Introduction

Fresh meat presents high protein and variable fat content together with high water activity (>0.95) and an acidic pH (around 5.5). These conditions make it an optimal media for microbial growth ¹. The preservation of fresh food with high water content, such as meat, for short periods of time is usually achieved by storage at low temperature ². In the case of “meat preparations” the European legislation ³ allows the addition of preservatives like antioxidants and seasonings.

Many bioactive compounds synthesized by plants are recognized as effective antimicrobials against a wide range of microorganisms ⁴. As well, it is possible to find other types of natural products, rich in bioactive compounds. One of these products is propolis, an elaborated by bees from natural resinous collected from plants ⁵. Several authors have demonstrated propolis to be a strong antimicrobial agent in many foods, such as sausages, beef patties, minced meat, minced Cyprinus carpio, poultry and burger meat ^5–13. This antimicrobial activity was attributed to the high phenolic compounds and flavonoids content in propolis ^14–17.

Propolis bactericidal and bacteriostatic effects depend on several influence factors, such as, extract concentration, propolis composition, its geographical and botanical origin and extraction method.  Ethanolic extraction method is the most efficient way of taking propolis out of the extract ¹². Nevertheless, it presents disadvantages like a strong residual flavor and the intolerance in sensitive consumers ¹⁸.

In summary, research on propolis indicates its potential to reduce spoilage and extend the shelf-life of some food products. However, propolis’ strong flavor is negatively influenced by the ethanol’s residual flavor, which may limit its use ¹⁹. Therefore, if propolis is to be used in the food industry, more research is needed in order to mitigate its effects on the organoleptic properties of the treated food ²⁰. In this sense, we hypothesize that it is possible to soften the strong flavor of the propolis extract whilst maintaining its preservative effects on refrigerated beef. Consequently, the objective of this work was to evaluate the preservative effects of three propolis-extracts solutions on beef during refrigerated storage. The effects on the sensory quality were also evaluated.

Materials and Methods

Raw Materials

In this study, twenty beef fillets (outside flat) were used. The meat was bought in a local market ten days after slaughter, stored overnight in original packaging (vacuum) at 4 ± 1 ºC and used one day after purchase. Unpasteurized raw propolis and honeydew (which is commonly known in the apiculture business as a bee’s secretion from sources different than flowers) were kindly provided by the Beekeepers Association of Navarre and stored in the dark at 20 ± 2 ºC until use. Propolis and honeydew come from northern Spain, a region called “Comunidad Foral de Navarra”. Regarding the botanical origin, propolis come from aromatic plants, fruit trees, holm oak and forest and honeydew comes from heather.

Propolis Extract and Treatment Solutions

The raw propolis resin was frozen, grinded and extracted using a 1:3 propolis:ethanol (70%, v/v) ratio, following the method described by Keskin ²¹. The obtained Propolis Ethanolic Extract (PEE) was homogenized with water using an Ultraturrax (Model T25 basic IKA, Staufen, Germay) at 11000-13000 rpm to prepare the three treatment solutions (8%, v/v) as follows: PE1 was directly packed into amber flasks after the homogenization. In order to diminish the negative effects of the ethanol flavor on the sensory properties of the meat, PE2 was evaporated for 5 min in a Büchi rotary evaporator R-200 which includes a heating bath (B-490, Flawil, Switzerland) at 38 ± 1 ºC. PE3 was prepared by adding honeydew at a concentration of 10% (w/v) to PE1.

Treatment Application

The beef fillets were cut into 10-g rectangular prisms and the fat and connective tissue were removed. For the physicochemical analyses, 24 pieces of meat/treatment were immersed for 10 min in one of the propolis solutions (PE1, PE2 and PE3) described above and untreated meat was used as the control. Two additional batches were prepared: 12 pieces/treatment for microbiological analyses and 27 pieces of 3x3x1 cm per treatment for the sensory analysis.

The solutions and the immersion time used in the study were selected from preliminary studies conducted in our laboratory (data not shown).

Packing and Storage

After the immersion in the treatment solutions, the samples for the shelf life studies were packed in polyamide/polyethylene bags (PA20/PE70 TR 300.0, Spain), thermally sealed (SACOPISA SB-400-ECO, Barcelona, Spain) and stored under atmospheric conditions at 4 ± 1 ºC for 14 days. For physicochemical analyses, three bags/treatment/evaluation day were used; each bag contained 2 pieces of meat. In addition, three bags/treatment/evaluation day with each bag containing one piece of meat were used for the microbiological analyses.

Analytical Methods

Physicochemical Parameters

The water content, soluble solids, free acidity, electrical conductivity and pH of the honeydew were determined in triplicate according to the official method of honey analysis ²². The pH of the PEE and the treatment solutions was measured in triplicate using a pH-meter (Sentron Europe B.V., Netherlands) equipped with a penetration probe.

Total Phenols Content

Total phenols content of raw materials and treatment solutions was determined following the Folin-Ciocalteu method ²³. The results were expressed as mg gallic acid equivalent per 100 g (honeydew), mL (PEE) or L solution (PE1, PE2, PE3).

Total Flavonoids Content

The flavonoids content of the raw materials and treatment solutions was determined using the aluminum chloride method described by Meda ²⁴. The results were expressed as mg quercetin per 100 g (honeydew), mL (PEE) or L solution (PE1, PE2, PE3).

Antioxidant Activity

Antioxidant activity was evaluated by the 2.2-diphenyl-1-picrylhydrazyl (DPPH) assay, following the method described by Bobo-García ²³. The results were expressed as µmol Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) equivalent per 100 g (honeydew), mL (PEE) or L solution (PE1, PE2, PE3).

Microbiological Analysis of Meat

Total aerobic mesophiles and Enterobacteria were analyzed on day 0, and after 7, 10 and 14 days of storage. For this purpose, 10 g of beef were homogenized in 90 mL sterile buffered peptone water (Cultimed, Spain) using a Stomacher 400 circulator (Seward, UK) for 120 s at high speed. Serial decimal dilutions of each homogenized sample were made in peptone water. From each dilution, 1-mL aliquots were aseptically pour-plated using the following media and culture conditions: (1) PCA (standard plate count) agar (Cultimed, Spain) incubated at 35 ± 2 °C for 48 h for total aerobic mesophiles ²⁵; and (2) VRBG (Cultimed, Spain) incubated at 35 ± 2 °C for 24 h for Enterobacteria ²⁶. All the samples were analyzed in triplicate and microbial counts were expressed as log (CFU/g) of meat.

Physicochemical Parameters of Meat

All the physicochemical parameters were determined in triplicate on day 0, and after 7, 10 and 14 days of storage. For texture, three subsamples per sample were taken with a cork borer; totalizing 9 measurements per treatment and evaluation date.

The pH of the meat was measured at different sites of each sample with a pH-meter (SENTRON Europe B.V. Netherlands) equipped with a penetration probe.

Color: the L*, a* and b* color coordinates were determined using a spectrophotometer (Minolta CM-2500d, Minolta CO, Japan), the CIELab color space ²⁷, the illuminant D₆₅ and a 10º observer.

Texture profile analyses: Texture Profile Analysis (TPA) was performed at room temperature using a TA.XT2icon texturometer and the Exponent lite V.6.1 software (Stable Micro System LTD., Surrey, UK). From each piece of meat, 3 cylindrical subsamples (14-16 mm diameter x 8-10 mm height) were taken and compressed twice to 50 and 75% deformation with a 35 mm-diameter probe. The following texture parameters were determined: hardness (N): resistance at maximum compression during the first compression cycle and cohesiveness (dimensionless): ratio of positive force during the second to that of the first compression cycle ²⁸.

Sensory Analysis of Meat

Immediately after the treatments, a sensorial analysis was carried out to evaluate the effect of the propolis solutions (PE1, PE2 and PE3) assayed on the organoleptic quality of the meat. One sample per treatment was evaluated by a sensory panel (15 female and 12 male) using a hedonic test. The analyses were carried out in individual booths and all the samples were presented coded with random numbers. Immediately before serving, the meat was cooked in a pan with olive oil for 3 minutes on each side.

The appearance (juiciness and general appearance) and global impression were rated from 1 (worst) to 7 (best quality). Color uniformity, from 1 (less uniform) to 7 (uniform). For aroma, the scale ranged from 1 (none) to 7 (full typical aroma). Tenderness was evaluated after the first and second bite with molar teeth, from 1 (least tender) to 7 (most tender). Finally, taste (full taste) was evaluated after eating the sample, from 1 (unpleasant taste) to 7 (pleasant taste). Panelists were asked to indicate if they detected off-flavors or off-odors that might impact their willingness to purchase the product. Scores below 4 in any of the attributes indicated the rejection of the product.

The study was reviewed and approved by the Public University of Navarre and informed consent was obtained from each subject prior to their participation in the study.

Statistical Analysis

Data were subjected to a one-way analysis of variance (α = 0.05) and when significant differences were observed, mean treatments were compared using Tukey’s test. A discriminant analysis (test of equality of means of Lambda Wilks groups; p ≤ 0.05) was also performed with the measured parameters to determinate which were useful in differentiating among the treatments applied. All the statistical analyses were performed with the IBM SPSS Statistics 25 software for Windows (IBM Corp., New York, U.S.A.).

Results and Discussion

Characterization of the Raw Materials and Treatment Solutions

The physicochemical attributes of the honeydew were as follows: water content of 12.69%; soluble solids of 84.39 °Bx free acidity of 22.42 meq/Kg honeydew; electric conductivity of 0.33 mS/cm and a pH of 4.10. Regarding the antioxidant activity and the total content of phenols and flavonoids of the honeydew, the values were 153.79 µmol Trolox, 88.46 mg gallic acid and 10.90 mg quercetin equivalents per 100 g of honeydew, respectively. After the ethanolic extraction, the solution had a pH of 5.10 and contained 13.46% of propolis. The propolis ethanolic extract (PEE) had an antioxidant activity of 677.07 µmol Trolox equivalents and contained 134.75 mg gallic acid (total phenols) and 26.55 mg quercetin equivalents (flavonoids) per mL PEE. The antioxidant activity and bioactive compounds content of the propolis used in this study were higher than those previously reported by Palomino ²⁹; Vargas ³⁰ and Vit ³¹.  The pH, the total phenols and flavonoids content, and the antioxidant activity of the three treatment solutions are listed in Table 1. The lower pH of the added honeydew in PE3 could have caused the pH in this treatment solution to be more acidic. At the same time, the contribution of the honeydew in PE3 is noteworthy, it provoked a significant increase in the antioxidant activity and in the phenolic compound and flavonoid contents of this solution.

Table 1: Characterization of Treatment Solutions.

^†Total phenols, flavonoids and antioxidant activity are expressed as mg gallic acid, mg quercetin and µmol Trolox equivalents per L solution. Values are the mean ± standard deviation (n=3). For each parameter, different letters indicate significant differences (p < 0.05) among treatment solutions.

Meat Quality Evaluation During Storage

Microbiological Analysis

All the applied treatments were effective at reducing the total aerobic mesophiles growth on day 7 with reductions of 0.24, 0.77 and 0.19 logarithmic units for PE1, PE2 and PE3, respectively (Figure 1 A). Furthermore, on day 10 the control samples almost reached the limit established in the European normative ³² of 5x10E5- 5x10E6 CFU/g expressed as 5.70 to 6.70 log CFU/g. On day 14 the mesophiles’ counts were above 7 log (CFU/g), exceeding the legal limit mentioned. During storage, the counts for this microbial group increased for all the samples. However, the tested solutions were effective in slowing down this increase; mesophiles’ counts of PE1-treated meat (6.60 log CFU/g), also complied during the whole storage period within the normative mentioned above. Finally, the samples treated with the PE2 and PE3 solutions exceeded this limit only on day 14.

Our results are in accordance with those of Istrati ³³, who reported a decrease in mesophilic aerobic and lactic acid bacteria until day 14 of storage in vacuum-packed beef marinated with honey solutions prepared at a concentration of 4%. In the same way, Rojo ³⁴ observed an antimicrobial effect of both, floral honey and honeydew against mesophilic aerobic and lactic acid bacteria in beef. The antimicrobial activity of propolis was also demonstrated in other types of meat like chicken breast ⁹ and Piaractus brachypomus fillets ³⁵.

Enterobacteria are considered to be indicators of potential fecal matter, food borne pathogen contamination and as bioindicators of food hygiene ³². The propolis and honeydew solutions were also effective in reducing the Enterobacteria speed growth in comparison with the control samples everyday these were tested (Figure 1 B). The greatest reductions (1.45 logarithmic units) were obtained in the PE2-treated samples.  Similar results were reported by Mehdizadeh and Langroodi ⁹ in chicken breasts treated with propolis extract and in in vitro studies carried out by Petruzzi ²⁰. According to these authors, propolis exerts its antibacterial activity by multiple effects, among which the inhibition of cell division and protein synthesis are included. Furthermore, Pobiega ¹⁸ and Petruzzi ²⁰, stated that the antimicrobial action of PEE is related to the propolis chemical composition, the solvent used for extraction and the PEE dose used in the different assays.

Figure 1: Total Aerobic Mesophiles (A) and Enterobacteria (B) Growth in Control (Untreated) and Treated Meat Samples. Click here to view Figure

pH and Color

Table 2 shows the pH and color evolution of the control and the treated samples during 14 days of refrigerated storage. On day 0, the pH ranged between 5.50 (control) and 5.40 (PE3); these values are similar to those reported by López-Gajardo ³⁶. The lower pH of the treated meat compared to the control samples can be explained by the pH of the solutions applied. Regardless of the treatment, the pH decreased significantly during storage, with the greatest decrease observed in the control samples. In all the evaluation dates, the PE3-treated meat presented the lowest, yet insignificant, pH values. Similar results were found in beef meat treated with either floral honey or honeydew ³⁴, in chicken meat treated with honey ³⁷ and in chicken patties treated with honey and a mixture of spices ³⁸. The general decrease in the pH during storage can be attributed to the increase in the Enterobacterias’ growth throughout the storage period.

Table 2: Changes in pH and Color Parameters of Raw Beef. (Control: Untreated; PE1: Propolis Extract Solution with Ethanol; PE2: Propolis Extract Solution without Ethanol; PE3: Propolis Extract Solution with Honeydew) During 14 Days of Refrigerated Storage.

Values are the mean ± standard deviation (n=3). For each evaluation date, different capital letters indicate significant differences (p < 0.05) among treatments; for each treatment, different lowercase letters indicate significant differences (p < 0.05) among evaluation dates.

Table 2 also illustrates the evolution of L, a* and b* color coordinates of the control and treated samples during refrigerated storage. Color is one of the main quality attributes influencing consumers’ buying decisions of fresh meat at the point of sale ³⁹. More specifically, the a* values that represent an index of redness have been used as an indicator of meat stability during storage ⁴⁰. In this study, the luminosity ranged between 41.78 and 46.86 and the b* values fluctuated between 13.43 and 16.60, which are in accordance with the results reported by Li ