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Incorporating nisin and grape seed extract in chitosan-gelatine edible coating and its effect on cold storage of fresh pork
Journal Pre-proof Incorporating nisin and grape seed extract in chitosan-gelatine edible coating and its effect on cold storage of fresh pork
Yun Xiong, Meng Chen, Robyn Dorothy Warner, Zhongxiang Fang PII:
S0956-7135(19)30607-3
DOI:
https://doi.org/10.1016/j.foodcont.2019.107018
Reference:
JFCO 107018
To appear in:
Food Control
Received Date:
05 August 2019
Accepted Date:
23 November 2019
Please cite this article as: Yun Xiong, Meng Chen, Robyn Dorothy Warner, Zhongxiang Fang, Incorporating nisin and grape seed extract in chitosan-gelatine edible coating and its effect on cold storage of fresh pork, Food Control (2019), https://doi.org/10.1016/j.foodcont.2019.107018
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Incorporating nisin and grape seed extract in chitosan-gelatine edible coating and its effect on cold storage of fresh pork
Yun Xionga, Meng Chena, Robyn Dorothy Warner a, Zhongxiang Fanga* a
School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The
University of Melbourne, Parkville, VIC 3010, Australia
* Corresponding author: Dr Zhongxiang Fang School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC 3010, Australia Email: [email protected]; Tel: +61 3 83445063
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Journal Pre-proof Abstract: Fresh pork is a highly perishable food product and susceptible to oxidation and microbial spoilage. The objective of this study was to develop a chitosan-gelatine edible coating system incorporating grape seed extract and/or nisin and investigate its effect on the preservation of fresh pork during cold storage at 4 ℃ for 20 days. Results showed that 1% chitosan (CHI) effectively inhibited pork oxidation and microbial spoilage; 1% chitosan combined with 3% gelatine (CHI-GEL) enhanced these preservative effects; and incorporating 0.5% grape seed extract (CHI-GEL-GSE) further enhanced antioxidant activity against meat oxidation. However, incorporating nisin (NIS) into the coating (CHI-GEL-NIS and CHI-GELNIS-GSE) did not further improve the antimicrobial and antioxidant effects. The CHI-GELGSE formulation had the best performance on pork preservation, which suggested that it could be developed as a hurdle technology to preserve fresh meat. Keywords: pork; chitosan; gelatine; nisin; grape seed extract; edible coating.
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Journal Pre-proof Abbreviations: CON = control group, non-coated pork samples NIS = nisin GSE = grape seed extract CHI = 1% chitosan GEL =3% gelatine CHI-GEL = 1% chitosan + 3% gelatine CHI-GEL-GSE = 1% chitosan + 3% gelatine + 0.5% GSE CHI-GEL-NIS = 1% chitosan + 3% gelatine + 0.1% nisin CHI-GEL-GSE-NIS = 1% chitosan + 3% gelatine + 0.5% GSE + 0.1% nisin TBARS = thiobarbituric acid reactive substances MDA = malondialdehyde TBA = thiobarbituric acid DTNB = 5,5’-dithiobis-(2-nitrobenzoic acid) SDS = sodium dodecyl sulfate TVC = total viable count
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Journal Pre-proof 1
Introduction
Pork is one of the most consumed meat products worldwide (McGlone, 2013). However, its high levels of polyunsaturated fatty acids make it susceptible to oxidation and microbial spoilage, which leads to colour changes, off-flavours, and rancidity (Huang et al., 2015). Therefore, preventing oxidation and microbial contamination has been an important task for fresh pork preservation. Numerous innovative packaging technologies have been developed in meat preservation, such as vacuum packaging, modified atmosphere packaging, intelligent packaging, active packaging and edible packaging (Fang, Zhao, Warner, & Johnson, 2017; Gertzou, Karabagias, Drosos, & Riganakos, 2017; Karabagias, Badeka, & Kontominas, 2011; McMillin, 2017). Recently, edible packaging (edible film or coating) has attracted great attention from researchers. Compared with conventional packaging, edible films or coatings are directly applied on the surface of food to maintain food quality and extend shelf life. The main advantage is that edible coatings or films can meet consumers’ demand for convenience, food quality and safety (Embuscado & Huber, 2009). Besides, the edible packaging materials are mainly derived from natural food-grade materials, which have edible, non-toxic, bioactive and biodegradable properties simultaneously, that may not found in synthetic packaging materials (Hassan, Chatha, Hussain, Zia, & Akhtar, 2018).
Various natural materials have been used to develop edible packaging, mainly polysaccharides (e.g. alginate, pectin and chitosan), proteins (e.g. whey, collagen, gelatine) and lipids (e.g. wax) (Hassan, Chatha, Hussain, Zia, & Akhtar, 2018). Among them, chitosan has been extensively studied and applied in the food industry owing to the unique film-forming properties, low gas permeability, antioxidant activity against lipid oxidation, and more importantly, antimicrobial activity against bacteria and fungi (Elsabee & Abdou, 2013; No, Meyers, Prinyawiwatkul, & Xu, 2007). However, the disadvantage is that it has a relatively high moisture permeability (Elsabee & Abdou, 2013). Gelatine, derived from the degradation of bone or skin collagen of piscine or bovine, is another coating material which has been extensively researched (GomezGuillen et al., 2009). It has been reported that chitosan and gelatine together can form a compact
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Journal Pre-proof structure by hydrogen bonding, which can effectively enhance the moisture permeability while improving the softness and flexibility of the film/coating (Pereda, Ponce, Marcovich, Ruseckaite, & Martucci, 2011). Several studies have shown that chitosan-gelatine film/coating can effectively improve the preservation of meat products. For example, Cardoso et al. (2016) found that a film composed of 0.5-1.0% chitosan and 3-6% gelatine preserved the colour and significantly inhibited the lipid oxidation of beef steaks in retail display. Farajzadeh, Motamedzadegan, Shahidi, & Hamzeh (2016) also observed that 1% chitosan-3% gelatine coating could extend the shelf-life of shrimps from 7 days to 13 days during cold storage.
In addition, incorporation of natural bioactive compounds such as gallic acid (Fang, Lin, Warner, & Ha, 2018), nisin (Cao, Warner, & Fang, 2019) and grape seed extract (Sivarooban, Hettiarachchy, & Johnson, 2008) has been an effective way to further enhance the preservation performance of the edible coating/film. Nisin is a bacteriocin produced by Lactococcus lactis and has been widely used in the food industry due to its ability to inhibit various of Grampositive bacteria such as lactic acid bacteria, which is a major cause of meat spoilage during cold storage (Punyauppa-path, Phumkhachorn, & Rattanachaikunsopon, 2015). Grape seed extract is another natural material and is well known for its high levels of phenolic contents such as phenolic acids, flavonoids and tannins, which possesses potent antimicrobial and antioxidant activities, and has a potential to be utilised as a functional food ingredient to improve food safety and quality (Nowshehri, Bhat, & Shah, 2015).
However, to our knowledge, no research has investigated the chitosan-gelatine edible coating incorporating grape seed extract and/or nisin on fresh meat preservation. Therefore, the objective of this study was to develop an edible coating system by incorporating grape seed extract and/or nisin into the chitosan-gelatine solution, and investigate its effect on the fresh pork quality during cold storage, which has the potential to be developed as a practical commercial method for fresh meat preservation.
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Journal Pre-proof 2 2.1
Material and methods Materials
Chitosan (degree of deacetylation > 90.0%), gelatine powder, and grape seed extract (proanthocyanidins > 95%) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China), Sciyu Biotech, Co. Ltd. (Xi’an, China) and Thermo Fisher Scientific (Scoresby, VIC, Australia) respectively. Plate count agar was obtained from Becton & Dickinson (North Ryde, NSW, Australia). Nisin (from Lactococcus lactis, 106 IU/g in 2.5% balance sodium chloride) and other chemicals used in the study were purchased from SigmaAldrich (Castle Hill, NSW, Australia). All reagents are of analytical grade.
2.2
Preparation of coating solutions
Chitosan was dissolved in 1.5% (v/v) acetic acid to prepare 1% (w/v) chitosan solution (CHI). Gelatine was soaked and swelled in distilled water overnight to prepare a 3% (w/v) gelatine solution (GEL). Nisin stock solution (105 IU/mL) (NIS) was prepared by dissolving nisin in 0.02 M HCl. Grape seed extract (GSE) powder was dissolved in distilled water to prepare a 0.5% (w/v) GSE solution. Five different coating solutions were prepared according to the formulations in Table 1. The chitosan-gelatine coating solutions were mixed at 70 ℃ with stirring for 10 min. After cooling to room temperature, NIS and/or GSE were added and thoroughly mixed. The pH of all coating solutions was adjusted to pH 5.6±0.1 by sodium bicarbonate to obtain the final coating solutions.
2.3
Preparation and coating of pork loin samples
Twenty fresh boneless pork loins were purchased from Shing Hing Wholesale (Keon Park, VIC, Australia) at 24 h post-mortem. Pork loins were trimmed to remove surrounding fat and connective tissues and then cut into 5 × 10 cm strips of 5 cm thickness. A total of 90 pork loin pieces were randomly chosen and divided into 6 treatment groups for the 5 sampling days, and three (n = 3) pieces were measured for each treatment on each sampling day. For the coating
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Journal Pre-proof process, each of the pork loin pieces was individually immersed in coating solutions for 30 s, removed from the solution using tongs, air-dried, and then packed in plastic trays with one absorbent pad placed underneath the sample. Pork loin pieces without any coating solution were used as control (CON). All trays were sealed with oxygen-impermeable film (Cryovac lid 1050/1051 film, Sealed Air, Fawkner, VIC, Australia) using a Multivac T200 packaging machine (Sepp Haggenmüller GmbH and Co., Germany). The headspace atmosphere in the plastic trays was air and not modified. All packed pork samples were stored in a double flat glass door upright fridge with LED display (Bromic Refrigeration, Ingleburn, NSW, Australia) at 4 °C, and rotated between shelves once a day to ensure consistent light exposure. Pork samples were measured on day 0, 5, 10, 15, 20 for pH, colour, lipid oxidation, protein oxidation, and total microbial account. The cutting, application of films, packaging and handling process of the pork loin samples was conducted at our Meat Science Lab with the room temperature of 6 ± 1 °C. Each coating formulation was repeated three times. 2.4
pH measurement
After removing the coating surface, the interior pH of pork samples was measured by an Ionode IJ44 spearhead pH probe with temperature compensation and WP-80 pH-mV meter (both from TPS Pty Ltd., Brisbane, QLD, Australia) (Cao et al. 2019). The pH meter was calibrated by pH 4 and 7 buffers before use.
2.5
Colour measurement
The colour of pork samples was tested by a colour sensor (Nix Pro Color Sensor, Nix Sensor Ltd., Ontario, Canada), followed the method of Holman, Collins, Kilgannon, & Hopkins (2018). The sensor had a 14.0 mm aperture and 45/0° measuring geometry. Illuminant D65 and 10° standard observer settings were used. The lens of the sensor was directly attached to the surface of the samples, and the CIE L* (lightness), a* (redness) and b* (yellowness) values were measured directly on the sample surface without removing the coating. For each treatment, 3
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Journal Pre-proof pieces of pork loin were measured, and each was measured in 3 different locations, so a total of 9 data were collected.
2.6
Lipid oxidation
Lipid oxidation of pork samples was evaluated based on the thiobarbituric acid reactive substances (TBARS) assay described by Sorensen & Jorgensen (1996) with some modification. Briefly, 5.0 g pork sample and 15 mL trichloroacetic acid (TCA) solution (7.5% TCA, 0.1% propyl gallate and 0.1% ethylenediaminetetraacetic acid) were blended using a homogeniser (Ultra-turrax T25 digital disperser, IKA Labortechnik, Germany) at 13500 rpm for 1 min. The mixture was then filtered through a Whatman No. 1 filter paper and 2 mL of the filtrate was mixed with 2 mL 0.02 M thiobarbituric acid (TBA), incubated at 100 ℃ for 40 min, and then cooled with cold water. The absorbance was measured by a Shimadzu UV-1800 UV-Vis spectrophotometer (Kyoto, Japan) at 532 nm against a blank sample (2 mL of the TCA solution with 2 mL 0.02 M TBA solution). A standard curve of 1,1,3,3-tetraethoxypropane (0 to 5 μM) was used to calculate the amount of malondialdehyde (MDA) produced. The TBARS value was reported as mg MDA (equivalent)/kg sample.
2.7
Protein oxidation
The protein oxidation was determined using 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB)based method proposed by Ellman (1959) and modified by Bao & Ertbjerg (2015). Briefly, 1.0 g pork sample was homogenised with 25 mL 5% (w/v) sodium dodecyl sulfate (SDS) in 0.10 M Tris buffer (pH 8.0) solution at 13500 rpm for 30 s. The homogenate was heated at 80 ℃ in a water bath for 30 min, and then cooled and filtered through a Whatman No. 1 filter paper. The filtrate was diluted five times with 5% (w/v) SDS in 0.10 M Tris buffer (pH 8.0) solution to determine the protein content; the absorbance was measured by the UV-Vis spectrophotometer at 280 nm, and bovine serum albumin was used as the standard (0 to 3 mg/mL). For the determination of the thiol group content, an aliquot of 0.5 mL filtrate was mixed with 2 mL 0.1 M Tris buffer (pH 8.0) and 0.5 mL 10 mM DTNB, and incubated in the 8
Journal Pre-proof dark at room temperature for 30 min. The absorbance was measured by the UV-Vis spectrophotometer at 412 nm against a blank sample (0.5 mL 5% SDS in 0.10 M Tris buffer, 2 mL 0.1 M Tris buffer (pH 8.0) and 0.5 mL 10 mM DTNB), and L-cysteine (0 to 1050 μM) was used as the standard. The protein oxidation was determined as the concentration of the thiol groups in the meat sample and was calculated by dividing the thiol group content by the protein content and reported as nmol thiol/mg protein.
2.8
Microbial analysis
The total viable count (TVC) was determined using plate count agar to evaluate the microbial growth in pork samples. About 25 g of pork and 225 mL 0.1% peptone water were transferred into a sterile stomacher bag and homogenised for 1 min by a stomacher (Interscience-BagMixer 400, St Nom, France). After stomaching, the liquid mixture was used to make ten-fold serial dilutions with 0.1% peptone solution, and 100 μL of each dilution was spread on an agar plate and then incubated in an aerobic condition at 37 °C for 48 h. All operations were conducted under aseptic conditions. The results were expressed as log CFU/g meat.
2.9
Statistical analysis
Three replicates (pork loin samples) were prepared for each coating treatment for each day. Unless otherwise stated, each sample was measured in duplicates (a total of 6 data was collected for each test). All data were expressed as mean ± standard error (SE), and the statistical analysis of data was performed using Minitab software (Minitab Windows, version 17, Sydney, Australia). The significant difference of means of each test (pH, colour, thiol groups, TBARS, and TVC) between six different treatments (five coating solutions and one control, Table 1) and days (0, 5, 10, 15, 20) were determined using one-way ANOVA with Turkey grouping at 95% confidence level.
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Journal Pre-proof 3 3.1
Results and discussion pH value
pH value is an important indicator of fresh meat quality. The pH of fresh pork normally ranges from 5.10 to 6.36, and a pH range of 5.7 to 6.1 is more likely to be favoured by consumers (Kim et al., 2016; Wright et al., 2005). The initial pH value (at day 0) of fresh pork was 5.80, and the value gradually decreased during the first 15 days of cold storage. However, no significant difference was observed among all samples for the first 10 days (Figure 1). The slow decline of the pH values may be due to the accumulation of lactic acid bacteria, and thus the production of lactic acid in pork samples (Pearce, Rosenvold, Andersen, & Hopkins, 2011).
However, pH changes occurred from day 15 to day 20, and the pH values of the pork loin were significantly influenced (P < 0.05) by the coating treatments (Figure 1). Results showed that the pH value of non-coated pork samples (CON, pH = 5.97) was dramatically increased at day 20, which could be due to the microbial action and accumulation of nitrogen compounds such as ammonia and trimethylamine in the pork samples (Arancibia, Lopez-Caballero, GomezGuillen, & Montero, 2015; Hebard, 1982). Pork with a pH above 5.8 is more susceptible to deterioration and contamination by microorganisms, and the increase in pH indicates that the pork begins to putrefy (Holmer et al., 2009). At day 20, the pH values of all coated pork samples were much lower than the CON sample, indicating that the pork was better preserved with the coating, probably due to the effective antimicrobial activity of chitosan toward various microorganisms including fungi, yeast and bacteria (No, Meyers, Prinyawiwatkul, & Xu, 2007). Chitosan or chitosan-gelatine based coating could minimise pH change and prevent meat deterioration, as was previously observed in chitosan-gelatine coated shrimp and gelatine coated rainbow trout fillet (Farajzadeh, Motamedzadegan, Shahidi, & Hamzeh, 2016; Hosseini, Rezaei, Zandi, & Ghavi, 2013).
However, no significant difference (P > 0.05) was observed among all the coated pork samples at day 20 (Figure 1), suggesting the incorporation of GSE and/or nisin did not affect the pH
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Journal Pre-proof value of the coated meat during storage. In addition, it should be noted that the pH values of nisin incorporated coating samples (CHI-GEL-NIS and CHI-GEL-GSE-NIS) were increased from day 15 to 20, though the mechanism requires further investigation.
3.2
Colour changes
Surface colour is an important visual quality indicator of fresh meat, and CIELAB colour space (L*, a* and b*) is the widely used method to assess the colour quality, with higher L*, a* and b* values indicating higher brightness, red and yellow, respectively, and vice versa (Pathare, Opara, & Al-Said, 2013). The initial colour of the uncoated pork loin samples was measured at day 0, which were L*= 54.0, a*= 3.7, b*= 3.8, and the colour of coated pork samples was measured from day 5 to 20 (Table 2). The results showed that the L* value of the uncoated pork sample was generally higher than that of the coated pork samples and had an increasing trend over the 20 days storage, which suggested a “paler” colour of the control sample (Cao et al., 2019). However, no obvious trend was found among different coating samples (Table 2).
Redness (a*) of meat is commonly used by consumers as an indicator of the freshness of the meat, and high redness values generally indicate fresher meat (AMSA, 2012). The a* values of the coated pork samples were significantly higher (P < 0.05) than that of the CON sample, and all coated samples had the highest a* values (6.4-10.0) at day 5 and then decreased thereafter, while the CON sample experienced a gradual decrease from 3.7 to 2.8 throughout the storage (Table 2). Similar findings were also reported on chitosan coated pork in modified atmosphere packaging (Fang, Lin, Warner, & Ha, 2018). The colour of the flesh meat is mainly determined by the haemoglobin and myoglobin pigments in meat, where myoglobin (deoxymyoglobin, oxymyoglobin and metmyoglobin) plays a key role in the colour of meat and is closely related to the a* value (Kim et al., 2010). During storage, as meat deteriorates, deoxymyoglobin (purple colour) and/or oxymyoglobin (red colour) are oxidised to metmyoglobin (brown colour), and thus meat colour shifts to lower redness and higher yellowness colour and become unattractive (Brewer, Zhu, Bidner, Meisinger, & McKeith, 2001;
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Journal Pre-proof Fang, Lin, Warner, & Ha, 2018). The results showed that the yellowness (b*) of the coated pork samples were higher than the CON sample, and all samples had higher b* values after 20 days of storage (Table 2). The higher b* values in the coated pork samples may be partially due to the colour of the coating itself, which was slightly yellow.
In addition, it was noticed that the pork samples coated with GSE (CHI-GEL-GSE and CHIGEL-GSE-NIS) had the highest a* and b* values amongst all groups, indicating that the sample colour is redder and yellower (Table 2). Consistent results were also reported in GSE incorporated chitosan edible films and GSE incorporated soy protein edible film (Rubilar et al., 2013; Sivarooban, Hettiarachchy, & Johnson, 2008). This may be due to the high levels of phenolic compounds in the GSE, and some phenolic compounds such as anthocyanins are well known natural pigments that are responsible for the red to blue colour (depending on pH), which may have contributed to the redder meat colour (He & Giusti, 2010; Shi, Yu, Pohorly, & Kakuda, 2003). In addition, the antioxidant activity of GSE phenolic compounds may inhibit the oxidation of meat, which could further enhance the colour (redness) of meat (Carpenter, O'Grady, O'Callaghan, O'Brien, & Kerry, 2007).
3.3
Lipid oxidation
The lipid oxidation of the pork sample was evaluated by the TBARS value, which measures the amount of secondary products produced during the lipid oxidation, and a higher TBARS value indicates a higher degree of lipid oxidation. The off-flavours in pork can generally be detectable by consumers when the TBARS value is higher than the threshold value of 0.5 mg MDA/kg (Sheard et al., 2000). In general, the TBARS values of all pork samples increased significantly over the 20 days’ storage period, particularly for the CON sample, whose TBARS value was dramatically increased from 0.053 mg MDA/kg on day 0 to 0.343 mg MDA/kg on day 20 (Figure 2). The TBARS value of CON sample was significantly higher (P < 0.05) than coated groups from day 5, and experienced an exponential growth thereafter, and was close to the threshold value at day 20, suggesting high lipid oxidation occurred in the CON sample. 12
Journal Pre-proof This is because the CON sample was directly exposed to the oxygen that was sealed in the packaging tray and lipid oxidation occurred (Bao & Ertbjerg, 2015), and the reactive oxygen species generated during the lipid oxidation would further accelerate the oxidation process (Burcham & Kuhan, 1996).
On the other hand, the TBARS values of all coated pork samples had much slower increase during the storage, and the values were well below the rancidity threshold at day 20 (Figure 2), indicating the coatings were effective to retard the lipid oxidation of pork samples. The antioxidant activity of chitosan-based coating in meat has been well documented. For instance, Wu et al. (2016) observed that 1.5 % chitosan coating could effectively inhibit lipid oxidation of refrigerated pacific mackerel fillets, and Farajzadeh, Motamedzadegan, Shahidi, & Hamzeh (2016) reported that 1% chitosan and 3% gelatine coating could significantly prevent lipid oxidation of shrimps under cold storage. It has been proposed that the antioxidant capacity of chitosan coatings may be attributed to its low oxygen permeability, which reduces or prevents the exposure of the samples to oxygen, as well as its ability to chelate metal ions such as copper and iron which inhibits catalytic activity and further delays the lipid oxidation (Chen, Zheng, Wang, Lee, & Park, 2002).
Among the coated samples, the GSE incorporated samples (CHI-GEL-GSE and CHI-GELGSE-NIS) had the lowest (P < 0.05) TBARS values at day 20 (Figure 2), indicating that addition of GSE into the coating significantly inhibited the lipid oxidation. The possible reason is that the high levels of phenolic compounds in the GSE are acting as a strong antioxidant, scavenging free radicals and hindering the oxidation chain reactions (Bagchi et al., 2000). This result is in accordance with the study of Li, Li, Hu, & Li (2013), who reported that incorporating 0.2% GSE into 1.5% chitosan coating significantly lowered the TBARS value in refrigerated red drum fillets. In contrast, there was no significant difference (P > 0.05) between the TBARS values of nisin incorporated coating (CHI-GEL-NIS and CHI-GEL-GSE-NIS) and CHI-GEL (Figure 2). This result suggests that addition of nisin into the CHI-GEL coating did
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Journal Pre-proof not affect the meat lipid oxidation, which is in line with those of previous studies (Cao, Warner, & Fang, 2019; Lu, Ding, Ye, & Liu, 2010).
3.4
Protein oxidation
The concentration of free thiol groups is commonly used as an indicator of protein oxidation in meat products. Amino acids such as L-cysteine are easily oxidised and form disulphide cross-links, resulting in the loss of the free thiol group. Therefore, a lower thiol group value indicates a greater reduction of the thiol group, and consequently greater protein oxidation (Winther & Thorpe, 2014). Figure 3 shows that the free thiol group values of all pork samples were significantly decreased during the 20 days of cold storage. Especially the CON sample, whose free thiol group value was decreased rapidly from 72.28 nmol thiol/mg protein on day 0 to 40.95 nmol thiol/mg protein on day 20, and was significantly lower than that of coated pork samples. This suggests that non-coated pork had a higher degree of protein oxidation than coating-protected samples, which may be due to the direct reaction of meat with the oxygen in the package. In other words, the coating treatments showed good protection against pork protein oxidation.
During the first five days of storage, the free thiol group values of all samples did not change much, but significant differences (P < 0.05) were observed between samples from day 10, and the difference was accelerated thereafter (Figure 3). This phenomenon was also found in lipid oxidation, as illustrated in Figure 2 and discussed above. This is because both lipid oxidation and protein oxidation are initiated by reactive oxygen species and highly correlated, and can influence each other (Lund, Heinonen, Baron, & Estevez, 2011; Min & Ahn, 2005). The link between lipid and myoglobin oxidation has been identified to be hydroxynonenal (HNE) where HNE is a product of lipid oxidation and can accelerate myoglobin oxidation (Suman & Joseph, 2013). Furthermore, the intermediate compounds generated during the protein and lipid oxidations could further accelerate oxidative chain reactions (Burcham & Kuhan, 1996).
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Journal Pre-proof In the coated pork samples, GSE and/or nisin incorporated coatings generally had higher thiol group values on day 15 and 20, and CHI-GEL-GSE coated pork sample had the highest thiol value (Figure 3), suggesting that incorporating GSE into the chitosan-gelatine coating could effectively prevent the pork protein oxidation. In contrast, Jongberg, Skov, Torngren, Skibsted, & Lund (2011) reported that beef samples treated with white grape extracts showed significantly lower thiol content compared to untreated samples. They proposed that polyphenols in grapes could interact with thiol groups to form adducts, which interfered with the results. Nevertheless, many studies have shown that incorporation of natural antioxidants such as gallic acid (Fang, Lin, Warner, & Ha, 2018), lemon extract (Lara, Gutierrez, Timón, & Andrés, 2011) and persimmon peel extract (Choe, Kim, & Kim, 2017) into meat or packaging could inhibit the protein oxidation. Combined with the above lipid oxidation results, it was demonstrated that the GSE incorporated chitosan-gelatine coating could effectively inhibit pork oxidation during cold storage.
3.5
Microbial analysis
The change of TVC in pork samples is shown in Figure 4. The TVC value of the fresh pork sample was 2.51 log CFU/g on day 0, and the TVC values of all coated and non-coated pork samples increased significantly during the 20 days’ cold storage. Not surprisingly, the TVC of non-coated sample (CON) showed the fastest increase during the storage and was significantly higher (P < 0.05) than all other samples since day 5. The elevated microbial count in pork samples during the cold storage may be caused by the growth of psychrotrophic bacteria such as Carnobacterium spp. and Pseudomonas spp. (Ercolini, Russo, Nasi, Ferranti, & Villani, 2009). According to Huang et al. (2014), the TVC value of 7 log CFU/g can be used as the threshold for pork quality, and pork may be spoiled by microorganisms if TVC is higher than this value. All the samples in this study were below this threshold after the 20-day storage, indicating the conventional tray packaging at 4 °C alone is effective to prevent the pork from microbial spoilage. However, it should be noted that the TVC of CON sample was 6.30 log
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Journal Pre-proof CFU/g on day 20, which was very close to the threshold and would likely have spoiled not long after 20 days.
Compared with the non-coated sample (CON), the chitosan coating (CHI) effectively inhibited the growth of microorganisms from the fifth day (Figure 4). This result is in agreement with Fang, Lin, Warner, & Ha (2018) and Cao, Warner, & Fang (2019), who examined 2% chitosan coating of fresh pork samples in a high-oxygen modified atmosphere packaging. Chitosan is an excellent natural antimicrobial agent. Goy, Britto, & Assis (2009) stated that chitosan is able to inhibit the growth of a broad spectrum of microorganisms, including Gram-negative (such as Escherichia coli and Pseudomonas spp.) and Gram-positive (such as Listeria monocytogenes and lactic acid bacteria) bacteria and fungi (such as Botrytis cinereal and Drechstera sorokiana). Elsabee & Abdou (2013) proposed that chitosan could interact with the negative charges located on the microbial cell membrane, causing the breakdown of the membrane and leaching of intracellular substances. Furthermore, the TVC of the chitosangelatine coated sample (CHI-GEL) was significantly lower (P < 0.05) than that of chitosan coating (CHI) alone on day 15 and 20 (Figure 4), indicating a lower antimicrobial activity in the CHI-GEL sample. Elsabee & Abdou (2013) explained that chitosan and gelatine could form a compact structure and reduce the moisture permeability, which may further enhance the antimicrobial effect of the coating, which most likely explains our results.
On the other hand, no TVC difference (P > 0.05) was observed between the chitosan-gelatine coated sample (CHI-GEL) and GSE (0.5%) and/or nisin (500 UI/mL) incorporated coating samples (CHI-GEL-GSE, CHI-GEL-NIS and CHI-GEL-GSE-NIS) on day 15 and 20 (Figure 4), indicating that addition of GSE and/or nisin did not further improve the antimicrobial effect of the coating. Similar results were also found by Lu, Ding, Ye, & Liu (2010) who used nisin (2000 IU/mL) incorporated into alginate-calcium coating on fresh northern snakehead fish fillets. However, it is worth noting that nisin is well known for its excellent antimicrobial activity, against various Gram-positive bacteria including Bacillus, Clostridium, Listeria,
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Journal Pre-proof Staphylococcus, and Enterococcus spp., and has been widely used in the food industry (Punyauppa-path, Phumkhachorn, & Rattanachaikunsopon, 2015). Also, incorporation of nisin (0.2%) into chitosan (2%) coating has been shown to have a significantly enhanced antimicrobial effect on fresh pork preservation (Cao, Warner, & Fang, 2019). The reason for such inconsistency is not clear, but it may be related to the coating matrix and that chitosan and gelatine form a compact structure which affects the functional property of nisin or GSE, as well as the environmental conditions and types of bacteria present, which requires further investigation.
4
Conclusion
The present study developed several chitosan and gelatine based edible coatings for fresh pork preservation. Chitosan coating (CHI) alone effectively extended the shelf-life of pork by minimising the pH change, preventing lipid and protein oxidation and inhibiting microbial growth during the 20 days of cold storage at 4 °C. These preservative effects were enhanced by combining chitosan with gelatine (CHI-GEL), possibly owing to the compact structure formed between chitosan and gelatine. Incorporating GSE into the chitosan-gelatine coating further enhanced antioxidant activity against protein and lipid oxidation, and also conferred meat with a redder and yellower colour, which may be attributed to the phenolic compounds present in the GSE. However, incorporating nisin into the coating had no significant effect on the antimicrobial and lipid and protein oxidation, and further work is required to uncover the underlying mechanism. Overall, the CHI-GEL-GSE formulation showed the best performance on fresh pork preservation and could be developed as a hurdle technology in preserving fresh meat products to extend the shelf life while improving the safety and quality.
5
Conflict of interest
The authors declare no conflict of interest.
17
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References
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Journal Pre-proof 838-846. doi:10.1016/j.bbagen.2013.03.031 Wright, L. I., Scanga, J. A., Belk, K. E., Engle, T. E., Tatum, J. D., Person, R. C., McKenna, D. R., Griffin, D. B., McKeith, F. K., Savell, J. W., & Smith, G. C. (2005). Benchmarking value in the pork supply chain: Characterization of US pork in the retail marketplace. Meat Science, 71(3), 451-463. doi:10.1016/j.meatsci.2005.04.024 Wu, C., Li, Y., Wang, L., Hu, Y., Chen, J., Liu, D., & Ye, X. (2016). Efficacy of chitosan-gallic acid coating on shelf life extension of refrigerated pacific mackerel fillets. Food and Bioprocess Technology, 9(4), 675-685.
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Journal Pre-proof Table captions: Table 1: Formulation of edible coating solutions. Table 2. Changes of colour attributes (L*, a*, b*) of pork loin samples during cold storage (4 ℃) for 20 days.
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Journal Pre-proof Table 1: Formulation of edible coating solutions. Treatments code
Formulae
CON
Control group, pork samples not coated with coating solutions
CHI
1% chitosan
CHI-GEL
1% chitosan + 3% gelatine
CHI-GEL-GSE
1% chitosan + 3% gelatine + 0.5% GSE
CHI-GEL-NIS
1% chitosan + 3% gelatine + 0.1% nisin
CHI-GEL-GSE-NIS
1% chitosan + 3% gelatine + 0.5% GSE + 0.1% nisin
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Journal Pre-proof Table 2. Changes of colour attributes (L*, a*, b*) of pork loin samples during cold storage (4 ℃) for 20 days. Attributes L*
a*
b*
a-d =
Coating treatment
Storage days 0
5
10
15
20
CON
54.0±0.5aB
58.4±1.1aA
58.5±1.8abA
57.8±1.2aA
57.6±0.9aA
CHI
54.0±0.5aA
49.8±1.1bA
55.7±1.8bcA
53.0±2.6abA
51.3±2.4bcA
CHI-GEL
54.0±0.5aB
50.0±1.9bB
61.0±1.9aA
53.2±2.4abB
58.2±1.3aA
CHI-GEL-GSE
54.0±0.5aA
55.0±1.7aA
56.5±1.3bcA
55.5±1.1abA
54.0±1.3abA
CHI-GEL-NIS
54.0±0.5aB
55.0±1.8aAB
58.6±1.3abA
52.6±1.0bC
46.3±3.5cD
CHI-GEL-GSE-NIS
54.0±0.5aA
54.9±0.7aA
53.8±0.7cA
53.6±1.6abA
49.4±2.2bcB
CON
3.7±0.2aA
3.2±0.2cB
2.6±0.3cC
2.0±0.3cC
2.8±0.4dC
CHI
3.7±0.2aB
6.4±0.4bA
6.1±0.7bA
6.4±0.4bcA
3.9±0.6dB
CHI-GEL
3.7±0.2aD
6.9±0.6bA
5.6±0.4bB
5.5±0.3bB
4.4±0.3cC
CHI-GEL-GSE
3.7±0.2aC
9.3±0.9aA
8.0±0.6aAB
7.5±0.3aB
7.6±0.3aB
CHI-GEL-NIS
3.7±0.2aB
6.2±0.4bA
6.3±0.5bA
6.5±0.7bcA
6.7±0.7abA
CHI-GEL-GSE-NIS
3.7±0.2aC
10.0±0.4aA
8.8±0.6aA
7.2±0.3aB
7.6±0.5aB
CON
3.8±0.2aB
4.6±0.2cA
4.7±0.4cA
4.5±0.3dA
4.4±0.6dA
CHI
3.8±0.2aC
4.8±0.2cB
6.5±0.7cA
6.3±0.2cA
6.0±0.3cA
CHI-GEL
3.8±0.2aB
6.3±0.2bcA
6.3±0.6cA
7.1±0.4bcA
6.3±0.3cA
CHI-GEL-GSE
3.8±0.2aB
9.7±1.0aA
10.1±0.9abA
9.1±0.4aA
9.5±0.6aA
CHI-GEL-NIS
3.8±0.2aB
7.0±1.1bA
8.5±0.5bA
8.1±0.7abA
7.8±0.5bA
CHI-GEL-GSE-NIS
3.8±0.2aC
11.6±0.5aA
10.7±0.7aA
8.6±0.7aB
8.2±0.3bB
Values with different superscripts in the same column are significantly different (P < 0.05).
A-D =
Values with different superscripts in the same row are significantly different (P < 0.05).
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Journal Pre-proof Figure captions: Figure 1: Changes of pH value of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
Figure 2: Changes of lipid oxidation (TBARS values, mg MDA/kg meat) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
Figure 3: Changes of protein oxidation (free thiol group values, nmol thiol/ mg protein) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
Figure 4. Changes of total viable counts (TVC) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
25
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Figure 1: Changes of pH value of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
26
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Figure 2: Changes of lipid oxidation (TBARS values, mg MDA/kg meat) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
27
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Figure 3: Changes of protein oxidation (free thiol group values, nmol thiol/ mg protein) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
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Figure 4. Changes of total viable counts (TVC) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
29
Journal Pre-proof Highlight:
Grape seed extract (GSE)/nisin were loaded in chitosan-gelatine edible coatings
The edible coatings were applied to fresh pork during cold storage
The GSE edible coating enhanced antioxidant activity against meat oxidation
Incorporating nisin did not improve the antimicrobial and antioxidant activity
CHI-GEL-GSE coating formulation showed the best performance on meat preservation
Yun Xiong, Meng Chen, Robyn Dorothy Warner, Zhongxiang Fang PII:
S0956-7135(19)30607-3
DOI:
https://doi.org/10.1016/j.foodcont.2019.107018
Reference:
JFCO 107018
To appear in:
Food Control
Received Date:
05 August 2019
Accepted Date:
23 November 2019
Please cite this article as: Yun Xiong, Meng Chen, Robyn Dorothy Warner, Zhongxiang Fang, Incorporating nisin and grape seed extract in chitosan-gelatine edible coating and its effect on cold storage of fresh pork, Food Control (2019), https://doi.org/10.1016/j.foodcont.2019.107018
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof Incorporating nisin and grape seed extract in chitosan-gelatine edible coating and its effect on cold storage of fresh pork
Yun Xionga, Meng Chena, Robyn Dorothy Warner a, Zhongxiang Fanga* a
School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The
University of Melbourne, Parkville, VIC 3010, Australia
* Corresponding author: Dr Zhongxiang Fang School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, VIC 3010, Australia Email: [email protected]; Tel: +61 3 83445063
1
Journal Pre-proof Abstract: Fresh pork is a highly perishable food product and susceptible to oxidation and microbial spoilage. The objective of this study was to develop a chitosan-gelatine edible coating system incorporating grape seed extract and/or nisin and investigate its effect on the preservation of fresh pork during cold storage at 4 ℃ for 20 days. Results showed that 1% chitosan (CHI) effectively inhibited pork oxidation and microbial spoilage; 1% chitosan combined with 3% gelatine (CHI-GEL) enhanced these preservative effects; and incorporating 0.5% grape seed extract (CHI-GEL-GSE) further enhanced antioxidant activity against meat oxidation. However, incorporating nisin (NIS) into the coating (CHI-GEL-NIS and CHI-GELNIS-GSE) did not further improve the antimicrobial and antioxidant effects. The CHI-GELGSE formulation had the best performance on pork preservation, which suggested that it could be developed as a hurdle technology to preserve fresh meat. Keywords: pork; chitosan; gelatine; nisin; grape seed extract; edible coating.
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Journal Pre-proof Abbreviations: CON = control group, non-coated pork samples NIS = nisin GSE = grape seed extract CHI = 1% chitosan GEL =3% gelatine CHI-GEL = 1% chitosan + 3% gelatine CHI-GEL-GSE = 1% chitosan + 3% gelatine + 0.5% GSE CHI-GEL-NIS = 1% chitosan + 3% gelatine + 0.1% nisin CHI-GEL-GSE-NIS = 1% chitosan + 3% gelatine + 0.5% GSE + 0.1% nisin TBARS = thiobarbituric acid reactive substances MDA = malondialdehyde TBA = thiobarbituric acid DTNB = 5,5’-dithiobis-(2-nitrobenzoic acid) SDS = sodium dodecyl sulfate TVC = total viable count
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Journal Pre-proof 1
Introduction
Pork is one of the most consumed meat products worldwide (McGlone, 2013). However, its high levels of polyunsaturated fatty acids make it susceptible to oxidation and microbial spoilage, which leads to colour changes, off-flavours, and rancidity (Huang et al., 2015). Therefore, preventing oxidation and microbial contamination has been an important task for fresh pork preservation. Numerous innovative packaging technologies have been developed in meat preservation, such as vacuum packaging, modified atmosphere packaging, intelligent packaging, active packaging and edible packaging (Fang, Zhao, Warner, & Johnson, 2017; Gertzou, Karabagias, Drosos, & Riganakos, 2017; Karabagias, Badeka, & Kontominas, 2011; McMillin, 2017). Recently, edible packaging (edible film or coating) has attracted great attention from researchers. Compared with conventional packaging, edible films or coatings are directly applied on the surface of food to maintain food quality and extend shelf life. The main advantage is that edible coatings or films can meet consumers’ demand for convenience, food quality and safety (Embuscado & Huber, 2009). Besides, the edible packaging materials are mainly derived from natural food-grade materials, which have edible, non-toxic, bioactive and biodegradable properties simultaneously, that may not found in synthetic packaging materials (Hassan, Chatha, Hussain, Zia, & Akhtar, 2018).
Various natural materials have been used to develop edible packaging, mainly polysaccharides (e.g. alginate, pectin and chitosan), proteins (e.g. whey, collagen, gelatine) and lipids (e.g. wax) (Hassan, Chatha, Hussain, Zia, & Akhtar, 2018). Among them, chitosan has been extensively studied and applied in the food industry owing to the unique film-forming properties, low gas permeability, antioxidant activity against lipid oxidation, and more importantly, antimicrobial activity against bacteria and fungi (Elsabee & Abdou, 2013; No, Meyers, Prinyawiwatkul, & Xu, 2007). However, the disadvantage is that it has a relatively high moisture permeability (Elsabee & Abdou, 2013). Gelatine, derived from the degradation of bone or skin collagen of piscine or bovine, is another coating material which has been extensively researched (GomezGuillen et al., 2009). It has been reported that chitosan and gelatine together can form a compact
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Journal Pre-proof structure by hydrogen bonding, which can effectively enhance the moisture permeability while improving the softness and flexibility of the film/coating (Pereda, Ponce, Marcovich, Ruseckaite, & Martucci, 2011). Several studies have shown that chitosan-gelatine film/coating can effectively improve the preservation of meat products. For example, Cardoso et al. (2016) found that a film composed of 0.5-1.0% chitosan and 3-6% gelatine preserved the colour and significantly inhibited the lipid oxidation of beef steaks in retail display. Farajzadeh, Motamedzadegan, Shahidi, & Hamzeh (2016) also observed that 1% chitosan-3% gelatine coating could extend the shelf-life of shrimps from 7 days to 13 days during cold storage.
In addition, incorporation of natural bioactive compounds such as gallic acid (Fang, Lin, Warner, & Ha, 2018), nisin (Cao, Warner, & Fang, 2019) and grape seed extract (Sivarooban, Hettiarachchy, & Johnson, 2008) has been an effective way to further enhance the preservation performance of the edible coating/film. Nisin is a bacteriocin produced by Lactococcus lactis and has been widely used in the food industry due to its ability to inhibit various of Grampositive bacteria such as lactic acid bacteria, which is a major cause of meat spoilage during cold storage (Punyauppa-path, Phumkhachorn, & Rattanachaikunsopon, 2015). Grape seed extract is another natural material and is well known for its high levels of phenolic contents such as phenolic acids, flavonoids and tannins, which possesses potent antimicrobial and antioxidant activities, and has a potential to be utilised as a functional food ingredient to improve food safety and quality (Nowshehri, Bhat, & Shah, 2015).
However, to our knowledge, no research has investigated the chitosan-gelatine edible coating incorporating grape seed extract and/or nisin on fresh meat preservation. Therefore, the objective of this study was to develop an edible coating system by incorporating grape seed extract and/or nisin into the chitosan-gelatine solution, and investigate its effect on the fresh pork quality during cold storage, which has the potential to be developed as a practical commercial method for fresh meat preservation.
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Journal Pre-proof 2 2.1
Material and methods Materials
Chitosan (degree of deacetylation > 90.0%), gelatine powder, and grape seed extract (proanthocyanidins > 95%) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China), Sciyu Biotech, Co. Ltd. (Xi’an, China) and Thermo Fisher Scientific (Scoresby, VIC, Australia) respectively. Plate count agar was obtained from Becton & Dickinson (North Ryde, NSW, Australia). Nisin (from Lactococcus lactis, 106 IU/g in 2.5% balance sodium chloride) and other chemicals used in the study were purchased from SigmaAldrich (Castle Hill, NSW, Australia). All reagents are of analytical grade.
2.2
Preparation of coating solutions
Chitosan was dissolved in 1.5% (v/v) acetic acid to prepare 1% (w/v) chitosan solution (CHI). Gelatine was soaked and swelled in distilled water overnight to prepare a 3% (w/v) gelatine solution (GEL). Nisin stock solution (105 IU/mL) (NIS) was prepared by dissolving nisin in 0.02 M HCl. Grape seed extract (GSE) powder was dissolved in distilled water to prepare a 0.5% (w/v) GSE solution. Five different coating solutions were prepared according to the formulations in Table 1. The chitosan-gelatine coating solutions were mixed at 70 ℃ with stirring for 10 min. After cooling to room temperature, NIS and/or GSE were added and thoroughly mixed. The pH of all coating solutions was adjusted to pH 5.6±0.1 by sodium bicarbonate to obtain the final coating solutions.
2.3
Preparation and coating of pork loin samples
Twenty fresh boneless pork loins were purchased from Shing Hing Wholesale (Keon Park, VIC, Australia) at 24 h post-mortem. Pork loins were trimmed to remove surrounding fat and connective tissues and then cut into 5 × 10 cm strips of 5 cm thickness. A total of 90 pork loin pieces were randomly chosen and divided into 6 treatment groups for the 5 sampling days, and three (n = 3) pieces were measured for each treatment on each sampling day. For the coating
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Journal Pre-proof process, each of the pork loin pieces was individually immersed in coating solutions for 30 s, removed from the solution using tongs, air-dried, and then packed in plastic trays with one absorbent pad placed underneath the sample. Pork loin pieces without any coating solution were used as control (CON). All trays were sealed with oxygen-impermeable film (Cryovac lid 1050/1051 film, Sealed Air, Fawkner, VIC, Australia) using a Multivac T200 packaging machine (Sepp Haggenmüller GmbH and Co., Germany). The headspace atmosphere in the plastic trays was air and not modified. All packed pork samples were stored in a double flat glass door upright fridge with LED display (Bromic Refrigeration, Ingleburn, NSW, Australia) at 4 °C, and rotated between shelves once a day to ensure consistent light exposure. Pork samples were measured on day 0, 5, 10, 15, 20 for pH, colour, lipid oxidation, protein oxidation, and total microbial account. The cutting, application of films, packaging and handling process of the pork loin samples was conducted at our Meat Science Lab with the room temperature of 6 ± 1 °C. Each coating formulation was repeated three times. 2.4
pH measurement
After removing the coating surface, the interior pH of pork samples was measured by an Ionode IJ44 spearhead pH probe with temperature compensation and WP-80 pH-mV meter (both from TPS Pty Ltd., Brisbane, QLD, Australia) (Cao et al. 2019). The pH meter was calibrated by pH 4 and 7 buffers before use.
2.5
Colour measurement
The colour of pork samples was tested by a colour sensor (Nix Pro Color Sensor, Nix Sensor Ltd., Ontario, Canada), followed the method of Holman, Collins, Kilgannon, & Hopkins (2018). The sensor had a 14.0 mm aperture and 45/0° measuring geometry. Illuminant D65 and 10° standard observer settings were used. The lens of the sensor was directly attached to the surface of the samples, and the CIE L* (lightness), a* (redness) and b* (yellowness) values were measured directly on the sample surface without removing the coating. For each treatment, 3
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Journal Pre-proof pieces of pork loin were measured, and each was measured in 3 different locations, so a total of 9 data were collected.
2.6
Lipid oxidation
Lipid oxidation of pork samples was evaluated based on the thiobarbituric acid reactive substances (TBARS) assay described by Sorensen & Jorgensen (1996) with some modification. Briefly, 5.0 g pork sample and 15 mL trichloroacetic acid (TCA) solution (7.5% TCA, 0.1% propyl gallate and 0.1% ethylenediaminetetraacetic acid) were blended using a homogeniser (Ultra-turrax T25 digital disperser, IKA Labortechnik, Germany) at 13500 rpm for 1 min. The mixture was then filtered through a Whatman No. 1 filter paper and 2 mL of the filtrate was mixed with 2 mL 0.02 M thiobarbituric acid (TBA), incubated at 100 ℃ for 40 min, and then cooled with cold water. The absorbance was measured by a Shimadzu UV-1800 UV-Vis spectrophotometer (Kyoto, Japan) at 532 nm against a blank sample (2 mL of the TCA solution with 2 mL 0.02 M TBA solution). A standard curve of 1,1,3,3-tetraethoxypropane (0 to 5 μM) was used to calculate the amount of malondialdehyde (MDA) produced. The TBARS value was reported as mg MDA (equivalent)/kg sample.
2.7
Protein oxidation
The protein oxidation was determined using 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB)based method proposed by Ellman (1959) and modified by Bao & Ertbjerg (2015). Briefly, 1.0 g pork sample was homogenised with 25 mL 5% (w/v) sodium dodecyl sulfate (SDS) in 0.10 M Tris buffer (pH 8.0) solution at 13500 rpm for 30 s. The homogenate was heated at 80 ℃ in a water bath for 30 min, and then cooled and filtered through a Whatman No. 1 filter paper. The filtrate was diluted five times with 5% (w/v) SDS in 0.10 M Tris buffer (pH 8.0) solution to determine the protein content; the absorbance was measured by the UV-Vis spectrophotometer at 280 nm, and bovine serum albumin was used as the standard (0 to 3 mg/mL). For the determination of the thiol group content, an aliquot of 0.5 mL filtrate was mixed with 2 mL 0.1 M Tris buffer (pH 8.0) and 0.5 mL 10 mM DTNB, and incubated in the 8
Journal Pre-proof dark at room temperature for 30 min. The absorbance was measured by the UV-Vis spectrophotometer at 412 nm against a blank sample (0.5 mL 5% SDS in 0.10 M Tris buffer, 2 mL 0.1 M Tris buffer (pH 8.0) and 0.5 mL 10 mM DTNB), and L-cysteine (0 to 1050 μM) was used as the standard. The protein oxidation was determined as the concentration of the thiol groups in the meat sample and was calculated by dividing the thiol group content by the protein content and reported as nmol thiol/mg protein.
2.8
Microbial analysis
The total viable count (TVC) was determined using plate count agar to evaluate the microbial growth in pork samples. About 25 g of pork and 225 mL 0.1% peptone water were transferred into a sterile stomacher bag and homogenised for 1 min by a stomacher (Interscience-BagMixer 400, St Nom, France). After stomaching, the liquid mixture was used to make ten-fold serial dilutions with 0.1% peptone solution, and 100 μL of each dilution was spread on an agar plate and then incubated in an aerobic condition at 37 °C for 48 h. All operations were conducted under aseptic conditions. The results were expressed as log CFU/g meat.
2.9
Statistical analysis
Three replicates (pork loin samples) were prepared for each coating treatment for each day. Unless otherwise stated, each sample was measured in duplicates (a total of 6 data was collected for each test). All data were expressed as mean ± standard error (SE), and the statistical analysis of data was performed using Minitab software (Minitab Windows, version 17, Sydney, Australia). The significant difference of means of each test (pH, colour, thiol groups, TBARS, and TVC) between six different treatments (five coating solutions and one control, Table 1) and days (0, 5, 10, 15, 20) were determined using one-way ANOVA with Turkey grouping at 95% confidence level.
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Journal Pre-proof 3 3.1
Results and discussion pH value
pH value is an important indicator of fresh meat quality. The pH of fresh pork normally ranges from 5.10 to 6.36, and a pH range of 5.7 to 6.1 is more likely to be favoured by consumers (Kim et al., 2016; Wright et al., 2005). The initial pH value (at day 0) of fresh pork was 5.80, and the value gradually decreased during the first 15 days of cold storage. However, no significant difference was observed among all samples for the first 10 days (Figure 1). The slow decline of the pH values may be due to the accumulation of lactic acid bacteria, and thus the production of lactic acid in pork samples (Pearce, Rosenvold, Andersen, & Hopkins, 2011).
However, pH changes occurred from day 15 to day 20, and the pH values of the pork loin were significantly influenced (P < 0.05) by the coating treatments (Figure 1). Results showed that the pH value of non-coated pork samples (CON, pH = 5.97) was dramatically increased at day 20, which could be due to the microbial action and accumulation of nitrogen compounds such as ammonia and trimethylamine in the pork samples (Arancibia, Lopez-Caballero, GomezGuillen, & Montero, 2015; Hebard, 1982). Pork with a pH above 5.8 is more susceptible to deterioration and contamination by microorganisms, and the increase in pH indicates that the pork begins to putrefy (Holmer et al., 2009). At day 20, the pH values of all coated pork samples were much lower than the CON sample, indicating that the pork was better preserved with the coating, probably due to the effective antimicrobial activity of chitosan toward various microorganisms including fungi, yeast and bacteria (No, Meyers, Prinyawiwatkul, & Xu, 2007). Chitosan or chitosan-gelatine based coating could minimise pH change and prevent meat deterioration, as was previously observed in chitosan-gelatine coated shrimp and gelatine coated rainbow trout fillet (Farajzadeh, Motamedzadegan, Shahidi, & Hamzeh, 2016; Hosseini, Rezaei, Zandi, & Ghavi, 2013).
However, no significant difference (P > 0.05) was observed among all the coated pork samples at day 20 (Figure 1), suggesting the incorporation of GSE and/or nisin did not affect the pH
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Journal Pre-proof value of the coated meat during storage. In addition, it should be noted that the pH values of nisin incorporated coating samples (CHI-GEL-NIS and CHI-GEL-GSE-NIS) were increased from day 15 to 20, though the mechanism requires further investigation.
3.2
Colour changes
Surface colour is an important visual quality indicator of fresh meat, and CIELAB colour space (L*, a* and b*) is the widely used method to assess the colour quality, with higher L*, a* and b* values indicating higher brightness, red and yellow, respectively, and vice versa (Pathare, Opara, & Al-Said, 2013). The initial colour of the uncoated pork loin samples was measured at day 0, which were L*= 54.0, a*= 3.7, b*= 3.8, and the colour of coated pork samples was measured from day 5 to 20 (Table 2). The results showed that the L* value of the uncoated pork sample was generally higher than that of the coated pork samples and had an increasing trend over the 20 days storage, which suggested a “paler” colour of the control sample (Cao et al., 2019). However, no obvious trend was found among different coating samples (Table 2).
Redness (a*) of meat is commonly used by consumers as an indicator of the freshness of the meat, and high redness values generally indicate fresher meat (AMSA, 2012). The a* values of the coated pork samples were significantly higher (P < 0.05) than that of the CON sample, and all coated samples had the highest a* values (6.4-10.0) at day 5 and then decreased thereafter, while the CON sample experienced a gradual decrease from 3.7 to 2.8 throughout the storage (Table 2). Similar findings were also reported on chitosan coated pork in modified atmosphere packaging (Fang, Lin, Warner, & Ha, 2018). The colour of the flesh meat is mainly determined by the haemoglobin and myoglobin pigments in meat, where myoglobin (deoxymyoglobin, oxymyoglobin and metmyoglobin) plays a key role in the colour of meat and is closely related to the a* value (Kim et al., 2010). During storage, as meat deteriorates, deoxymyoglobin (purple colour) and/or oxymyoglobin (red colour) are oxidised to metmyoglobin (brown colour), and thus meat colour shifts to lower redness and higher yellowness colour and become unattractive (Brewer, Zhu, Bidner, Meisinger, & McKeith, 2001;
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Journal Pre-proof Fang, Lin, Warner, & Ha, 2018). The results showed that the yellowness (b*) of the coated pork samples were higher than the CON sample, and all samples had higher b* values after 20 days of storage (Table 2). The higher b* values in the coated pork samples may be partially due to the colour of the coating itself, which was slightly yellow.
In addition, it was noticed that the pork samples coated with GSE (CHI-GEL-GSE and CHIGEL-GSE-NIS) had the highest a* and b* values amongst all groups, indicating that the sample colour is redder and yellower (Table 2). Consistent results were also reported in GSE incorporated chitosan edible films and GSE incorporated soy protein edible film (Rubilar et al., 2013; Sivarooban, Hettiarachchy, & Johnson, 2008). This may be due to the high levels of phenolic compounds in the GSE, and some phenolic compounds such as anthocyanins are well known natural pigments that are responsible for the red to blue colour (depending on pH), which may have contributed to the redder meat colour (He & Giusti, 2010; Shi, Yu, Pohorly, & Kakuda, 2003). In addition, the antioxidant activity of GSE phenolic compounds may inhibit the oxidation of meat, which could further enhance the colour (redness) of meat (Carpenter, O'Grady, O'Callaghan, O'Brien, & Kerry, 2007).
3.3
Lipid oxidation
The lipid oxidation of the pork sample was evaluated by the TBARS value, which measures the amount of secondary products produced during the lipid oxidation, and a higher TBARS value indicates a higher degree of lipid oxidation. The off-flavours in pork can generally be detectable by consumers when the TBARS value is higher than the threshold value of 0.5 mg MDA/kg (Sheard et al., 2000). In general, the TBARS values of all pork samples increased significantly over the 20 days’ storage period, particularly for the CON sample, whose TBARS value was dramatically increased from 0.053 mg MDA/kg on day 0 to 0.343 mg MDA/kg on day 20 (Figure 2). The TBARS value of CON sample was significantly higher (P < 0.05) than coated groups from day 5, and experienced an exponential growth thereafter, and was close to the threshold value at day 20, suggesting high lipid oxidation occurred in the CON sample. 12
Journal Pre-proof This is because the CON sample was directly exposed to the oxygen that was sealed in the packaging tray and lipid oxidation occurred (Bao & Ertbjerg, 2015), and the reactive oxygen species generated during the lipid oxidation would further accelerate the oxidation process (Burcham & Kuhan, 1996).
On the other hand, the TBARS values of all coated pork samples had much slower increase during the storage, and the values were well below the rancidity threshold at day 20 (Figure 2), indicating the coatings were effective to retard the lipid oxidation of pork samples. The antioxidant activity of chitosan-based coating in meat has been well documented. For instance, Wu et al. (2016) observed that 1.5 % chitosan coating could effectively inhibit lipid oxidation of refrigerated pacific mackerel fillets, and Farajzadeh, Motamedzadegan, Shahidi, & Hamzeh (2016) reported that 1% chitosan and 3% gelatine coating could significantly prevent lipid oxidation of shrimps under cold storage. It has been proposed that the antioxidant capacity of chitosan coatings may be attributed to its low oxygen permeability, which reduces or prevents the exposure of the samples to oxygen, as well as its ability to chelate metal ions such as copper and iron which inhibits catalytic activity and further delays the lipid oxidation (Chen, Zheng, Wang, Lee, & Park, 2002).
Among the coated samples, the GSE incorporated samples (CHI-GEL-GSE and CHI-GELGSE-NIS) had the lowest (P < 0.05) TBARS values at day 20 (Figure 2), indicating that addition of GSE into the coating significantly inhibited the lipid oxidation. The possible reason is that the high levels of phenolic compounds in the GSE are acting as a strong antioxidant, scavenging free radicals and hindering the oxidation chain reactions (Bagchi et al., 2000). This result is in accordance with the study of Li, Li, Hu, & Li (2013), who reported that incorporating 0.2% GSE into 1.5% chitosan coating significantly lowered the TBARS value in refrigerated red drum fillets. In contrast, there was no significant difference (P > 0.05) between the TBARS values of nisin incorporated coating (CHI-GEL-NIS and CHI-GEL-GSE-NIS) and CHI-GEL (Figure 2). This result suggests that addition of nisin into the CHI-GEL coating did
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Journal Pre-proof not affect the meat lipid oxidation, which is in line with those of previous studies (Cao, Warner, & Fang, 2019; Lu, Ding, Ye, & Liu, 2010).
3.4
Protein oxidation
The concentration of free thiol groups is commonly used as an indicator of protein oxidation in meat products. Amino acids such as L-cysteine are easily oxidised and form disulphide cross-links, resulting in the loss of the free thiol group. Therefore, a lower thiol group value indicates a greater reduction of the thiol group, and consequently greater protein oxidation (Winther & Thorpe, 2014). Figure 3 shows that the free thiol group values of all pork samples were significantly decreased during the 20 days of cold storage. Especially the CON sample, whose free thiol group value was decreased rapidly from 72.28 nmol thiol/mg protein on day 0 to 40.95 nmol thiol/mg protein on day 20, and was significantly lower than that of coated pork samples. This suggests that non-coated pork had a higher degree of protein oxidation than coating-protected samples, which may be due to the direct reaction of meat with the oxygen in the package. In other words, the coating treatments showed good protection against pork protein oxidation.
During the first five days of storage, the free thiol group values of all samples did not change much, but significant differences (P < 0.05) were observed between samples from day 10, and the difference was accelerated thereafter (Figure 3). This phenomenon was also found in lipid oxidation, as illustrated in Figure 2 and discussed above. This is because both lipid oxidation and protein oxidation are initiated by reactive oxygen species and highly correlated, and can influence each other (Lund, Heinonen, Baron, & Estevez, 2011; Min & Ahn, 2005). The link between lipid and myoglobin oxidation has been identified to be hydroxynonenal (HNE) where HNE is a product of lipid oxidation and can accelerate myoglobin oxidation (Suman & Joseph, 2013). Furthermore, the intermediate compounds generated during the protein and lipid oxidations could further accelerate oxidative chain reactions (Burcham & Kuhan, 1996).
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Journal Pre-proof In the coated pork samples, GSE and/or nisin incorporated coatings generally had higher thiol group values on day 15 and 20, and CHI-GEL-GSE coated pork sample had the highest thiol value (Figure 3), suggesting that incorporating GSE into the chitosan-gelatine coating could effectively prevent the pork protein oxidation. In contrast, Jongberg, Skov, Torngren, Skibsted, & Lund (2011) reported that beef samples treated with white grape extracts showed significantly lower thiol content compared to untreated samples. They proposed that polyphenols in grapes could interact with thiol groups to form adducts, which interfered with the results. Nevertheless, many studies have shown that incorporation of natural antioxidants such as gallic acid (Fang, Lin, Warner, & Ha, 2018), lemon extract (Lara, Gutierrez, Timón, & Andrés, 2011) and persimmon peel extract (Choe, Kim, & Kim, 2017) into meat or packaging could inhibit the protein oxidation. Combined with the above lipid oxidation results, it was demonstrated that the GSE incorporated chitosan-gelatine coating could effectively inhibit pork oxidation during cold storage.
3.5
Microbial analysis
The change of TVC in pork samples is shown in Figure 4. The TVC value of the fresh pork sample was 2.51 log CFU/g on day 0, and the TVC values of all coated and non-coated pork samples increased significantly during the 20 days’ cold storage. Not surprisingly, the TVC of non-coated sample (CON) showed the fastest increase during the storage and was significantly higher (P < 0.05) than all other samples since day 5. The elevated microbial count in pork samples during the cold storage may be caused by the growth of psychrotrophic bacteria such as Carnobacterium spp. and Pseudomonas spp. (Ercolini, Russo, Nasi, Ferranti, & Villani, 2009). According to Huang et al. (2014), the TVC value of 7 log CFU/g can be used as the threshold for pork quality, and pork may be spoiled by microorganisms if TVC is higher than this value. All the samples in this study were below this threshold after the 20-day storage, indicating the conventional tray packaging at 4 °C alone is effective to prevent the pork from microbial spoilage. However, it should be noted that the TVC of CON sample was 6.30 log
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Journal Pre-proof CFU/g on day 20, which was very close to the threshold and would likely have spoiled not long after 20 days.
Compared with the non-coated sample (CON), the chitosan coating (CHI) effectively inhibited the growth of microorganisms from the fifth day (Figure 4). This result is in agreement with Fang, Lin, Warner, & Ha (2018) and Cao, Warner, & Fang (2019), who examined 2% chitosan coating of fresh pork samples in a high-oxygen modified atmosphere packaging. Chitosan is an excellent natural antimicrobial agent. Goy, Britto, & Assis (2009) stated that chitosan is able to inhibit the growth of a broad spectrum of microorganisms, including Gram-negative (such as Escherichia coli and Pseudomonas spp.) and Gram-positive (such as Listeria monocytogenes and lactic acid bacteria) bacteria and fungi (such as Botrytis cinereal and Drechstera sorokiana). Elsabee & Abdou (2013) proposed that chitosan could interact with the negative charges located on the microbial cell membrane, causing the breakdown of the membrane and leaching of intracellular substances. Furthermore, the TVC of the chitosangelatine coated sample (CHI-GEL) was significantly lower (P < 0.05) than that of chitosan coating (CHI) alone on day 15 and 20 (Figure 4), indicating a lower antimicrobial activity in the CHI-GEL sample. Elsabee & Abdou (2013) explained that chitosan and gelatine could form a compact structure and reduce the moisture permeability, which may further enhance the antimicrobial effect of the coating, which most likely explains our results.
On the other hand, no TVC difference (P > 0.05) was observed between the chitosan-gelatine coated sample (CHI-GEL) and GSE (0.5%) and/or nisin (500 UI/mL) incorporated coating samples (CHI-GEL-GSE, CHI-GEL-NIS and CHI-GEL-GSE-NIS) on day 15 and 20 (Figure 4), indicating that addition of GSE and/or nisin did not further improve the antimicrobial effect of the coating. Similar results were also found by Lu, Ding, Ye, & Liu (2010) who used nisin (2000 IU/mL) incorporated into alginate-calcium coating on fresh northern snakehead fish fillets. However, it is worth noting that nisin is well known for its excellent antimicrobial activity, against various Gram-positive bacteria including Bacillus, Clostridium, Listeria,
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Journal Pre-proof Staphylococcus, and Enterococcus spp., and has been widely used in the food industry (Punyauppa-path, Phumkhachorn, & Rattanachaikunsopon, 2015). Also, incorporation of nisin (0.2%) into chitosan (2%) coating has been shown to have a significantly enhanced antimicrobial effect on fresh pork preservation (Cao, Warner, & Fang, 2019). The reason for such inconsistency is not clear, but it may be related to the coating matrix and that chitosan and gelatine form a compact structure which affects the functional property of nisin or GSE, as well as the environmental conditions and types of bacteria present, which requires further investigation.
4
Conclusion
The present study developed several chitosan and gelatine based edible coatings for fresh pork preservation. Chitosan coating (CHI) alone effectively extended the shelf-life of pork by minimising the pH change, preventing lipid and protein oxidation and inhibiting microbial growth during the 20 days of cold storage at 4 °C. These preservative effects were enhanced by combining chitosan with gelatine (CHI-GEL), possibly owing to the compact structure formed between chitosan and gelatine. Incorporating GSE into the chitosan-gelatine coating further enhanced antioxidant activity against protein and lipid oxidation, and also conferred meat with a redder and yellower colour, which may be attributed to the phenolic compounds present in the GSE. However, incorporating nisin into the coating had no significant effect on the antimicrobial and lipid and protein oxidation, and further work is required to uncover the underlying mechanism. Overall, the CHI-GEL-GSE formulation showed the best performance on fresh pork preservation and could be developed as a hurdle technology in preserving fresh meat products to extend the shelf life while improving the safety and quality.
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Conflict of interest
The authors declare no conflict of interest.
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Journal Pre-proof 6
References
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Journal Pre-proof Gertzou, I. N., Karabagias, I. K., Drosos, P. E., & Riganakos, K. A. (2017). Effect of combination of ozonation and vacuum packaging on shelf life extension of fresh chicken legs during storage under refrigeration. Journal of Food Engineering, 213, 18-26. Gomez-Guillen, M. C., Perez-Mateos, M., Gomez-Estaca, J., Lopez-Caballero, E., Gimenez, B., & Montero, P. (2009). Fish gelatin: a renewable material for developing active biodegradable films. Trends in Food Science & Technology, 20(1), 3-16. Goy, R. C., Britto, D. d., & Assis, O. B. G. (2009). A review of the antimicrobial activity of chitosan. Polímeros, 19(3), 241-247. Hassan, B., Chatha, S. A. S., Hussain, A. I., Zia, K. M., & Akhtar, N. (2018). Recent advances on polysaccharides, lipids and protein based edible films and coatings: A review. International Journal of Biological Macromolecules, 109, 1095-1107. doi:10.1016/j.ijbiomac.2017.11.097 He, J., & Giusti, M. M. (2010). Anthocyanins: natural colorants with health-promoting properties. Annual review of food science and technology, 1, 163-187. doi:10.1146/annurev.food.080708.100754 Hebard, C. (1982). Occurrence and significance of trimethylamine oxide and its derivatives in fish and shellfish. Chemistry & Biochemistry of Marine Food Products, 149-304. Holman, B. W., Collins, D., Kilgannon, A. K., & Hopkins, D. L. (2018). The effect of technical replicate (repeats) on Nix Pro Color Sensor™ measurement precision for meat: A case-study on aged beef colour stability. Meat Science, 135, 42-45. Holmer, S. F., McKeith, R. O., Boler, D. D., Dilger, A. C., Eggert, J. M., Petry, D. B., McKeith, F. K., Jones, K. L., & Killefer, J. (2009). The effect of pH on shelf-life of pork during aging and simulated retail display. Meat Science, 82(1), 86-93. doi:10.1016/j.meatsci.2008.12.008 Hosseini, S. F., Rezaei, M., Zandi, M., & Ghavi, F. F. (2013). Preparation and functional properties of fish gelatin– chitosan blend edible films. Food Chemistry, 136(3--4), 1490-1495. Huang, X. W., Zou, X. B., Shi, J. Y., Guo, Y., Zhao, J. W., Zhang, J., & Hao, L. (2014). Determination of pork spoilage by colorimetric gas sensor array based on natural pigments. Food Chemistry, 145, 549-554. doi:10.1016/j.foodchem.2013.08.101 Huang, Y., Gan, Y., Li, F., Yan, C., Li, H., & Feng, Q. (2015). Effects of high pressure in combination with thermal treatment on lipid hydrolysis and oxidation in pork. LWT-Food Science and Technology, 63(1), 136-143. Jongberg, S., Skov, S. H., Torngren, M. A., Skibsted, L. H., & Lund, M. N. (2011). Effect of white grape extract and modified atmosphere packaging on lipid and protein oxidation in chill stored beef patties. Food Chemistry, 128(2), 276-283. doi:10.1016/j.foodchem.2011.03.015 Karabagias, I., Badeka, A., & Kontominas, M. G. (2011). Shelf life extension of lamb meat using thyme or oregano essential oils and modified atmosphere packaging. Meat Science, 88(1), 109-116. Kim, G.-D., Jeong, J.-Y., Hur, S.-J., Yang, H.-S., Jeon, J.-T., & Joo, S.-T. (2010). The relationship between meat color (CIE L* and a*), myoglobin content, and their influence on muscle fiber characteristics and pork quality. Korean Journal for Food Science of Animal Resources, 30(4), 626-633. Kim, T. W., Kim, C. W., Kwon, S. G., Hwang, J. H., Park, D. H., Kang, D. G., Ha, J., Yang, M. R., Kim, S. W., & Kim, I.-S. (2016). pH as Analytical Indicator for Managing Pork Meat Quality. Sains Malaysiana, 45(7), 10971103. Lara, M. S., Gutierrez, J. I., Timón, M., & Andrés, A. I. (2011). Evaluation of two natural extracts (Rosmarinus officinalis L. and Melissa officinalis L.) as antioxidants in cooked pork patties packed in MAP. Meat Science, 88(3), 481-488.
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Journal Pre-proof 838-846. doi:10.1016/j.bbagen.2013.03.031 Wright, L. I., Scanga, J. A., Belk, K. E., Engle, T. E., Tatum, J. D., Person, R. C., McKenna, D. R., Griffin, D. B., McKeith, F. K., Savell, J. W., & Smith, G. C. (2005). Benchmarking value in the pork supply chain: Characterization of US pork in the retail marketplace. Meat Science, 71(3), 451-463. doi:10.1016/j.meatsci.2005.04.024 Wu, C., Li, Y., Wang, L., Hu, Y., Chen, J., Liu, D., & Ye, X. (2016). Efficacy of chitosan-gallic acid coating on shelf life extension of refrigerated pacific mackerel fillets. Food and Bioprocess Technology, 9(4), 675-685.
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Journal Pre-proof Table captions: Table 1: Formulation of edible coating solutions. Table 2. Changes of colour attributes (L*, a*, b*) of pork loin samples during cold storage (4 ℃) for 20 days.
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Journal Pre-proof Table 1: Formulation of edible coating solutions. Treatments code
Formulae
CON
Control group, pork samples not coated with coating solutions
CHI
1% chitosan
CHI-GEL
1% chitosan + 3% gelatine
CHI-GEL-GSE
1% chitosan + 3% gelatine + 0.5% GSE
CHI-GEL-NIS
1% chitosan + 3% gelatine + 0.1% nisin
CHI-GEL-GSE-NIS
1% chitosan + 3% gelatine + 0.5% GSE + 0.1% nisin
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Journal Pre-proof Table 2. Changes of colour attributes (L*, a*, b*) of pork loin samples during cold storage (4 ℃) for 20 days. Attributes L*
a*
b*
a-d =
Coating treatment
Storage days 0
5
10
15
20
CON
54.0±0.5aB
58.4±1.1aA
58.5±1.8abA
57.8±1.2aA
57.6±0.9aA
CHI
54.0±0.5aA
49.8±1.1bA
55.7±1.8bcA
53.0±2.6abA
51.3±2.4bcA
CHI-GEL
54.0±0.5aB
50.0±1.9bB
61.0±1.9aA
53.2±2.4abB
58.2±1.3aA
CHI-GEL-GSE
54.0±0.5aA
55.0±1.7aA
56.5±1.3bcA
55.5±1.1abA
54.0±1.3abA
CHI-GEL-NIS
54.0±0.5aB
55.0±1.8aAB
58.6±1.3abA
52.6±1.0bC
46.3±3.5cD
CHI-GEL-GSE-NIS
54.0±0.5aA
54.9±0.7aA
53.8±0.7cA
53.6±1.6abA
49.4±2.2bcB
CON
3.7±0.2aA
3.2±0.2cB
2.6±0.3cC
2.0±0.3cC
2.8±0.4dC
CHI
3.7±0.2aB
6.4±0.4bA
6.1±0.7bA
6.4±0.4bcA
3.9±0.6dB
CHI-GEL
3.7±0.2aD
6.9±0.6bA
5.6±0.4bB
5.5±0.3bB
4.4±0.3cC
CHI-GEL-GSE
3.7±0.2aC
9.3±0.9aA
8.0±0.6aAB
7.5±0.3aB
7.6±0.3aB
CHI-GEL-NIS
3.7±0.2aB
6.2±0.4bA
6.3±0.5bA
6.5±0.7bcA
6.7±0.7abA
CHI-GEL-GSE-NIS
3.7±0.2aC
10.0±0.4aA
8.8±0.6aA
7.2±0.3aB
7.6±0.5aB
CON
3.8±0.2aB
4.6±0.2cA
4.7±0.4cA
4.5±0.3dA
4.4±0.6dA
CHI
3.8±0.2aC
4.8±0.2cB
6.5±0.7cA
6.3±0.2cA
6.0±0.3cA
CHI-GEL
3.8±0.2aB
6.3±0.2bcA
6.3±0.6cA
7.1±0.4bcA
6.3±0.3cA
CHI-GEL-GSE
3.8±0.2aB
9.7±1.0aA
10.1±0.9abA
9.1±0.4aA
9.5±0.6aA
CHI-GEL-NIS
3.8±0.2aB
7.0±1.1bA
8.5±0.5bA
8.1±0.7abA
7.8±0.5bA
CHI-GEL-GSE-NIS
3.8±0.2aC
11.6±0.5aA
10.7±0.7aA
8.6±0.7aB
8.2±0.3bB
Values with different superscripts in the same column are significantly different (P < 0.05).
A-D =
Values with different superscripts in the same row are significantly different (P < 0.05).
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Journal Pre-proof Figure captions: Figure 1: Changes of pH value of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
Figure 2: Changes of lipid oxidation (TBARS values, mg MDA/kg meat) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
Figure 3: Changes of protein oxidation (free thiol group values, nmol thiol/ mg protein) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
Figure 4. Changes of total viable counts (TVC) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
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Figure 1: Changes of pH value of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
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Figure 2: Changes of lipid oxidation (TBARS values, mg MDA/kg meat) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
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Figure 3: Changes of protein oxidation (free thiol group values, nmol thiol/ mg protein) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
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Figure 4. Changes of total viable counts (TVC) of pork loin samples during cold storage (4 ℃) for 20 days. Bars with different letters in the same day are significantly different (P < 0.05).
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Journal Pre-proof Highlight:
Grape seed extract (GSE)/nisin were loaded in chitosan-gelatine edible coatings
The edible coatings were applied to fresh pork during cold storage
The GSE edible coating enhanced antioxidant activity against meat oxidation
Incorporating nisin did not improve the antimicrobial and antioxidant activity
CHI-GEL-GSE coating formulation showed the best performance on meat preservation