracts as antioxidant films materials to wrap pork meat, and the prepared film had good antioxidant activity and prolonged the shelf life of fresh pork. However, distiller dried grains protein was difficult to extract and had limited applications, so it was not a suitable coating material for industrial production. Thus, choosing suitable raw materials for preparing edible coating is the key to obtain wide application in pork preservation, especially the selection of renewable, abundant and low cost raw materials. Sodium alginate (SA) as a linear polysaccharide is an attractive edible coating material in food industry due to its good film forming property [5]. In addition, SA has a wide range of sources, low prices and great advantages [11]. It is reported that the edible coatings made of SA have high antimicrobial and oil barrier property [12]. SA-based edible coating has many applications in food industry [13,14]. Hamedi et al. reported SA edible coating containing Ziziphora essential oil could improve the quality of chicken fillet and extend its shelf life during cold storage
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[15]. Carboxymethyl cellulose (CMC) is a biopolymer derived from cellulose. The edible coating prepared by CMC had good barrier property [16,17]. It can be used in meat packaging to block oxygen and reduce oxidation. Muppalla et al. [18] used CMC with clove oil to coat chicken meat and they found meat samples coated by CMC with clove oil displayed a shelf life of 12 days, 8 days longer than control samples during refrigerated storage. Despite the fact that there were some studies on antioxidant films or coatings of SA-CMC [5,19], little is known about the effect of adding EGCG to SA-CMC on practical food, especially meat applications. In previous studies, we successfully prepared antioxidant films with SA-CMC and EGCG and evaluated the composite films properties [5]. Therefore, the purpose of this study was to investigate the effect of SA-CMC edible coating with EGCG on prolonging the shelf life of fresh pork stored at 4 ± 1 °C. The influence of SA-CMC edible coating with different EGCG concentrations on the quality attributes of fresh pork was assessed to determine whether the antioxidant edible coating could extend the shelf life of pork. 2. Materials and methods 2.1. Materials SA was obtained from Qingdao Bright Moon Seaweed Group Co., Ltd. (China). CMC was purchased from Shanghai Shenguang Food Chemicals Co., Ltd. (China). EGCG (98% purity) were offered by Huzhou Rongkai Foliage Extract Co., Ltd. (China). Fresh pork loins were purchased from the local market (Hefei, China). The other chemicals were analytical grade. 2.2. Preparation of SA-CMC edible coating and pork samples The SA-CMC edible coating preparation method has been described in detail in our previous study [5]. Briefly, the EGCG solution with different concentrations (0, 0.8, 1.2 and 1.6 g/100 mL) was mixed with SACMC solution at a volume ratio of 1:1. The resulted edible coating solutions were obtained by stirring for 30 min at 50 °C and degassing. The fresh pork samples were prepared according to previous report [1], with some modifications. The external visible connective tissue and fat of pork loins were removed and about 5.5 kg pork loins were cut into small pieces with an approximate weight of 50 g. Then, pork loins were randomly and equally divided into five groups and each group contained 21 pieces of pork loins: uncoated pork (None), pork with SA-CMC edible coating (SA-CMC), pork with SA-CMC-0.8EGCG edible coating (SA-CMC-0.8EGCG), pork with SA-CMC-1.2EGCG edible coating (SA-CMC-1.2EGCG), pork with SA-CMC-1.6EGCG edible coating (SACMC-1.6EGCG). After that, pork loins were completely immersed into the coating solutions (SA-CMC with or without EGCG) for 5 s. After immersion, the pork loins were dried on grills at room temperature for 15 min, and the process was repeated twice. After coating, each sample was placed in an individual polystyrene tray over-wrapped with a polyethylene plastic film and stored refrigerated at 4 ± 1 °C for a period of 7 days. Weight loss, pH, color, lipid oxidation, protein oxidation, microbial changes and sensory evaluation of pork samples were tested every day to study the effects of different edible coatings on the quality indices over the storage time. 2.3. Weight loss measurement To measure the weight loss during the storage time, pork samples were taken to weight on an automatic electro-balance (Yuyao Jiming Equipment Co. Ltd., China), with a precision of 0.01 g, at each sampling point. Three readings of each pork sample were assessed. For each sample, results were expressed as a percentage of weight loss relative to its
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initial weight. The weight loss was calculated as followed: Weight loss ð%Þ ¼ ðW 0 −W Þ=W 0 100 where W0 is the initial weight and W is the final weight at each sampling point. 2.4. Determination of pH The pH of pork samples were determined based on the previous method reported by Zhang et al. [1], with some modification. 10 g of each pork sample was chopped and then homogenized thoroughly with 100 mL distilled water at 6000 rpm for 5 min. The pH of the pork sample solution was measured using a pH meter (OHAUS, USA). The pH test was repeated three times for each sample. 2.5. Color measurement The pork sample colors of L* (lightness/darkness), a* (redness/ greenness) and b* (yellowness/blueness) were measured directly on the sample surface by Minolta colorimeter (CR 400; Minolta, Japan) with a 10° observer angle, D65 illumination and an 8 mm aperture. Besides, white board calibration and background were used. The analysis was performed on three samples from each treatment and five measurements at randomly selected points were recorded per sample. 2.6. Total viable counts (TVC) The total viable counts (TVC) measurements were performed for microbiological analysis, according to the method of Feng et al. [20]. A minced pork sample (25 g) was mixed with 225 mL sterile normal saline in a sterile plastic bag and homogenized for 1–2 min. Serial dilutions (1:10 v/v each time) were made. TVC was determined using spread plate technique on standard plate count agar (PCA) after 48 h incubation at 37 °C. TVC values were expressed as log CFU/g meat. All the experiments were carried out in triplicate. 2.7. Lipid oxidation measurement (TBARS) Lipid oxidation of pork samples was assessed by the 2-thiobarbituric acid (TBA) assay method as described by Zhang et al. [1], with some modifications. In brief, a minced pork sample (5 g) was added to 50 mL of TCA solution (7.5% trichloroacetic acid and 0.1% ethylenediaminetetraacetic acid) and mixed well. The mixture was oscillated on a thermostatic oscillator (Guoyu Instrument Manufacturing Co. Ltd., China) at 50 °C for 30 min. After cooling to room temperature, the mixture was filtered through two Whatman No.1 filter papers and 5 mL of filtrate was mixed with 5 mL 20 mM 2-thiobarbituric acid (TBA) and incubated at 90 °C for 30 min. After that, the mixture was cooled to room temperature with running tap water. The absorbance of the solution was determined at 532 nm with Lambda 35 UV–Vis spectrophotometer (PerkinElmer Ltd., USA). A standard curve was prepared using 1, 1, 3, 3tetramethoxypropane (MDA) and TBARS values were expressed as mg of MDA equivalent per kg of meat. All the experiments were repeated in triplicate. 2.8. Total volatile basic nitrogen (TVB-N) TVB-N was determined according to previous method [4] with some modifications. A minced pork sample (20 g) was added to 100 mL distilled water in a beaker. The beaker was mixed by gently shaking for 30 min to obtain a homogeneous solution. The solution was filtered through a Whatman No.1 filter paper. Afterwards, 5 mL filtrate was alkalinized by adding 5 mL of MgO solution (10 g/L). A Kjeldahl distillation unit was used to carry out the steam distillation for 5 min. The distillate was collected in a receiving flask with 10 mL boric acid
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(20 g/L) and a few drops of methyl red (0.1%) and bromocresol green (0.1%) indicators. The distillate solution was finally titrated with HCl (0.01 mol/L) and TVB-N values were expressed as mg/100 g meat. All the experiments were repeated in triplicate.
