Shelf life evaluation of fresh chicken burgers based on the combination of chitosan dip and vacuum packaging under refrigerated storage


The aim of the present study was to investigate the combined effect of chitosan dip (1% w/v) and vacuum packaging on the shelf life of fresh chicken burgers packaged in LDPE/PA/LDPE bags and stored at 4 ± 1 °C for up to 12 days. Furthermore, the possible correlation among microbiological, physico-chemical and sensory indices was investigated. Burger treatments included: aerobic packaging (AP, control), vacuum packaging (VP), chitosan dipping (CHI), and vacuum packaging plus chitosan dipping (VP + CHI). Microbiological [Total viable count (TVC), Pseudomonas spp., Brochothrix thermosphacta, Enterobacteriaceae, Lactic acid bacteria (LAB)], physicochemical [color, pH, total volatile basic Nitrogen (TVB-N), and Thiobarbituric acid (TBA)] and sensory (odor, taste, and texture) analyses were carried out. Results showed that the majority of microbiological, physico-chemical, and sensory analysis parameters varied significantly (p < 0.05) depending on treatment. Based primarily on sensory, followed by microbiological and physico-chemical data, the shelf life of chicken burgers was 4 days for AP samples, 8 days for VP samples, 10 days for CHI treated samples, and 12 days for the VP + CHI treated samples. Finally, a positive and significant correlation (p < 0.05) was observed among most microbiological, sensory, and physico-chemical data, introducing new data relating initial TVC to TVB-N values regarding alternative treatments of minced chicken meat for its optimum preservation.


In recent years, significant concerns have been raised with regard to, the protection and safety of foods. The increasing consumer demand for fresh-unprocessed, nutritious foods, that do not contain synthetic additives, pose challenges to scientists. Recent advances in meat processing and packaging, play a key role in its preservation with parallel quality and safety retention. The quality and safety of fresh meat depends greatly on the effectiveness of emerging preservation technologies applied along with specific packaging materials used. The primary objective of packaging fresh and processed meat products is to delay spoilage, prevent contamination, reduce weight loss and avoid lipid oxidation, thus, maintaining meat attractive color (Mondry 1996; Brody 1997).

At the same time there is a growing interest in using packaging materials originating from renewable energy sources. However, the food industry has to balance the potential protection effect of packaging with other issues, including the energy consumption involved, cost of packaging materials, increased social and environmental awareness and the strict regulations on pollutants and disposal of municipal waste along with consumer preference (McMillin 2017). With regard to the latter issues, the interest in the use of biopolymers in packaging applications has increased, also due to the depletion of fossil fuel reserves as well as the environmental impact caused by the accumulation of non-biodegradable plastic-based packaging materials.

Many biopolymers have been isolated from food waste products to be used in the development of new, biodegradable food packaging materials. Chitosan is such an alternative to synthetic polymers, produced from the shells of crustaceans (Kerch and Korkhov 2011). Chitosan has been classified as GRAS by the USFDA in 2001 (Sagoo et al. 2002). Furthermore, with regard to health claims related to chitosan consumption, EFSA has concluded that a cause and effect relationship has been established between the consumption of chitosan and maintenance of blood LDL-cholesterol levels. In contrast, no cause and effect relationship has been established between the consumption of chitosan and (i) reduction in body weight, (ii) reduction of intestinal transit time and (iii) reduction of body inflammation (EFSA 2011). Desirable properties of chitosan include its film-forming capabilities, mechanical and barrier properties as well as antimicrobial/antioxidant properties for the development of packaging and active packaging containers.

More specifically, mechanical properties allow predicting the behavior of films or rigid containers during transportation, handling and storage of packaged foods. Barrier properties to gases and moisture, in turn, play a key role in maintaining the food product quality (Gertzou et al. 2017). Specific mechanical and barrier properties of pure chitosan films for a given food packaging application can be achieved by combining chitosan with additives such as plasticizers, other polysaccharides, proteins, organic acids, lipids, etc. (Bordenave et al. 2007; Kerch and Korkhov 2011; Šuput et al. 2015). Such additives can modify the properties of the final chitosan biopolymer in relation to the specific needs of the particular food studied in order to extend its commercial shelf life, while maintaining product quality and the biodegradability of the polymer.

Regarding its antimicrobial properties, it has been well documented, that chitosan displays antimicrobial activity against a wide range of food borne filamentous fungi, yeast, gram-negative and gram-positive bacteria (Bordenave et al. 2007). This unique attribute along with its film-forming capacity has made chitosan a reference polymer to develop active packaging with the ability to inhibit microbial growth and improve food safety. Regarding optical properties, pure chitosan films exhibit high light transmittance values in the visible range. This is an important parameter related to the acceptability of packaged goods in chitosan films by the consumer. In addition, chitosan-based films exhibit remarkable UV absorbance, which allows protection of foodstuffs from lipid oxidation induced by UV radiation (Yen et al. 2008).

Current fresh meat packaging practices range from the use simple conventional films [e.g. polyvinyl chloride cling films) for short-term refrigeration (5–6 days)] to VP and modified atmosphere packaging (MAP) for an extended product shelf life (10–12 days) (Kerry et al. 2006). However, of the two, VP is simpler and less expensive than MAP, thus, enjoying wide preference by the food industry and consumers (Van Wezemael et al. 2011). Based on the aforementioned, the objective of the present study was to evaluate the potential shelf life extension of chicken burgers stored at 4 °C using VP, chitosan, and VP + chitosan by monitoring microbiological, sensory, and physico-chemical parameters during storage. A second objective was to investigate whether a positive correlation exists among microbiological, sensory, and physico-chemical parameters of minced chicken meat under the most effective packaging treatment.

Materials and methods

Chicken samples

Fresh chicken fillets were donated by the Agricultural Poultry Cooperative of Pindos Ioannina (Pindos S.A., Ioannina, Greece). Fillet samples were commercially packaged (1 kg/package) in the poultry processing plant and transferred to the laboratory in polystyrene insulated boxes in ice within 1 h post slaughter. Fillets were fed through a clean mincer to produce minced meat. Subsequently, minced chicken meat was used to prepare chicken burgers (90 g per burger) under sterile conditions. All experiments were replicated twice on different occasions with different minced chicken samples Analyses were run in duplicate samples per replicate on each sampling day (n = 2 × 2 = 4).

Packaging conditions, treatments, and materials

Chicken burgers were then placed in low density polyethylene/polyamide/polyethylene (LDPE/PA/LDPE) barrier pouches of dimensions, 29.5 × 29.5 cm, 90 μm in thickness having an oxygen permeability < 15 cm3 m−2 day−1 atm−1, nitrogen permeability < 15 cm3 m−2 day−1 atm−1, carbon dioxide permeability < 100 cm3 m−2 day−1 atm−1 at 75% relative humidity (RH), 23 °C (DIN 53380-2, Germany) and a water vapor permeability of < 1 g m−2 day−1 at 85% RH, 23 °C (DIN 53122-2, Germany)(Gertzou et al. 2017). The pouches were donated by Kapelis Packaging S.A. (Athens, Greece). The chicken burgers were subjected to the following treatments: (a) burgers packaged in air (control) (AP), (b) burgers packaged under vacuum (VP), (c) burgers packaged in air after double immersion in a 1% aqueous chitosan solution (each immersion lasting for 60 s)(CHI), and (d) burgers double immersed in chitosan solution and packaged under vacuum (VP + CHI). Finally, pouches were heat-sealed using a BOSS model N48 vacuum sealer (BOSS, Bad Homburg, Germany) and kept under refrigerated storage (4 ± 1 °C). Sampling was carried out on 0, 2, 4, 6, 8, 10, and 12 days of storage. Eight samples [2 AP, 2 VP, 2 CHI and 2 (VP + CHI)] were analyzed on each sampling day. To ensure aerobic conditions in the control sample, the LDPE/PA/LDPE pouch was punctured with a 23 gauge needle all over its surface. For the preparation of the chitosan solution, chitosan of low molecular weight (MW = 50,000–190,000 Da) and 75–85% degree of deacetylation (Sigma-Aldrich Co., USA) was used. The solution was formed by adding 1 g of chitosan to 1 mL of acetic acid plus 99 ml of deionized water and mixing on a magnetic plate overnight.

Microbiological analysis

TVC, Pseudomonas spp, Brochothrix thermosphacta, Enterobacteriaceae, and LAB were monitored as a function of storage time. Ten grams of chicken burger were removed aseptically from the package using a spoon, transferred to a stomacher bag (Seward Medical, Worthing, West Sussex, UK), containing 90 mL of sterile buffered peptone water (ΒPW, Merck 1.07228.0500, Darmstadt, Germany) (0.1 g/.100 mL of distilled water) and homogenized using a stomacher (LAB Blender 400, Seward Medical, UK) for 90 s at room temperature. For microbial enumeration, 0.1 mL samples of serial dilutions (1:10 diluents, buffered peptone water) of chicken burger meat homogenates were spread on the surface of the following agar plates.

TVC were determined using plate count agar (PCA, LAB010, Heywood UK), after incubation for 2 days at 30 °C. Pseudomonads were determined on cetrimide fusidin cephaloridine agar (CFC, Oxoid CM0559, Basingstoke, UK) after incubation at 30 °C for 2 days. During the preparation of the CFC nutrient agar, an antibody was added (Pseudomonas C-N supplement, Oxoid SR0102E, Basingstoke, England), while colony counting was monitored using the oxidase reaction sticks (Bactident Oxidase, Merck, 1.13300.0001, Darmstadt, Germany).

Brochothrix thermosphacta was determined on Streptomycin Thallous Acetate Agar substrate (STAA, Oxoid CM0881, Basingstoke, UK), after incubation for 2 days at 30 °C. As in the case of pseudomonads, an antibiotic was added (STAA Selective Supplement, Oxoid SR0151E, Basingstoke, UK).

For members of the Enterobacteriaceae family, 1.0 mL of sample was inoculated into 10 mL of molten (45 °C) violet red bile glucose agar (VRGBA, LAB088, Heywood, UK) after incubation for 24 h at 37 °C. The large colonies with purple haloes were counted.

Finally, LAB were determined on Man Rogosa Sharpe Agar (MRS, LAB223, Heywood, UK), after inoculation of the sample in MRS plate and incubation at 37 °C for 3 days. All plates were examined visually for typical colony types and morphological characteristics associated with each medium. Duplicate analyses (n = 2) were performed.

Sensory evaluation

After each sampling day, all samples were frozen at − 18 °C for sensory evaluation. Chicken burgers (ca. 90 g) after defrosting, were cooked in a microwave oven set at high power (700 W) for 100 s. A panel of 7 experienced judges consisting of researchers and faculty of the Food Chemistry and Technology laboratory was used for sensory evaluation (acceptability study). Panelists were asked to evaluate odor, taste, and texture of samples. Each panelist was given 10 g of sample for each treatment, a glass of water and crackers to rinse their mouth between tests. Acceptability of odor, taste, and texture was evaluated using an acceptability scale ranging from 0 to 5 with 5 corresponding to a most liked sample and 0 corresponding to a least liked sample. A score of 3 was taken as the lower limit of acceptability. During each sensory evaluation session eight samples [2 AP, 2 VP, 2 CHI and 2 (VP + CHI)] were evaluated. There were 7 sensory evaluation sessions carried out (days: 0, 2, 4, 6, 8, 10 and 12).

Physico-chemical analysis

Determination of color

The determination of color parameters (L*-lightness, a*-redness, b*-yellowness) was carried out on the surface of chicken burgers (ca. 90 g), which were placed into a cylindrical optical cell of a Hunter Lab model DP-9000 colorimeter coupled to a D25 L optical sensor (Hunter Associates Laboratory, Reston VA, USA). Reflectance values were obtained using manual rotation of plate at 45 mm viewing aperture. Five measurements were recorded for each sample.

Determination of pH

Ten grams of sample were diluted with 100 ml of deionized water in a Stomacher bag, and then homogenized in Lab Blender 400 Stomacher (Seward Medical, UK) for 90 s. The pH was determined in the homogenate using a Delta OHM, model HD 3456.2, pH-meter (Padova, Italy) with a precision of ± 0.002, at room temperature. Each sample was measured in duplicate (n = 2).

Determination of total volatile basic nitrogen (TVB-N)

The determination of TVB-N was carried out according to the method 999.01 of AOAC (2002). Ten grams of chicken burger were homogenized with 100 mL of distilled water in a beaker for 10 min and then centrifuged for 10 min at 3000 rpm. The obtained solution was then filtered and 5 mL of the filtrate along with 5 mL of magnesium oxide 5% (w/v) MgO, 10 mL 10% (w/v) of H3BO3 boric acid, and a few boiling stones were placed in a simple distillation flask. Finally, 5 mL of the resulting distillate were titrated within 5 min with a solution of 0.01 M HCl using an equilibrium mixture of 0.1 g methyl-red and 0.05 g methyl-blue in 100 mL of ethanol as indicator. A blank sample consisting of distilled water and the titration indices was also titrated. The results were expressed in mg N2/100 g of sample.

Determination of thiobarbituric acid (TBA)

TBA was determined according to the method of Chouliara et al. (2007). TBA content was expressed as mg malondialdehyde (MDA)/kg chicken meat.

Statistical analysis

ANOVA and Tukey’s honestly significant test (HSD) (p = 0.05) were used to show differences between treatments during storage, concerning microbiological, sensory, and physicochemical data. Microbiological data were converted to decimal logs of colony forming units (cfu/g). Correlations among the determined parameters were obtained using Pearson’s bivariate statistics. The average and standard deviation values of the microbiological, physicochemical and sensory data were calculated and subjected to statistical analysis. Statistical analysis was carried out using the SPSS software package version 26.0 (2019).

Results and discussion

Microbiological changes

TVC gives a quantitative estimate of the population of microorganisms such as bacteria, yeasts and moulds in a food sample capable of forming visible colonies. The majority of microorganisms present in chicken flesh, either as part of its natural microflora, or as the result of cross contamination from other sources, are mostly aerobic microorganisms and their population is an indicator of product microbiological quality. Figure 1a shows the changes in TVC of chicken burgers as a function of treatment and storage time. As shown in Fig. 1a, the initial value of TVC was 3.19 log cfu/g, indicative of a very good microbiological quality chicken meat. TVC reached 7 log cfu/g, the upper microbiological limit for acceptable quality poultry meat (Chouliara et al. 2007) on day 6 for AP samples. Similar results were reported by Latou et al. (2014). In VP samples, TVC reached the limit of 7 log cfu/g on day 12 of storage. For CHI treated samples and VP + CHI treated the respective TVC values on day 12 were 4.77 and 4.30 log cfu/g. It is obvious that TVC values were significantly higher (p < 0.05) in AP samples compared to all other treatments. This is mainly due to the inhibition of the pseudomonads, which are strictly aerobic by the creation of vacuum and oxygen barrier provided by the chitosan coating formed on the chicken meat surface (Šuput et al. 2015). Furthermore, the exhibited antimicrobial activity of chitosan is due to its positively charged macromolecules which interact with the negatively charged cell membrane of microorganisms altering cell permeability and blocking the transcription of RNA from DNA (Severino et al. 2015). There were no statistically significant differences (p > 0.05) between the CHI and VP + CHI treatments regarding the microbiological shelf life of chicken burgers. This is due to the fact that the effect of chitosan was much greater than that of vacuum. In conclusion, VP increased the microbiological shelf life of chicken burgers by 5–6 days, while chitosan and its combination with vacuum packaging extended the shelf life of minced chicken meat by substantially more than 6 days. Gertzou et al. (2017) reported that TVC of chicken breast meat reached 7 log cfu/g on day 10 of storage using vacuum packaging. In addition, Latou et al. (2014) reported that chicken fillets dipped in 1% chitosan solution and stored for 14 days under refrigeration, maintained TVC values lower than 7 log cfu/g.

Fig. 1

a Growth of TVC in chicken burgers during refrigerated storage. b Growth of Pseudomonads in chicken burgers during refrigerated storage. c Growth of Brochothrix thermosphacta in chicken burgers during refrigerated storage. d Growth of lactic acid bacteria in chicken burgers during refrigerated storage. e Growth of Enterobacteriaceae in chicken burgers during refrigerated storage

The pseudomonads are the major specific spoilage microorganisms of meat, chicken and seafood (Koutsoumanis and Taoukis 2005; Karam et al. 2019). Figure 1b shows the changes in the pseudomonads as a function of treatment and storage time. The initial population of pseudomonads in the control samples was 1.74 log cfu/g. On day 6 of storage, corresponding to the microbiological shelf life of chicken burgers packaged aerobically, the pseudomonads reached a population of 6.09 log cfu/g, while for the VP, CHI, and the combined use of VP + CHI, the respective population was 3.77, 1.39 and 1.17 log cfu/g, manifesting statistically significant differences (p < 0.05) among the different (AP, VP and CHI) treatments. The use of VP significantly (p < 0.05), inhibited the growth of the pseudomonads. Complete removal of air cannot be achieved by VP leading to the partial growth of the pseudomonads (Jay et al. 2005). It has been reported that chitosan binds to the cell membrane of Gram-negative bacteria when the pH is above pKa (Bassi et al. 1999). This phenomenon leads to an altered cell membrane which inhibits the intake of nutrients and trace elements by bacteria (Goy et al. 2009). Another possible mechanism involved is the electrostatic interaction of chitosan with the negatively charged lipo-polysaccharide in the outer membrane which disrupts the cell membrane function (Helander et al. 2001). Latou et al. (2014) reported that the combination of 1% chitosan solution along with MAP, contributed to the reduction of the pseudomonads by 3.3 log cfu/g compared to aerobically packaged chicken breast meat during 14 days of storage.

Brochothrix thermosphacta is a facultative anaerobe, considered along with the pseudomonads, a spoilage specific microorganism for meats packaged aerobically or under modified atmospheres (Remenant et al. 2015). Figure 1c shows the changes of Brochothrix thermosphacta in chicken burgers as a function of treatment and storage time. The population of Br. thermosphacta had an initial value of 1.47 log cfu/g. During early storage the population of Br. thermosphacta increased fairly quickly in AP samples reaching a value of 6.35 log cfu/g on day 6 of storage. On the same day, in VP samples the Br. thermosphacta count reached 4.27 log cfu/g. The treatments with CHI and VP + CHI gave significantly lower (p < 0.05) populations (1.50 log cfu/g) of Br. thermosphacta compared to AP and VP samples. In Gram-positive bacteria, chitosan binds non-covalently to anionic fatty acids of the cell membrane (Raafat et al. 2008). The electrostatic interaction created between the positively charged chitosan and the negatively charged acids, disturbs their function and hence the function of the cell. Sirocchi et al. (2017) stored beef under AP, under VP, and MAP, in the presence and absence of rosemary oil for 21 days. The authors reported that under VP and MAP, in the absence of rosemary oil, the Br. thermosphacta count was lower by 2.0 and 2.7 log cfu/g respectively compared to the AP an on day 10 of storage.

LAB are fermentative facultative anaerobes classified as homo- or hetero-fermenters depending on the nature of fermentation products (Gänzle 2015). They are recognized as important spoilage microorganisms producing mainly sour flavors and butter odors due to the production of lactic acid (Castellano et al. 2004). Figure 1d shows the changes in LAB as a function of treatment and storage time. The LAB initial count was 3.00 log cfu/g reaching 3.52 log cfu/g on day 6 of storage. The same holds for the VP in which the LAB population reached 3.51 log cfu/g on the same day. LAB in both CHI treatments showed a small decrease (p < 0.05) with their population reaching 2.81 and 2.87 log cfu/g respectively on day 6 of storage. An observation to be made is that in all treatments the growth rate of LAB was similar reaching a maximum value of 5.69 log cfu/g in the VP samples. The lowest LAB count (2.6 log cfu/g) was recorded in the CHI treatment with a statistically significant (p < 0.05) difference from the other treatments, between day 8–12 of storage. Based on the above, VP and its combination with CHI had no positive effect on the inhibition of LAB. As mentioned previously, these bacteria are facultative anaerobes and grow easily under vacuum. Regarding the chitosan treatment, the only one that had a positive effect on LAB, this is due to phenomena described in the case of B. thermosphacta.

Given the antimicrobial activity of chitosan, a low population of LAB would be expected in the VP +CHI treatment which was not the case. Therefore, the combination of PV + CHI should be further studied in relation to the growth of LAB. An increase in the population of LAB in VP chicken thighs was also observed by Gertzou et al. (2017). According to Jääskeläinen et al. (2016), after two weeks of beef storage under VP and AP, the genera Lactococcus and Lactobacillus were the predominant ones under VP and the genus Leuconostoc under AP conditions. In both cases, the LAB population exceeded 8 log cfu/g on day 9 of storage.

Finally, Paparella et al. (2016) showed that the use of chitosan and its combination with 2 and 4% oregano oil in pork meat, reduced the LAB count by 2 log cfu/g compared to control samples (modified atmosphere: 70% O2, 20% CO2, 10% N2) on day 13 of storage.

Enterobacteriaceae are facultative anaerobes considered as hygiene indicators in the food industry along with TVC, total coliforms and E. coli. This is of utmost importance in the poultry industry given that muscle foods provide ideal conditions for the growth of both spoilage microorganisms as well as pathogens through contamination during bird slaughtering and subsequent processing (Dias et al. 2017). Enterobacteriaceae grow easily under aerobic conditions, while under anaerobic conditions their growth is largely dependent on the availability of fermentable sugars (Pandey et al. 1999). The initial population of Enterobacteriaceae was 2.27 log cfu/g, as shown in Fig. 1e. In AP samples, their population reached 5.00 log cfu/g on day 6 of storage, while respective values for the VP, CHI and VP + CHI treatments were 4.05, 1.92 and 2.02 logcfu/g. Enterobacteriaceae followed a similar increasing trend both in air and under vacuum (p < 0.05), with AP samples showing a higher population. This finding is reasonable, as these microorganisms grow both in the presence and absence of oxygen. In the treatments of CHI and VP + CHI it is evident that the population of Enterobacteriaceae was kept at low levels throughout storage. Statistically significant differences (p < 0.05). were recorded between AP, VP and both CHI treatments. These results are consistent with those of other researchers. Pavelková et al. (2014) studied the effect of vacuum individually, but also in combination with 1.5%, EDTA, 0.2% oregano oil and 0.2% thyme oil on the shelf life of chicken breast meat. It was observed that in AP (control) and VP samples, Enterobacteriaceae showed a similar growth rate, with their final population reaching 5.66 and 5.60 log cfu/g respectively, after 18 days of refrigerated storage. Likewise, Higueras et al. (2013) reported a reduced growth of Enterobacteriaceae in chicken breast meat packaged in chitosan/cyclodextrin films containing carvacrol. Mahdavi et al. (2017) studied, the effect of chitosan film (CF) with different concentrations of anise essential oil (AEO), (0, 0.5, 1, 1.5 and 2%) on the quality of chicken burgers during chilled storage (4 + 1 °C) for a period of 12 days. CF with AEO, delayed product lipid oxidation while improving chemical propertiy indicators of product quality. Likewise, microbial spoilage parameter values, decreased significantly compared to the control sample. Samples treated with AEO (1.5% and 2%) had acceptable biochemical, bacteriological attributes up to the end of storage. Finally, Petrou et al. (2012) investigated the effect of natural antimicrobials: chitosan (CHI), oregano essential (O) and their combination, on the shelf-life of modified atmosphere packaged chicken breast meat stored at 4 °C for up to 21 days. Results showed decreasing population of all microorganism groups monitored and improvement of all physico-chemical parameter values after treatment with CHI or O individually and their combination.

Sensory changes

Table 1 shows significant (p < 0.05) differences in odor among the different treatments of chicken burgers, throughout storage. Samples AP, VP, CHI treated and VP + CHI reached the lower limit of acceptability on day 4, 8, 10 and at least day 12 respectively.

Table 1 Sensory analysis data of chicken burgers with respect to treatment during refrigerated storage

Taste proved to be an equally sensitive sensory property as odor behaving exactly like odor with regard to the limit of acceptability. The AP samples were not tested beginning with day 6 of storage, based on TVC values exceeding 7 log cfu/g. The same holds for VP samples on day 12 of storage. CHI treated samples exhibited an acidic aftertaste due to the acetic acid used as the chitosan solvent. Finally, the VP + CHI treated samples exhibited a less strong aftertaste than that of chitosan, being acceptable up to day 12 of storage probably due to the partial loss of acidic flavor compounds during the drawing of a vacuum.

Finally, texture showed the same trend as odor and taste. Changes in texture can be affected by changes in insoluble proteins in the muscle fibers as well as in the connective tissue of the chicken (Murphy and Marks 2000). Based on sensory analysis results, the shelf life of the AP, VP, CHI, and VP + CHI treated chicken burgers, was 4, 8, 10, and 12 days, respectively.

Present sensory data are in close agreement with those of Mexis et al. (2012) reporting a shelf life of 4 days for AP ground chicken meat and 7 days for samples containing an oxygen absorber. They are also in good agreement with those of Latou et al. (2014) who reported a shelf life of AP and chitosan treated chicken breast fillets of 5 and 11 days respectively.

Present sensory data are not in perfect line with those of the microbiological analysis (TVC) but in agreement with the reports of other researchers (Chouliara et al. 2007; Khanjari et al. 2013). Differences between the two may be justified by the fact that it is not the TVC, but specific spoilage microorganisms that are responsible for the spoilage of chicken during storage (Jay et al. 2005). Mahdavi et al. (2017) reported an increased shelf life of at least 12 days for chicken burgers packaged in a chitosan film with the addition of anise essential oil at a concentration of 1.5%. Finally, based on sensory data Petrou et al. (2012) reported that both chitosan (CHI) and oregano essential oil (O) individually applied extended the shelf-life of chicken fillets by 6 days, while the combined treatment of (CHI + O) resulted in a product with a shelf-life of 14 days.

Physico-chemical changes

The initial color parameter values for the minced chicken samples were: L* = 60.00 ± 0.32, a* = 8.18 ± 0.22 and b* = 16.44 ± 0.29 (Table 2). In general all three color parameters showed rather large variations as a function of time and treatment. Lightness (L*) decreased (p < 0.05) in AP samples after day 6 of storage indicating that chicken meat became more dull, probably due to decomposition of muscle proteins. L* in VP and CHI treated samples were higher, indicative of the protection provided to minced chicken meat by these treatments. Parameter a* values, related to the degree of oxymyoglobin oxidation varied significantly with no particular pattern in any of the treatments. Such phenomena are of minor importance in chicken meat made up of white rather than red muscle fibers. The same general pattern holds for color parameter b*, expressing the degree of yellowness. Present color parameter data are in general agreement with those reported by Chouliara et al. (2007) and Mexis et al. (2012) for chicken breast meat.

Table 2 Color of chicken burgers with respect to treatment during refrigerated storage

The pH values are shown in Fig. 2. Chicken burger initial pH was 5.5. The pH of AP samples ranged from 5.5 to 5.77; for VP samples from 5.5 to 5.53; for CHI treated samples from 5.5 to 4.92 and for VP + CHI treated samples from 5.5 to 4.99. As shown in Fig. 2, the pH of the AP samples was higher than all other treatments. The chicken burger samples packaged in air showed statistically significant differences (p < 0.05) with those of VP and both chitosan treatments. During storage under AP, aerobic bacteria, mainly the pseudomonads decompose proteins for the production of aminoacids which further, through deamination, produce ammonia related compounds. Such compounds, in turn, raise the pH. In the absence of air (i.e. under VP) the pseudomonads are inhibited leading to reduced protein decomposition and thus to lower pH values. Under VP conditions, preferential growth of LAB occurs leading to the production of lactic acid (Jimenez et al. 1997) which reduces pH. The final substrate pH value is the net result of the two above opposing mechanisms. The result of above two phenomena clearly explain the relative position of pH curves for AP and VP in Fig. 2. Finally, under VP the growth of Br. thermosphacta, is favored which in turn reduces the pH due to the production of lactic acid (Pin et al. 2002), thus, creating an unfavorable environment for the growth of pathogens and Gram (−) bacteria (Jay et al. 2005). The lowest pH values were recorded in the samples containing chitosan. Such a reduction in pH was anticipated given that chitosan was dissolved in acetic acid.

Fig. 2

Variation of pH of chicken burgers in relation to treatment during refrigerated storage

Present results are in agreement with those of Latou et al. (2014) who reported that the combination of chitosan + MAP in chicken fillets gave pH values between 5.85 and 5.96 over 14 days of storage. In AP samples the pH values were higher (6.13–6.28) the first 6 days of storage. Likewise, Mexis et al. (2012) working with ground chicken meat preserved with an oxygen absorber and citrus extract reported an increase in pH for AP samples from 6.38 (initial pH value) to 6.73 on day 6 of storage. Respective pH values for samples treated with citrus extract plus oxygen absorber was 5.83 on day 14 of storage.

As shown in Fig. 3 the initial value for TVB-N (day 0) in chicken burgers chicken was 5.6 mg N2/100 g and reached 32.2 mg N2/100 g for the AP samples, 22.4 mg N2/100 g for the VP samples, 15.2 mg N2/100 g for the CHI treated samples and 12.6 mg N2/100 g for the VP + CHI samples, on day 12 of storage. In all cases, there were statistically significant differences (p < 0.05) among AP samples and all other sample treatments. The two chitosan treatments showed significant differences (p < 0.05) between them only after the 8th day of storage. Significant differences (p < 0.05) were also observed among AP, VP and both chitosan treated samples. According to sensory analysis, the limit of acceptability was reached on day 4 for AP samples, day 8 for VP samples, day 10 for CHI treated samples, and day 12 for VP + CHI treated samples. The corresponding values of TVB-N for these treatments were 16.8, 15.4, 14.5 and 12.6 mg N2/100 g. Based on these values, the limit range of 15–20 mg N2/100 g is suggested as an indicator of freshness for chicken burgers. This proposed acceptability limit for TVB-N, is substantially different than that of 30 mg N2/100 g suggested for pork by Byun et al. (2003). On the other hand, Mexis et al. (2012) working with ground chicken meat reported that the above proposed acceptability limit of 30 mg N2/100 g proved to be a non-dependable freshness indicator since this value was already exceeded on day 0 of storage. An explanation for this is most probably related to the initial TVC of ground chicken meat used (5.9 log cfu/g). Finally, Economou et al. (2009), packaged chicken fillets (initial TVC of 4.3 log cfu/g) under AP and under different packaging conditions (nisin, EDTA, MAP, etc.) and reported TVB-N values in the range of 23.6–40 mg N2/100 g. Based on sensory analysis results, these authors proposed a TVB-N value of 40 mg N2/100 g as an indicator of chicken fillet freshness. The MDA values in chicken burgers were maintained in all treatments and throughout storage at low levels, with a higher value of 0.56 mg MDA/kg (data not shown). This value is substantially lower than the reported perception threshold of 2 mg/kg for oxidized odors (Byun et al. 2003). The very low MDA values recorded can be justified by the very low fat content (less than 4%) of chicken breast meat used in the study. Present MDA values are in excellent agreement with those of Latou et al. (2014) reporting MDA values between 0.19 and 0.80 mg/kg of chicken breast meat and those of Chouliara et al. (2007) reporting MDA values between 0.10 and 0.90 mg/kg of chicken breast meat.

Fig. 3

Variation in TVB-N values of chicken burgers in relation to treatment and storage time

Correlation among microbiological, sensory, and physicochemical parameters

Given that the shelf life of chicken burgers was extended mostly in the VP + CHI treatment, an effort was made to correlate microbiological, sensory, and physico-chemical parameter data. Pearson’s bivariate statistics showed positive and significant correlations (p < 0.05) between specific parameters (Table 3). What is worth mentioning is the negative correlation among sensory parameters and TVC supporting further the role of specific spoilage microorganisms in the deterioration of chicken meat (Jay et al. 2005). TVC was positively correlated to LAB and TVB-N, whereas the pseudomonads were positively correlated to Enterobacteriaceae. At the same time, TVB-N apart from TVC, was positively correlated to LAB, odor, taste, and texture of chicken burgers. The use of color parameters did not provide any supporting information. Taste was positively correlated to Enterobacteriaceae, pH, and TVB-N. Finally, pH was positively correlated to Enterobacteriaceae, odor, taste, and texture.

Table 3 Correlation of microbiological, sensory, and physicochemical parameters of fresh chicken burgers in VP + CHI treatment


With regard to the achievement of the study objectives, the present work has shown that (i) based on sensory but also microbiological, and physicochemical data, a shelf life extension of 4 days, 6 days, and 8 days was achieved for VP, CHI treated and VP + CHI treated chicken burgers respectively, and (ii) a positive and significant correlation (p < 0.05) was found between most microbiological, sensory, and physico-chemical parameters determined. In conclusion, CHI dip combined with VP may be used as an alternative commercial practice for shelf life extension of chicken burgers.



Low density polyethylene/polyamide/low density polyethylene


Aerobic packaging


Vacuum packaging




Total viable count


Total volatile basic nitrogen


Thiobarbituric acid


Lactic acid Bacteria


Modified atmosphere packaging


Analysis of variance


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The authors are grateful to the Agricultural Poultry Cooperative of Pindos Ioannina (Pindos S.A., Ioannina, Greece) for the donation of chicken fillets.

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Assanti, E., Karabagias, V.K., Karabagias, I.K. et al. Shelf life evaluation of fresh chicken burgers based on the combination of chitosan dip and vacuum packaging under refrigerated storage. J Food Sci Technol 58, 870–883 (2021).

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  • Chicken burgers
  • Vacuum packaging
  • Chitosan treatment
  • Shelf life extension