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The effect of chitosan coating and vacuum packaging on the microbiological and chemical properties of beef
Meat Science xxx (xxxx) xxxx
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The effect of chitosan coating and vacuum packaging on the microbiological and chemical properties of beef ⁎
Ayhan Duran , Halil Ibrahim Kahve Food Engineering Department, Aksaray University, Aksaray, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Vacuum packaging Beef Storage
Beef is an animal food sensitive to deterioration due to its rich nutrient content. Therefore, some preservation techniques are applied. These include vacuum packaging, a modified atmosphere, a controlled atmosphere and an edible film coating. In this study, it was aimed to extend the shelf life of beef using vacuum packaging (VP) and chitosan coating with vacuum packaging (CC + VP). For this purpose, total mesophilic aerobic bacteria (TMAB), Stapylococcus aureus, lactic acid bacteria (LAB) counts, thiobarbituric acid (TBA) values and total volatile basic nitrogen (TVB-N) content were analyzed in beef obtained from local markets. As a result, it was found that the chitosan coating reduced the TMAB, LAB and TVB-N values and inhibited all S. aureus up to day 15 of storage. In addition, it was seen that the application of CC + VP was significantly more effective (p˂0.05) on the reduction of the TBA value, compared to the VP application over a long period of storage (45 days). The combined use of the two technologies is more effective on TVB-N. According to the data obtained from this study, because of the antimicrobial and antioxidant properties of chitosan, it has been concluded that it can be used as a bio-preservative in the meat industry.
1. Introduction Beef is quickly perishable by nature, and the spoilage of beef is a complicated process involving the interactions of physical, chemical and microbial changes. Microbial contamination and lipid oxidation in beef during processing and storage are the major causes of foodborne illnesses and reduction in shelf-life (Rao, Chander, & Sharma, 2008). The predominant bacteria associated with spoilage of refrigerated beef are Brochothrix thermosphacta, Carnobacterium spp., Enterobacteriaceae, Lactobacillus spp., Leuconostoc spp., Pseudomonas spp. and Shewanella putrefaciens (Borch, Muermans, & Blixt, 1996). Lactic acid bacteria, Brochothrix thermosphacta, Aeromonas (Lambert, Smith, & Dodds, 1991) and Clostridium (Moschonas, Bolton, Sheridan, & McDowell, 2010; Reid, Fanning, Whyte, Kerry, & Bolton, 2017) species are found in vacuum-packed and cold-stored meats. The microorganisms that will affect meat depend on the pH values of the meat, the oxygen permeability of the packaging material and the storage temperature. Meat products and beef provide a favorable environment for the growth of Staphylococcus aureus, and further secondary contamination affects the beef and is distributed throughout the product during grinding and mixing operations used during milled-beef production (Lambert et al., 1991). In recent years, research into fresh meat preservation has led to new ⁎
non-thermal deactivation techniques such as edible film coatings, vacuum applications and high-pressure applications (Aymerich, Picouet, & Monfort, 2008; Zhou, Xu, & Liu, 2010). Vacuum packaging is a commonly used packaging method in the meat industry, especially for the transport and sale of fresh meat and for the preservation of quality and shelf-life extension (Jeremiah, 2001). The greatest advantage comes from the consumer's desire to consume meat with the least heat treatment, followed closely by good, fresh meat. When the vacuum packaging applied in meat studies was examined, it was found that the storage period varies between 40 days and 55 days (Fregonesi et al., 2014; Polkinghorne et al., 2018; Sun, Sun, Zheng, Kong, & Liu, 2019). In this study, the storage period was selected to be 45 days. Edible films have become popular in the meat industry due to their low-waste characteristics, low cost and their ability to provide protection after the packaging has been opened (Cha & Chinnan, 2004; Morris, Padmanabhan, Cruz-Romero, Cummins, & Kerry, 2017). Chitosan films have been successfully used as a packaging material to preserve the quality of meat and meat products (Chantarasataporn et al., 2014; Kanatt, Rao, Chawla, & Sharma, 2013). Chitosan has an antimicrobial effect due to its polycationic properties. As a matter of fact, it is effective against mold, yeast and bacteria because it interacts to counteract negatively charged substances. The pKa value of the amine group of the glucose monomer of chitosan, under a pH value of
Corresponding author. E-mail address: [email protected] (A. Duran).
https://doi.org/10.1016/j.meatsci.2019.107961 Received 12 March 2019; Received in revised form 27 September 2019; Accepted 30 September 2019 0309-1740/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Ayhan Duran and Halil Ibrahim Kahve, Meat Science, https://doi.org/10.1016/j.meatsci.2019.107961
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sample of meat was weighed and homogenized with 90 ml of Maximum Recovery Diluent (Merck, 112,535) using a stomacher (MAYO, HG-400, Australia). Then, a series of dilutions up to 10−7 were prepared. Plate Count Agar (PCA, Sigma Aldrich) for Total Mesophilic Aerobic Bacteria (TMAB) (ISO, 4833–2, 2013), Baird Parker Agar (BPA, Sigma Aldrich) for Staphylococcus aureus (ISO, 6888–1, 1999) and de Man Rogosa Sharpe Agar (MRS agar, Merck) for lactic acid bacteria were used for colony counts (ISO 13721:1995/Cor.1, 1996). The selected colonies were subjected to a coagulase assay and verified. For the TMAB analysis, the incubation temperature was 30 ± 2 °C for 48 h. After incubation, colonies on the PCA were counted. In the Staphylococcus aureus analysis, the incubation temperature was 37 ± 2 °C for 48 h. After incubation, the black colonies with a clear zone were identified to be Staphylococcus aureus. For the lactic acid bacteria count, the incubation was at 30 ± 2 °C for 72 h under anaerobic conditions. After incubation the white and opaque zones were identified to be lactic acid bacteria. In addition, as a result of each microbiological analysis, colonies were randomly selected from the Petri dishes and examined microscopically and the results were checked. All the results were given as log cfu (colony forming units)/g values.
6.3, electrostatically interacts with negatively charged microbial cell membranes. As a result, the integrity of the microbial membranes deteriorates, and this leads to leakage of the cell components from the inner and outer membranes (Helander, Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001; Liu, Du, Wang, & Sun, 2004; Papineau, Hoover, Dnorr, & Farkas, 1991; Sudarshan, Hoover, & Knorr, 1992). Chitosan also has antioxidant properties. Lipid oxidation in beef is one of the most important factors limiting the storage period, and thiobarbituric acid extraction (TBA) and total volatile basic nitrogen (TVB-N) is an indicator of this. Albeit at a low level, lipid oxidation persists in cold-stored and vacuum-packed meat and products (Djenane, Beltran, Camo, & Roncales, 2016; Jouki & Yazdi, 2014). Primer and secondary antioxidants are used to prevent this oxidation. Primer antioxidants react before oxidation because of their phenolic groups, while secondary antioxidants form chelates with the metal ions that catalyze oxidation. Chitosan has secondary antioxidant activity (Agullo, Rodriguez, Ramos, & Albertengo, 2003). In this study, vacuum packaging (VP) and chitosan coating with vacuum packaging (CC + VP) were applied to beef. The VP meat and (CC + VP) meat were stored for 45 days. In addition, the beef was stored in the refrigerator at 4 ± 1 °C. The aim of the study was to examine the microbiological and chemical changes caused by the vacuum packing and chitosan coating on the beef.
2.5. Chemical analyses During the preservation of meats, the reagents formed as a result of lipid peroxidation are determined by the thiobarbituric acid (TBA) method and show the level of oxidative degradation. The analyses were performed using the method proposed by Tarladgis, Watts, Younathan, & Dugan, 1960Ten gram samples were transferred to a stomacher (MAYO, HG-400, Australia) bag and homogenized. Then, an HCl solution was added for the distillation process and 5 ml of TBA reactive substances was added after the distillation. After that, the distillates were transferred to spectrophotometer tubes to measure the optical density. Finally, the density value obtained was multiplied by 7.80 and the results were given as mg of malondialdehyde in a 1000 g sample. During the preservation process of meats, the total volatile basic nitrogen (TVB-N) method was used to determine the proteolytic degradation. The TVB-N content was determined by distillation after the addition of MgO to the minced sample. The distillate was collected in a flask containing a 3% (w/v) aqueous solution of boric acid and a mixed indicator produced by dissolving 0.1 g of methyl red and 0.05 g of methylene blue to 100 ml of ethanol. The boric acid solution turned green when the distilled TVB-N made it alkaline. Finally, the boric acid solution was titrated with an 0.01 mol/L HCl solution until it turned pink (Goulas & Kontominas, 2005). The quantity of TVB-N in mg/100 g sample was then calculated.
2. Material and methods 2.1. Preparation of meat samples This study used meat prepared from strips of loin (longissimus lumborum). Beef was provided by a local market. For each packaging treatment (VP and CC + VP), 12 samples of 100 g of meat cubes (2 × 2 cm) were prepared. The average shelf life of VP meat and CC + VP meat in the refrigerator (4 ± 1 °C) has been reported to be 45 days in published literature. Therefore, the storage period of the VP and CC + VP treated meats was selected to be 45 days and analyses were carried out on days 0, 15, 30 and 45. 2.2. Preparation of the chitosan solution and its application The chitosan used in the study was obtained from Sigma Aldrich (448,877, USA). Chitosan has a medium molecular weight and is soluble character in organic acids (sorbic acid, p-aminobenzoic acid, lactic acid, acetic acid etc.) In order to prepare the chitosan solution, 10 g of chitosan was weighed and mixed with 500 ml of acetic acid. Then the chitosan solution was stirred with a magnetic stirrer (Velp Scientifica, F20520165, EU) for two days, the first day at high speed and the second day at moderate speed. In this way, the concentration of chitosan used was set to 2% (Cruz-Romero, Murphy, Morris, Cummins, & Kerry, 2013). The beef samples weighing 100 g were immersed in the chitosan solution for 5 s to completely cover them. The meat coated with the solution was placed in a cooling cabinet (Alveo KT, ASE.01, Turkey) at 4 ± 1 °C for 30 min (Cardoso et al., 2019). Following the drying procedure, the film thickness on the meat was measured to be 15 ± 3 μm.
2.6. Statistical analyses Two-factor repeated-measures analysis of variance (ANOVA) was performed using SPSS 22.0.0 software (SPSS Inc., Chicago, USA) to analyze the effects of surface treatments and storage time on microbial quality and lipid oxidation. A completely randomized design included study replication, treatment groups (VP, CC + VP) and storage times (0, 15, 30 and 45 days) as fixed variables. For each storage time, three samples in a treatment group were evaluated. The study was replicated for confirmation later under the same conditions. Means were compared by Duncan's multiple range test at a p value which was calculated for each analysis (Duncan, 1955). Differences among mean values were considered significant when p < .05.