2.9. Sensory evaluation The sensory quality of pork sample during storage time was evaluated by 10 trained members from the Anhui Engineering Laboratory for Agro-products Processing (Anhui Agricultural University). According to the method of Mohammadi et al. [4], pork samples with different treatments were independently presented in white polyethylene trays to each panellist and a fresh pork sample was offered to panellists for comparison purposes along with the test samples. Panellists gave scores for sensory characteristics, including color, odor, and overall acceptance using a 5-point descriptive scale [7]. The color value expressed the degree of color darkening in pork samples: 5 = none; 4 = slight; 3 = small; 2 = moderate; and 1 = extreme. The odor value represented the degree of spoilage odor in pork sample: 5 = none; 4 = slight; 3 = small; 2 = moderate; and 1 = extreme. The overall acceptance value indicated the degree to which panellists liked the pork sample as a whole, where 5 = like extremely and 1 = dislike extremely. Pork was considered unacceptable when the score was below 3.
2.10. Statistical analysis All indicators in the experiment were performed in triplicate. The results were presented as means ± standard deviation of three parallel assays. The data were analyzed by IBM SPSS Statistics V17.0 and Origin 9.0. Statistics on a completely randomized design were performed with the analysis of variance (ANOVA) procedure in SPSS software. Duncan's multiple range test (P b 0.05) was used to detect differences among mean values of pork samples' properties in all tests.
3. Results and discussion 3.1. Weight loss Weight loss is an important indicator of fresh meat quality, because it can change the color, flavor and texture of fresh meat, affect the taste and sensory quality [21]. Table 1 showed the effects of SA-CMC edible coating containing EGCG on the weight loss of fresh pork samples. SACMC edible coating significantly reduced the weight loss of pork samples during storage. In particular, the weight loss of pork decreased with the increase of EGCG content. The relevant research in recent years showed that the less weight loss by SA-CMC edible coating might be caused by water barrier property of SA-CMC [20]. With the increase of EGCG, the structure of SA-CMC edible coating became denser [5] and water barrier property improved, which reduced the weight loss of pork samples. This was consistent with previous studies [2,22].
3.2. pH value The pH changes of fresh pork samples with SA-CMC-EGCG edible coating during storage were shown in Fig. 1. As shown in Fig. 1, the pH of all samples generally showed an overall upward trend during storage, in line with the previous research [23]. The pH value increased first, then decreased slightly, and then increased gradually with time. This might be due to the fact that there were some aerobic bacteria on the surface of pork samples at the early stage of storage. Under sufficient oxygen and nutrient conditions, the proteins in pork samples were rapidly decomposed and alkaline substances were produced, which increased the pH value of pork [20]. With the increase of storage time, the internal environment of packaging gradually became hypoxia and high carbon dioxide, which inhibited the growth of aerobic bacteria and created a suitable environment for the growth of lactic acid bacteria. The decrease in pH value of pork samples might be due to the accumulation of lactic acid bacteria [24]. At the later stage of storage, proteins in pork samples were degraded into volatile alkaline nitrogen molecules, including ammonia and primary amines, secondary amines and tertiary amines, through microbial activity and meat endogenous proteases (e.g. calpains), resulting in an increase in the pH of pork samples [2,25]. Especially, the pH value of pork samples coated with SA-CMC and SA-CMCEGCG was lower than that of uncoated pork samples. With the increase of EGCG content, the pH value gradually decreased. This might because protein degradation was significantly inhibited by edible coatings, while EGCG as an active antioxidant and antimicrobial component inhibited microbial activity and produced fewer amines [25]. Similar results were reported by Zhang et al. [1] and Siripatrawan et al. [26]. Therefore, the pH value of pork samples coated with SA-CMC-EGCG was more stable than that of uncoated samples. It indicated that the quality of fresh pork samples by using edible coating treatment was maintained during storage. 3.3. Color The changes of color parameters L*, a* and b* were shown in Fig. 2. The L* value increased on the first day, probably due to changes in pork structure, such as protein denaturation and protein conformation, which might increase light dispersion [27]. At the later stage of storage, pork samples were further oxidized by enzymes and microbial reproductive activities, which oxidized myoglobin to brown metmyoglobin (MetMb) and reduced L* value [28]. In particular, uncoated pork samples had the lowest L* value, while those coated with SA-CMC and SACMC-EGCG had higher L* value at the end of storage. This might be because the edible coating could block oxygen, while EGCG as an antioxidant reduced oxidation, so the L* values of SA-CMC and SA-CMC-EGCG pork samples were relatively high. Huang et al. [29] and Cardoso et al. [30] also reported similar results. The a* represents the freshness of pork sample. As shown in Fig. 2b, all pork samples had the highest a* values on the first day and then decreased during storage. This was mainly because most of the surface myoglobin would bind with oxygen on the first day to form the red color pigment of oxymyoglobin and a* values increased [25]. In
Table 1 Effect of SA-CMC based coating solutions with EGCG at different concentration on weight loss of pork samples during 4 ± 1 °C storage. Sample
Storage time (d) 1
None SA-CMC SA-CMC-0.8EGCG SA-CMC-1.2EGCG SA-CMC-1.6EGCG
2 a
0.87 ± 0.58 0.53 ± 0.09a 0.52 ± 0.09a 0.51 ± 0.17a 0.51 ± 0.20a
3 a
8.27 ± 0.61 2.81 ± 0.67b 2.50 ± 1.88b 1.70 ± 1.00b 1.32 ± 0.92b
4 a
10.92 ± 0.83 4.55 ± 1.25b 3.27 ± 0.82b,c 2.64 ± 1.10b,c 2.00 ± 1.17c
5 a
15.05 ± 0.93 7.57 ± 0.70b 6.27 ± 1.55b,c 4.05 ± 1.08c,d 3.19 ± 0.90d
6 a
24.32 ± 0.58 12.12 ± 0.75b 7.93 ± 1.56c 5.45 ± 0.82d 4.41 ± 0.98d
7 a
36.98 ± 1.45 16.06 ± 1.30b 9.79 ± 0.90c 7.29 ± 1.22c,d 6.05 ± 0.83d
All values are presented as mean ± standard deviation. Data in the same line with different superscript letters are significantly different (P b 0.05).
39.82 ± 1.36a 18.05 ± 1.45b 11.69 ± 0.70c 9.32 ± 0.92c,d 7.72 ± 1.33d
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Fig. 1. Effect of SA-CMC based coating solutions with EGCG at different concentration on pH values of pork samples during 4 ± 1 °C storage.
addition, the a* value of uncoated pork samples on the first day was the highest because uncoated pork was directly combined with more oxygen. After the first day, the a* values of all pork samples continued to decline, although a* values of SA-CMC-EGCG pork samples decreased more slowly than uncoated and SA-CMC samples. The decrease of a* might be due to the formation of MetMb, which was related to the oxidation of oxymyoglobin or deoxymyoglobin [31]. In our previous studies, SA-CMC-EGCG edible film showed good antioxidant activity [5], which might lead to its effective delay of pork myoglobin oxidation and discoloration. In some previous studies, the b* value was not discussed or showed no significant change [22,30]. However, our results showed that the b* value increased gradually during the storage shown in Fig. 2c. This might be due to the formation of hydrogen sulfide (H2S), which was produced by microorganisms and enzymes that degraded proteins, thus binding to hemoglobin to form yellow complexes [32]. Especially compared with SA-CMC and SA-CMC-EGCG pork samples, the b* value of uncoated pork samples increased more. The b* value of SA-CMC and SA-CMC-EGCG pork samples increased slowly because edible coating could block oxygen and EGCG could reduce oxidation as an antioxidant. Therefore, adding EGCG in SA-CMC edible coating had better color protection effect on fresh pork. 3.4. Total viable counts In order to study the inhibitory effect of edible coating on the growth of microorganisms in pork samples, the total viable count (TVC) was determined. Fig. 3 showed the TVC changes of uncoated and coated pork samples. From 0 to 7 days, it increased gradually in all groups, especially in the uncoated pork samples. The initial microorganism counts in the pork samples were around 3.48 log CFU/g, which was a relatively low, similar with the previous report [1]. However, on the seventh day, TVC reached at 9.79, 10.77, 8.92, 8.17 and 6.13 log CFU/g for uncoated, SA-CMC, SA-CMC-0.8EGCG, SA-CMC-1.2EGCG and SA-CMC-1.6EGCG pork samples, respectively. At the end of storage, the TVC value of SACMC pork samples was higher than that of uncoated pork samples. This might attribute to the good barrier property of SA-CMC edible coating. The uncoated pork samples lost more weight than that of SA-CMC coated, thus losing more water and nutrients, making microorganisms difficult to survive in the last period [20,33]. Fresh pork with TVC b6 log CFU/g is acceptable to consumers [4]. Our experiments showed that the TVC value of uncoated pork samples exceeded 6 log CFU/g on the third day. SA-CMC and SA-CMC-0.8 EGCG exceeded the threshold on the fourth day. However, SA-CMC-1.2 EGCG and SA-CMC-1.6 EGCG
Fig. 2. Effect of SA-CMC based coating solutions with EGCG at different concentration on changes in color parameters of pork samples during 4 ± 1 °C storage. a: L* for lightness; b: a* for redness; c: b* for yellowness.
exceeded the threshold on day 5 and 7, respectively. These results indicated that SA-CMC edible coating with EGCG could significantly inhibit the microbial growth and extend the shelf life of pork. The enhanced
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feel unpleasant flavor [34]. This indicated that consumers could detect severe oxidation and decay in uncoated and SA-CMC pork samples on the third day. However, with the increase of EGCG, the TBARS values of pork samples at 4, 5 and 7 days were N0.6 mg MDA/kg meat respectively, indicating that the SA-CMC edible coating with EGCG could prolong the shelf life of fresh pork for 1–3 days. Compared with the SA-CMC pork samples, the TBARS value during storage was lower when EGCG was added to the SA-CMC edible coating. This might be attributed to the antioxidant property of EGCG. The incorporation of EGCG into SACMC edible coating made the structure of SA-CMC edible coating dense and improved oxygen barrier property, further reducing the exposure of pork samples to oxygen, thus retarding lipid oxidation. Similar report showed that pork meat wrapped with protein films containing tea extracts decreased the TBARS value and had the slowest lipid oxidation during storage [10]. These results indicated that the addition of EGCG in SA-CMC edible coating had a strong inhibitory effect on lipid oxidation of fresh pork during storage. 3.6. Total volatile basic nitrogen (TVB-N) Fig. 3. Effect of SA-CMC based coating solutions with EGCG at different concentration on TVC values of pork samples during 4 ± 1 °C storage.
Lipid oxidation is a main factor of quality deterioration in pork. As an index of lipid oxidation, the TBARS values of pork samples were measured. As shown in Fig. 4, it increased continuously during storage. Compared with uncoated pork samples, the TBARS values of pork samples coated with SA-CMC or SA-CMC-EGCG decreased significantly during storage (P b 0.05). The TBARS value of uncoated pork samples increased rapidly during storage, reaching 0.63 ± 0.02 mg MDA/kg meat on the third day and 1.94 ± 0.03 mg MDA/kg meat on the seventh day. While the TBARS value of SA-CMC pork samples increased relatively slowly, reaching 0.61 ± 0.01 mg MDA/kg meat on the third day and 1.71 ± 0.06 mg MDA/kg meat on the seventh day. It has been reported that when TBARS value exceeds 0.6 mg MDA/kg meat, customers could
TVB-N is the main product of protein decomposition by spoilage bacteria in pork. Protein provides abundant nutrition for microbial growth [35], which is an index of pork quality evaluation. Fig. 5 showed the changes of TVB-N values of all pork samples with storage time. The initial TVB-N value of fresh pork was 4.36 mg/100 g, and its value increased with the prolongation of storage time. It was speculated that this upward trend might be due to the growth and reproduction of microorganisms, which degraded protein in pork samples and destroyed the integrity of muscle cell structure [36]. Therefore, a large number of endogenous enzymes was released from broken cells, which accelerated protein degradation and the release of amino acids [37]. It was noteworthy that during storage, the TVB-N values of uncoated pork samples were significantly higher than those of SA-CMC and SA-CMC-EGCG pork samples. This might be because the oxygen barrier of SA-CMC and SA-CMC-EGCG pork samples had been improved, which delayed the oxidation of pork samples and reduced the protein decomposition caused by microorganisms. When EGCG was present, the TVB-N value of SA-CMC-EGCG pork samples was lower during storage. This effect was related to the concentration of EGCG, especially the highest concentration (SA-CMC-1.6 EGCG) could more effectively delay the oxidation process of pork samples. The result was consistent with previous study [4], which showed that the concentration of okra mucilage in CMC edible coating would influence the TVB-N values of chicken breast
Fig. 4. Effect of SA-CMC based coating solutions with EGCG at different concentration on TBARS values of pork samples during 4 ± 1 °C storage.
Fig. 5. Effect of SA-CMC based coating solutions with EGCG at different concentration on TVB-N values of pork samples during 4 ± 1 °C storage.
antimicrobial activity might be attributed to EGCG, which reduced negative charges and led to cell rupture or stomatal formation, leading to microbial death [25]. Similar reports also showed that tea polyphenol incorporated into chitosan coating had lower microbial counts compared to that without coating [23]. 3.5. Lipid oxidation
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Table 2 Effect of SA-CMC based coating solutions with EGCG at different concentration on sensory attributes of pork samples during 4 ± 1 °C storage. Parameter
Color scores
Odor scores
Overall acceptance
Sample
None SA-CMC SA-CMC-0.8EGCG SA-CMC-1.2EGCG SA-CMC-1.6EGCG None SA-CMC SA-CMC-0.8EGCG SA-CMC-1.2EGCG SA-CMC-1.6EGCG None SA-CMC SA-CMC-0.8EGCG SA-CMC-1.2EGCG SA-CMC-1.6EGCG
Storage time (d) 1
2
3
4
5
6
7
4.83 ± 0.24a 4.53 ± 0.17a 4.63 ± 0.19a 4.60 ± 0.08a 4.67 ± 0.12a 4.43 ± 0.33a 4.57 ± 0.12a 4.70 ± 0.16a 4.73 ± 0.12a 4.80 ± 0.08a 4.17 ± 0.12b 4.60 ± 0.08a 4.70 ± 0.16a 4.77 ± 0.12a 4.83 ± 0.09a
4.73 ± 0.21a 4.43 ± 0.33a 4.33 ± 0.29a 4.40 ± 0.22a 4.63 ± 0.17a 3.43 ± 0.33b 3.80 ± 0.16b 4.47 ± 0.12a 4.60 ± 0.08a 4.63 ± 0.12a 3.17 ± 0.24c 3.83 ± 0.17b 4.33 ± 0.12a 4.60 ± 0.14a 4.73 ± 0.17a
2.50 ± 0.41b 2.63 ± 0.54b 3.80 ± 0.24a 4.17 ± 0.29a 4.40 ± 0.14a 2.17 ± 0.24b 2.50 ± 0.36b 3.67 ± 0.12a 4.07 ± 0.12a 4.10 ± 0.16a 2.23 ± 0.17b 2.57 ± 0.42b 3.67 ± 0.26a 4.00 ± 0.16a 4.07 ± 0.19a
2.33 ± 0.47b 2.40 ± 0.22b 2.83 ± 0.29b 3.80 ± 0.22a 4.10 ± 0.08a 1.43 ± 0.33c 2.33 ± 0.12b 2.83 ± 0.29b 3.43 ± 0.17a 3.77 ± 0.21a 1.23 ± 0.21d 2.00 ± 0.16c 2.27 ± 0.21c 3.27 ± 0.21b 3.73 ± 0.17a
1.57 ± 0.17c 1.87 ± 0.29c 1.93 ± 0.33c 2.87 ± 0.12b 3.47 ± 0.21a \ 1.73 ± 0.17c 2.00 ± 0.16c 2.87 ± 0.21b 3.37 ± 0.19a \ 1.03 ± 0.12d 1.77 ± 0.21c 2.80 ± 0.16b 3.37 ± 0.21a
\ 1.47 ± 0.21c 1.67 ± 0.29c 2.37 ± 0.33b 3.03 ± 0.17a \ 1.03 ± 0.12c 1.40 ± 0.14c 2.27 ± 0.21b 3.03 ± 0.21a \ \ 1.23 ± 0.17c 1.97 ± 0.12b 2.43 ± 0.17a
\ \ 1.10 ± 0.29b,c 1.57 ± 0.33b 2.57 ± 0.25a \ \ \ 1.13 ± 0.09b 2.50 ± 0.22a \ \ 1.03 ± 0.17b 1.07 ± 0.09b 2.00 ± 0.16a
“\” means the score is below 1. All values are presented as mean ± standard deviation. Data in the same line with different superscript letters are significantly different (P b 0.05).
meat and high concentration decreased the TVB-N values more efficiently. It has been reported that the upper limit of pork deterioration TVB-N value is 20 mg/100 g [38]. As shown in Fig. 5, the TVB-N value of uncoated and SA-CMC pork samples exceeded 20 mg/100 g on the third day. However, the TVB-N values of the other three pork samples exceeded 20 mg/100 g on the fourth, fifth and seventh days, respectively. Therefore, the SA-CMC edible coating added with EGCG could effectively prolong the shelf life of fresh pork.
solutions significantly inhibited the growth of microorganism and lipid oxidation of fresh pork during storage at 4 ± 1 °C. Besides, SACMC-EGCG edible coating could reduce the weight loss of pork samples and have protective effect on color of pork samples. The sensory evaluation results also showed that the treatment of SA-CMC-EGCG active coating solution had no adverse effects on the sensory attributes of pork samples, and improved the color, odor and overall acceptance of pork samples. Therefore, SA-CMC-EGCG edible coatings could effectively prolong the shelf life of fresh pork.
3.7. Sensory evaluation Acknowledgements Changes in all sensory properties (color, odor and overall acceptability) of pork samples were shown in Table 2. The color, odor and overall acceptance scores of pork samples, whether coated or not, showed a similar downward trend during storage. On the third day, the color scores of uncoated and SA-CMC pork samples were significantly lower than those of pork samples coated with SA-CMC-EGCG solution (P b 0.05), and reached unacceptable scores. However, the other three pork samples coated with SA-CMC-EGCG solution showed a color scores of b3 points and became unacceptable on the fourth, fifth and seventh days, respectively. This could be due to the antioxidant and antimicrobial properties of EGCG and the inhibitory effect of SA-CMC edible coating on microbial spoilage and lipid oxidation during pork storage. The unacceptable odors were mainly caused by lipid oxidation and microbial spoilage. On the first day, there was no significant difference in odor score between uncoated pork and coated pork samples. However, from the second day, the odor scores of uncoated and SA-CMC pork samples were significantly lower than those of other pork samples. In particular, at the end of storage period, SA-CMC-1.6EGCG pork samples had the highest scores and became unacceptable on the last day of storage. Furthermore, the overall acceptance scores of uncoated and SACMC pork samples were significantly (P b 0.05) lower than those of other three pork samples from the second day of storage and became unacceptable on the third day. With the increase of EGCG, SA-CMCEGCG pork samples reached unacceptable overall acceptance scores (below 3) on the fourth, fifth and sixth day, respectively. It showed that the SA-CMC edible coating with EGCG could prolong the shelf life of fresh pork by 1–3 days, compared with the pork without coating. These results were consistent with the values of pH, TVB-N, TBARS and TVC of pork samples. 4. Conclusion An active edible coating solution for pork preservation was developed by mixing EGCG with SA-CMC. SA-CMC-EGCG edible coating
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