2.3. Applying vacuum packaging For the preparation of vacuum-packed beef, a Turkish Stilea Rimini VS2140 brand vacuum device was used. A 100-g portion of meat was used to fill the vacuum bags (Eurobag & Film S.L., Spain). Vacuum bag permeability is 40 cm3/m2 of O2 under this conditions; 24 h at 23 °C, 75% relative humidity.
3. Results The results obtained in the study are given in Tables 1 and 2. The samples were divided into two groups as vacuum-packed meat and vacuum-packed meat with a chitosan coating according to the application type, and the changes – both within the groups and against time
2.4. Microbiological analyses For microbiological analyzes to be made during storage, a 10-g 2
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Table 1 The microbial changes in the beef during storage (4 ± 1 °C). 0. Day
15. Day
30. Day
45. Day
Total mesophilic aerobic bacteria (log cfu/g) VP meat 4.44 ± 0.12d1 CC + VP meat 4.44 ± 0.12d1
5.05 ± 0.02c1 4.51 ± 0.07c2
5.15 ± 0.22b1 4.58 ± 0.23b2
5.25 ± 0.10a1 4.69 ± 0.21a2
Stapylococcus aureus (log cfu/g) VP meat CC + VP meat
2.00 ± 1.15b1 2.00 ± 1.15b1
2.30 ± 1.31a1 ND
2.00 ± 0.40b1 ND
2.30 ± 0.15a1 ND
Lactic acid bacteria (log cfu/g) VP meat CC + VP meat
2.18 ± 0.01d1 2.18 ± 0.01d1
3.47 ± 0.25c1 2.56 ± 0.42c2
4.48 ± 0.11b1 3.11 ± 0.36b2
6.02 ± 0.98a1 3.67 ± 0.23a2
ND not detected. Each value represents the mean of 6 samples ± standard deviation. Bearing different superscripts row wise (alphabet) and column wise (numeral) differ significantly (P < .05).
– were investigated. During the storage, the TMAB count in the vacuum-packed meat with a chitosan coating was lower than that in the vacuum-packed group. After 45 days, TMAB counts in vacuum-packed meat (5.25 ± 0.1 log cfu/g) were statistically higher than that of vacuumpacked meat with a chitosan coating (4.69 ± 0.21 log cfu/g). During storage, TMAB counts increased steadily in both groups. S. aureus counts in vacuum-packed beef were statistically different from the value on the first day. On day 15 of storage, chitosan coated, vacuum-packed meat inhibited all S. aureus. In both groups, lactic acid bacteria counts increased continuously. At the end of storage, lactic acid bacteria counts were statistically lower in the vacuum-packed meat with a chitosan coating group (3.67 ± 0.23 log cfu/g) compared to that in the vacuum-packed meat group (6.02 ± 0.9 log cfu/g). When the TBA numbers were examined in the table, it was determined that TBA numbers were close to the values of the first day in both groups. As shown in the Table 2, the TVB-N content of samples constantly increased.
TMAB counts in meat by using additional methods. The increase in the TMAB count was lower in the group coated with chitosan. In a study in which a 2% chitosan solution was used to coat ready-to-cook mutton, the samples were stored for 14 days, and TBA, TMAB and S. aureus analyses were carried out. In the meat samples, the initial value of 6.1 log cfu/g was reduced to a range from 3.4 to 6.1 log cfu/g at the end of the storage (Kanatt et al., 2013). In a study in which the shelf-life of chitosan-coated sausage was investigated, the TMAB count at the end of day 15 of storage was close to the initial count (Bostan & Mahan, 2011). As seen in studies on chitosan, it is successful at halting increases in the TMAB count, which agrees with the results obtained in this study. The most important effect of chitosan in halting increases in the TMAB count is attributed to its strong antimicrobial and antifungal properties. Although the antimicrobial property of chitosan was attributed to its polycationic properties, it may also be due to its chelator-like behavior, water-binding properties, activation of the defense processes in the host tissue and inhibiting mRNA synthesis by entering the nucleus (Liu et al., 2004). When the results of the study are examined, it is seen that vacuum packaging suppresses the increase in the S. aureus count. This is thought to be due to S. aureus being a facultative anaerobic organism. However, S. aureus completely disappeared in the group with meat covered by chitosan. Chitosan naturally inhibits the growth of a broad spectrum of microorganisms (Vu, Hollingsworth, Leroux, Salmier, & Lacroix, 2011). Its antimicrobial activity is greatly influenced by its molecular weight with values below 300 kDa reportedly increasing its inhibitory effect on S. aureus, while its suppressive effect on E. coli decreased (Zheng & Zhu, 2003). In a study on vacuum-packed sliced ham, it was found that vacuum packaging reduced the development of S. aureus (Christiansen & Foster, 1965). In another study on chitosan-coated meats, the meats were stored for 48 h at 30 °C and 10 days at 4 °C; the Bacillus subtilis IFO 3025, Escherichia coli RB, Pseudomonas fragi IFO 3458 and Staphylococcus aureus IAM 1011 bacteria were analyzed, and the results showed that chitosan inhibited the growth of these species of bacteria (Darmadji & Izumimoto, 1994).
4. Discussion During storage, the TMAB count in the vacuum-packed meat with a chitosan coating was lower than that in the vacuum-packed meat group. Vacuum packing is capable of effectively improving the quality and shelf-life of packaged meat compared to previously employed methods. However, compared to the VP application, it was found that the single CC + VP application was more effective in reducing the TMAB's proliferation rate. Because the activities of anaerobic microorganisms continue, it is an important factor that the oxygen-free environment is insufficient to stop the growth of microorganisms. In a study on vacuum-packed beef, the samples were stored at +4 °C. The TMAB count was 6.99 log cfu/g on the first day and the count increased continuously to 8.23 log cfu/g after 7 days of storage (Çiçek, Karabıyıklı, Kılınçer, Yıldırım, & Cevahiroğlu, 2014). This study showed that vacuum packaging will be more effective in reducing the rate of proliferation of Table 2 The chemical changes in the beef during storage (4 ± 1 °C). 0. Day
15. Day
30. Day
45. Day
0.22 ± 0.01b1 0.22 ± 0.01b1
0.33 ± 0.02b1 0.24 ± 0.05a2
0.19 ± 0.10c1 0.17 ± 0.01c1
0.22 ± 0.13b1 0.19 ± 0.01c1
Total volatile basic nitrogen (mg/100 g) VP meat 5.41 ± 0.71d1 CC + VP meat 5.41 ± 0.71d1
7.25 ± 0.69c1 6.64 ± 0.80c2
10.66 ± 0.92b1 7.48 ± 1.01b2
14.9 3 ± 1.21a1 8.31 ± 1.55a2
Thiobarbituric acid (mg/1000 g) VP meat CC + VP meat
Each value represents the mean of 6 samples ± standard deviation. Bearing different superscripts row wise (alphabet) and column wise (numeral) differ significantly (P < .05). 3
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Future studies should focus on its use and applications on an industrial scale.
When the results are examined, lactic acid bacteria counts increased during the storage period. However, the increase in lactic acid bacteria counts was lower in the group coated with chitosan. Generally lactic acid bacteria are anaerobic and microaerophilic. When the growth of aerobic spoilage bacteria is inhibited, lactic acid bacteria may become the dominant component of the microbial flora of meats. It has been concluded from the results indicated in Table 1 that the dominant microflora of CC + VP meat is mostly composed of LAB after 15 days. Yet the TMAB counts seems to be more than that of LAB, which is thought to be caused by the inhibition of some LAB species, such as Carnobacterium spp., on MRS agar (Leisner, Laursen, Prevost, Drider, & Dalgaard, 2007). The wide applicability of chitosan allows for the development of various packaging systems for fresh and processed meat. By adding nanocellulose to a chitosan film, Dehnad et al. achieved improved mechanical film properties, as well as a reduction of lactic acid bacteria on ground meat packed in it by 3.1 log cfu/g compared to the control (Dehnad, Mirzaei, Emam-Djomeh, Jafari, & Dadashi, 2014). In other studies, researchers studied Greek-style fresh pork sausages. Samples were coated with chitosan and stored for 28 days. In the control group, the LAB number increased from 4.73 log cfu to 7.90 log cfu, and in the chitosan-coated group, the LAB number increased from 3.34 to 6.18 (Soultos, Tzikas, Abrahim, Georgantelis, & Ambrosiadis, 2008). When the TBA results are examined in the table, it is seen that the TBA numbers are the same in both groups at the end of storage. TBA content is a parameter that can measure lipid oxidation in meats. In addition, TBA count is directly related to oxygen. The fluctuation in the number of TBAs can be thought to be due to the presence of oxygen in the package, albeit at a minimal level after the meat is vacuum packed. At the end of 15 days, after the oxygen is completely exhausted, it can be said that the vacuum bag has a very small amount of oxygen permeability. Normally, chitosan is formed as a barrier that covers the surface of the red meat and thus isolates the food from the air and reduces the oxidation of the lipids in meat (Liu et al., 2004). However, it has only been effective in reducing TBA counts in vacuum packaging. In a study of the effect of vacuum packaging and modified atmosphere in beef, researchers found the lowest TBA value in vacuum-packed meat (John et al., 2005). It is thought that the lipid oxidation is delayed due to the removal of the oxygen in the environment whereas the vacuum packaging is effective on the TBA. As shown in Table 2, the TVB-N content of samples was 5.41 mg N/ 100 g at the beginning, but this value increased to 14.93 mg N/100 g on day 45. Up to day 15, the freshness and appearance was good but it spoiled very quickly thereafter. The TVB-N value is used as an indicator of degradation in meat products. This value is eventually increased by the activities of endogenous enzymes and spoilage bacteria (Kyrana, Lougovois, & Valsamis, 1997). The TVB-N value in chitosan-coated samples on days 15, 30 and 45 was 6.64, 7.48 and 8.31 respectively. The results show that the TVB-N count was lower in chitosan-coated groups (p < .05). In one study, the silver carp fish was coated with chitosan and the TVB-N values were lower (Fan et al., 2009). In another similar study, a 35 to 50% reduction in TVB-N formation in codfish filaments coated with different types of chitosan at the end of day 12 storage period was reported (Jeon, Kamil, & Shahidi, 2002).
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The effect of chitosan coating and vacuum packaging on the microbiological and chemical properties of beef ⁎
Ayhan Duran , Halil Ibrahim Kahve Food Engineering Department, Aksaray University, Aksaray, Turkey
A R T I C LE I N FO
A B S T R A C T
Keywords: Chitosan Vacuum packaging Beef Storage
Beef is an animal food sensitive to deterioration due to its rich nutrient content. Therefore, some preservation techniques are applied. These include vacuum packaging, a modified atmosphere, a controlled atmosphere and an edible film coating. In this study, it was aimed to extend the shelf life of beef using vacuum packaging (VP) and chitosan coating with vacuum packaging (CC + VP). For this purpose, total mesophilic aerobic bacteria (TMAB), Stapylococcus aureus, lactic acid bacteria (LAB) counts, thiobarbituric acid (TBA) values and total volatile basic nitrogen (TVB-N) content were analyzed in beef obtained from local markets. As a result, it was found that the chitosan coating reduced the TMAB, LAB and TVB-N values and inhibited all S. aureus up to day 15 of storage. In addition, it was seen that the application of CC + VP was significantly more effective (p˂0.05) on the reduction of the TBA value, compared to the VP application over a long period of storage (45 days). The combined use of the two technologies is more effective on TVB-N. According to the data obtained from this study, because of the antimicrobial and antioxidant properties of chitosan, it has been concluded that it can be used as a bio-preservative in the meat industry.
1. Introduction Beef is quickly perishable by nature, and the spoilage of beef is a complicated process involving the interactions of physical, chemical and microbial changes. Microbial contamination and lipid oxidation in beef during processing and storage are the major causes of foodborne illnesses and reduction in shelf-life (Rao, Chander, & Sharma, 2008). The predominant bacteria associated with spoilage of refrigerated beef are Brochothrix thermosphacta, Carnobacterium spp., Enterobacteriaceae, Lactobacillus spp., Leuconostoc spp., Pseudomonas spp. and Shewanella putrefaciens (Borch, Muermans, & Blixt, 1996). Lactic acid bacteria, Brochothrix thermosphacta, Aeromonas (Lambert, Smith, & Dodds, 1991) and Clostridium (Moschonas, Bolton, Sheridan, & McDowell, 2010; Reid, Fanning, Whyte, Kerry, & Bolton, 2017) species are found in vacuum-packed and cold-stored meats. The microorganisms that will affect meat depend on the pH values of the meat, the oxygen permeability of the packaging material and the storage temperature. Meat products and beef provide a favorable environment for the growth of Staphylococcus aureus, and further secondary contamination affects the beef and is distributed throughout the product during grinding and mixing operations used during milled-beef production (Lambert et al., 1991). In recent years, research into fresh meat preservation has led to new ⁎
non-thermal deactivation techniques such as edible film coatings, vacuum applications and high-pressure applications (Aymerich, Picouet, & Monfort, 2008; Zhou, Xu, & Liu, 2010). Vacuum packaging is a commonly used packaging method in the meat industry, especially for the transport and sale of fresh meat and for the preservation of quality and shelf-life extension (Jeremiah, 2001). The greatest advantage comes from the consumer's desire to consume meat with the least heat treatment, followed closely by good, fresh meat. When the vacuum packaging applied in meat studies was examined, it was found that the storage period varies between 40 days and 55 days (Fregonesi et al., 2014; Polkinghorne et al., 2018; Sun, Sun, Zheng, Kong, & Liu, 2019). In this study, the storage period was selected to be 45 days. Edible films have become popular in the meat industry due to their low-waste characteristics, low cost and their ability to provide protection after the packaging has been opened (Cha & Chinnan, 2004; Morris, Padmanabhan, Cruz-Romero, Cummins, & Kerry, 2017). Chitosan films have been successfully used as a packaging material to preserve the quality of meat and meat products (Chantarasataporn et al., 2014; Kanatt, Rao, Chawla, & Sharma, 2013). Chitosan has an antimicrobial effect due to its polycationic properties. As a matter of fact, it is effective against mold, yeast and bacteria because it interacts to counteract negatively charged substances. The pKa value of the amine group of the glucose monomer of chitosan, under a pH value of
Corresponding author. E-mail address: [email protected] (A. Duran).
https://doi.org/10.1016/j.meatsci.2019.107961 Received 12 March 2019; Received in revised form 27 September 2019; Accepted 30 September 2019 0309-1740/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Ayhan Duran and Halil Ibrahim Kahve, Meat Science, https://doi.org/10.1016/j.meatsci.2019.107961
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sample of meat was weighed and homogenized with 90 ml of Maximum Recovery Diluent (Merck, 112,535) using a stomacher (MAYO, HG-400, Australia). Then, a series of dilutions up to 10−7 were prepared. Plate Count Agar (PCA, Sigma Aldrich) for Total Mesophilic Aerobic Bacteria (TMAB) (ISO, 4833–2, 2013), Baird Parker Agar (BPA, Sigma Aldrich) for Staphylococcus aureus (ISO, 6888–1, 1999) and de Man Rogosa Sharpe Agar (MRS agar, Merck) for lactic acid bacteria were used for colony counts (ISO 13721:1995/Cor.1, 1996). The selected colonies were subjected to a coagulase assay and verified. For the TMAB analysis, the incubation temperature was 30 ± 2 °C for 48 h. After incubation, colonies on the PCA were counted. In the Staphylococcus aureus analysis, the incubation temperature was 37 ± 2 °C for 48 h. After incubation, the black colonies with a clear zone were identified to be Staphylococcus aureus. For the lactic acid bacteria count, the incubation was at 30 ± 2 °C for 72 h under anaerobic conditions. After incubation the white and opaque zones were identified to be lactic acid bacteria. In addition, as a result of each microbiological analysis, colonies were randomly selected from the Petri dishes and examined microscopically and the results were checked. All the results were given as log cfu (colony forming units)/g values.
6.3, electrostatically interacts with negatively charged microbial cell membranes. As a result, the integrity of the microbial membranes deteriorates, and this leads to leakage of the cell components from the inner and outer membranes (Helander, Nurmiaho-Lassila, Ahvenainen, Rhoades, & Roller, 2001; Liu, Du, Wang, & Sun, 2004; Papineau, Hoover, Dnorr, & Farkas, 1991; Sudarshan, Hoover, & Knorr, 1992). Chitosan also has antioxidant properties. Lipid oxidation in beef is one of the most important factors limiting the storage period, and thiobarbituric acid extraction (TBA) and total volatile basic nitrogen (TVB-N) is an indicator of this. Albeit at a low level, lipid oxidation persists in cold-stored and vacuum-packed meat and products (Djenane, Beltran, Camo, & Roncales, 2016; Jouki & Yazdi, 2014). Primer and secondary antioxidants are used to prevent this oxidation. Primer antioxidants react before oxidation because of their phenolic groups, while secondary antioxidants form chelates with the metal ions that catalyze oxidation. Chitosan has secondary antioxidant activity (Agullo, Rodriguez, Ramos, & Albertengo, 2003). In this study, vacuum packaging (VP) and chitosan coating with vacuum packaging (CC + VP) were applied to beef. The VP meat and (CC + VP) meat were stored for 45 days. In addition, the beef was stored in the refrigerator at 4 ± 1 °C. The aim of the study was to examine the microbiological and chemical changes caused by the vacuum packing and chitosan coating on the beef.
2.5. Chemical analyses During the preservation of meats, the reagents formed as a result of lipid peroxidation are determined by the thiobarbituric acid (TBA) method and show the level of oxidative degradation. The analyses were performed using the method proposed by Tarladgis, Watts, Younathan, & Dugan, 1960Ten gram samples were transferred to a stomacher (MAYO, HG-400, Australia) bag and homogenized. Then, an HCl solution was added for the distillation process and 5 ml of TBA reactive substances was added after the distillation. After that, the distillates were transferred to spectrophotometer tubes to measure the optical density. Finally, the density value obtained was multiplied by 7.80 and the results were given as mg of malondialdehyde in a 1000 g sample. During the preservation process of meats, the total volatile basic nitrogen (TVB-N) method was used to determine the proteolytic degradation. The TVB-N content was determined by distillation after the addition of MgO to the minced sample. The distillate was collected in a flask containing a 3% (w/v) aqueous solution of boric acid and a mixed indicator produced by dissolving 0.1 g of methyl red and 0.05 g of methylene blue to 100 ml of ethanol. The boric acid solution turned green when the distilled TVB-N made it alkaline. Finally, the boric acid solution was titrated with an 0.01 mol/L HCl solution until it turned pink (Goulas & Kontominas, 2005). The quantity of TVB-N in mg/100 g sample was then calculated.
2. Material and methods 2.1. Preparation of meat samples This study used meat prepared from strips of loin (longissimus lumborum). Beef was provided by a local market. For each packaging treatment (VP and CC + VP), 12 samples of 100 g of meat cubes (2 × 2 cm) were prepared. The average shelf life of VP meat and CC + VP meat in the refrigerator (4 ± 1 °C) has been reported to be 45 days in published literature. Therefore, the storage period of the VP and CC + VP treated meats was selected to be 45 days and analyses were carried out on days 0, 15, 30 and 45. 2.2. Preparation of the chitosan solution and its application The chitosan used in the study was obtained from Sigma Aldrich (448,877, USA). Chitosan has a medium molecular weight and is soluble character in organic acids (sorbic acid, p-aminobenzoic acid, lactic acid, acetic acid etc.) In order to prepare the chitosan solution, 10 g of chitosan was weighed and mixed with 500 ml of acetic acid. Then the chitosan solution was stirred with a magnetic stirrer (Velp Scientifica, F20520165, EU) for two days, the first day at high speed and the second day at moderate speed. In this way, the concentration of chitosan used was set to 2% (Cruz-Romero, Murphy, Morris, Cummins, & Kerry, 2013). The beef samples weighing 100 g were immersed in the chitosan solution for 5 s to completely cover them. The meat coated with the solution was placed in a cooling cabinet (Alveo KT, ASE.01, Turkey) at 4 ± 1 °C for 30 min (Cardoso et al., 2019). Following the drying procedure, the film thickness on the meat was measured to be 15 ± 3 μm.
2.6. Statistical analyses Two-factor repeated-measures analysis of variance (ANOVA) was performed using SPSS 22.0.0 software (SPSS Inc., Chicago, USA) to analyze the effects of surface treatments and storage time on microbial quality and lipid oxidation. A completely randomized design included study replication, treatment groups (VP, CC + VP) and storage times (0, 15, 30 and 45 days) as fixed variables. For each storage time, three samples in a treatment group were evaluated. The study was replicated for confirmation later under the same conditions. Means were compared by Duncan's multiple range test at a p value which was calculated for each analysis (Duncan, 1955). Differences among mean values were considered significant when p < .05.
2.3. Applying vacuum packaging For the preparation of vacuum-packed beef, a Turkish Stilea Rimini VS2140 brand vacuum device was used. A 100-g portion of meat was used to fill the vacuum bags (Eurobag & Film S.L., Spain). Vacuum bag permeability is 40 cm3/m2 of O2 under this conditions; 24 h at 23 °C, 75% relative humidity.
3. Results The results obtained in the study are given in Tables 1 and 2. The samples were divided into two groups as vacuum-packed meat and vacuum-packed meat with a chitosan coating according to the application type, and the changes – both within the groups and against time
2.4. Microbiological analyses For microbiological analyzes to be made during storage, a 10-g 2
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Table 1 The microbial changes in the beef during storage (4 ± 1 °C). 0. Day
15. Day
30. Day
45. Day
Total mesophilic aerobic bacteria (log cfu/g) VP meat 4.44 ± 0.12d1 CC + VP meat 4.44 ± 0.12d1
5.05 ± 0.02c1 4.51 ± 0.07c2
5.15 ± 0.22b1 4.58 ± 0.23b2
5.25 ± 0.10a1 4.69 ± 0.21a2
Stapylococcus aureus (log cfu/g) VP meat CC + VP meat
2.00 ± 1.15b1 2.00 ± 1.15b1
2.30 ± 1.31a1 ND
2.00 ± 0.40b1 ND
2.30 ± 0.15a1 ND
Lactic acid bacteria (log cfu/g) VP meat CC + VP meat
2.18 ± 0.01d1 2.18 ± 0.01d1
3.47 ± 0.25c1 2.56 ± 0.42c2
4.48 ± 0.11b1 3.11 ± 0.36b2
6.02 ± 0.98a1 3.67 ± 0.23a2
ND not detected. Each value represents the mean of 6 samples ± standard deviation. Bearing different superscripts row wise (alphabet) and column wise (numeral) differ significantly (P < .05).
– were investigated. During the storage, the TMAB count in the vacuum-packed meat with a chitosan coating was lower than that in the vacuum-packed group. After 45 days, TMAB counts in vacuum-packed meat (5.25 ± 0.1 log cfu/g) were statistically higher than that of vacuumpacked meat with a chitosan coating (4.69 ± 0.21 log cfu/g). During storage, TMAB counts increased steadily in both groups. S. aureus counts in vacuum-packed beef were statistically different from the value on the first day. On day 15 of storage, chitosan coated, vacuum-packed meat inhibited all S. aureus. In both groups, lactic acid bacteria counts increased continuously. At the end of storage, lactic acid bacteria counts were statistically lower in the vacuum-packed meat with a chitosan coating group (3.67 ± 0.23 log cfu/g) compared to that in the vacuum-packed meat group (6.02 ± 0.9 log cfu/g). When the TBA numbers were examined in the table, it was determined that TBA numbers were close to the values of the first day in both groups. As shown in the Table 2, the TVB-N content of samples constantly increased.
TMAB counts in meat by using additional methods. The increase in the TMAB count was lower in the group coated with chitosan. In a study in which a 2% chitosan solution was used to coat ready-to-cook mutton, the samples were stored for 14 days, and TBA, TMAB and S. aureus analyses were carried out. In the meat samples, the initial value of 6.1 log cfu/g was reduced to a range from 3.4 to 6.1 log cfu/g at the end of the storage (Kanatt et al., 2013). In a study in which the shelf-life of chitosan-coated sausage was investigated, the TMAB count at the end of day 15 of storage was close to the initial count (Bostan & Mahan, 2011). As seen in studies on chitosan, it is successful at halting increases in the TMAB count, which agrees with the results obtained in this study. The most important effect of chitosan in halting increases in the TMAB count is attributed to its strong antimicrobial and antifungal properties. Although the antimicrobial property of chitosan was attributed to its polycationic properties, it may also be due to its chelator-like behavior, water-binding properties, activation of the defense processes in the host tissue and inhibiting mRNA synthesis by entering the nucleus (Liu et al., 2004). When the results of the study are examined, it is seen that vacuum packaging suppresses the increase in the S. aureus count. This is thought to be due to S. aureus being a facultative anaerobic organism. However, S. aureus completely disappeared in the group with meat covered by chitosan. Chitosan naturally inhibits the growth of a broad spectrum of microorganisms (Vu, Hollingsworth, Leroux, Salmier, & Lacroix, 2011). Its antimicrobial activity is greatly influenced by its molecular weight with values below 300 kDa reportedly increasing its inhibitory effect on S. aureus, while its suppressive effect on E. coli decreased (Zheng & Zhu, 2003). In a study on vacuum-packed sliced ham, it was found that vacuum packaging reduced the development of S. aureus (Christiansen & Foster, 1965). In another study on chitosan-coated meats, the meats were stored for 48 h at 30 °C and 10 days at 4 °C; the Bacillus subtilis IFO 3025, Escherichia coli RB, Pseudomonas fragi IFO 3458 and Staphylococcus aureus IAM 1011 bacteria were analyzed, and the results showed that chitosan inhibited the growth of these species of bacteria (Darmadji & Izumimoto, 1994).
4. Discussion During storage, the TMAB count in the vacuum-packed meat with a chitosan coating was lower than that in the vacuum-packed meat group. Vacuum packing is capable of effectively improving the quality and shelf-life of packaged meat compared to previously employed methods. However, compared to the VP application, it was found that the single CC + VP application was more effective in reducing the TMAB's proliferation rate. Because the activities of anaerobic microorganisms continue, it is an important factor that the oxygen-free environment is insufficient to stop the growth of microorganisms. In a study on vacuum-packed beef, the samples were stored at +4 °C. The TMAB count was 6.99 log cfu/g on the first day and the count increased continuously to 8.23 log cfu/g after 7 days of storage (Çiçek, Karabıyıklı, Kılınçer, Yıldırım, & Cevahiroğlu, 2014). This study showed that vacuum packaging will be more effective in reducing the rate of proliferation of Table 2 The chemical changes in the beef during storage (4 ± 1 °C). 0. Day
15. Day
30. Day
45. Day
0.22 ± 0.01b1 0.22 ± 0.01b1
0.33 ± 0.02b1 0.24 ± 0.05a2
0.19 ± 0.10c1 0.17 ± 0.01c1
0.22 ± 0.13b1 0.19 ± 0.01c1
Total volatile basic nitrogen (mg/100 g) VP meat 5.41 ± 0.71d1 CC + VP meat 5.41 ± 0.71d1
7.25 ± 0.69c1 6.64 ± 0.80c2
10.66 ± 0.92b1 7.48 ± 1.01b2
14.9 3 ± 1.21a1 8.31 ± 1.55a2
Thiobarbituric acid (mg/1000 g) VP meat CC + VP meat
Each value represents the mean of 6 samples ± standard deviation. Bearing different superscripts row wise (alphabet) and column wise (numeral) differ significantly (P < .05). 3
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Future studies should focus on its use and applications on an industrial scale.
When the results are examined, lactic acid bacteria counts increased during the storage period. However, the increase in lactic acid bacteria counts was lower in the group coated with chitosan. Generally lactic acid bacteria are anaerobic and microaerophilic. When the growth of aerobic spoilage bacteria is inhibited, lactic acid bacteria may become the dominant component of the microbial flora of meats. It has been concluded from the results indicated in Table 1 that the dominant microflora of CC + VP meat is mostly composed of LAB after 15 days. Yet the TMAB counts seems to be more than that of LAB, which is thought to be caused by the inhibition of some LAB species, such as Carnobacterium spp., on MRS agar (Leisner, Laursen, Prevost, Drider, & Dalgaard, 2007). The wide applicability of chitosan allows for the development of various packaging systems for fresh and processed meat. By adding nanocellulose to a chitosan film, Dehnad et al. achieved improved mechanical film properties, as well as a reduction of lactic acid bacteria on ground meat packed in it by 3.1 log cfu/g compared to the control (Dehnad, Mirzaei, Emam-Djomeh, Jafari, & Dadashi, 2014). In other studies, researchers studied Greek-style fresh pork sausages. Samples were coated with chitosan and stored for 28 days. In the control group, the LAB number increased from 4.73 log cfu to 7.90 log cfu, and in the chitosan-coated group, the LAB number increased from 3.34 to 6.18 (Soultos, Tzikas, Abrahim, Georgantelis, & Ambrosiadis, 2008). When the TBA results are examined in the table, it is seen that the TBA numbers are the same in both groups at the end of storage. TBA content is a parameter that can measure lipid oxidation in meats. In addition, TBA count is directly related to oxygen. The fluctuation in the number of TBAs can be thought to be due to the presence of oxygen in the package, albeit at a minimal level after the meat is vacuum packed. At the end of 15 days, after the oxygen is completely exhausted, it can be said that the vacuum bag has a very small amount of oxygen permeability. Normally, chitosan is formed as a barrier that covers the surface of the red meat and thus isolates the food from the air and reduces the oxidation of the lipids in meat (Liu et al., 2004). However, it has only been effective in reducing TBA counts in vacuum packaging. In a study of the effect of vacuum packaging and modified atmosphere in beef, researchers found the lowest TBA value in vacuum-packed meat (John et al., 2005). It is thought that the lipid oxidation is delayed due to the removal of the oxygen in the environment whereas the vacuum packaging is effective on the TBA. As shown in Table 2, the TVB-N content of samples was 5.41 mg N/ 100 g at the beginning, but this value increased to 14.93 mg N/100 g on day 45. Up to day 15, the freshness and appearance was good but it spoiled very quickly thereafter. The TVB-N value is used as an indicator of degradation in meat products. This value is eventually increased by the activities of endogenous enzymes and spoilage bacteria (Kyrana, Lougovois, & Valsamis, 1997). The TVB-N value in chitosan-coated samples on days 15, 30 and 45 was 6.64, 7.48 and 8.31 respectively. The results show that the TVB-N count was lower in chitosan-coated groups (p < .05). In one study, the silver carp fish was coated with chitosan and the TVB-N values were lower (Fan et al., 2009). In another similar study, a 35 to 50% reduction in TVB-N formation in codfish filaments coated with different types of chitosan at the end of day 12 storage period was reported (Jeon, Kamil, & Shahidi, 2002).
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5. Conclusions This study investigated the effect of vacuum packaging, which is a non-thermal food preservation method, and an edible film called chitosan, on the beef during the storage period. The results showed that chitosan prevented the increase of TMAB and lactic acid bacteria counts. Also, chitosan completely inhibited the growth of Staphylococcus aureus. It is understood that vacuum packaging is more effective when the TBA count was influential, but that chitosan is more effective on TVB-N. In conclusion, the study provided proof of both the antimicrobial and barrier properties of chitosan. However, the studies on the production of chitosan films are still at the developmental stage. 4
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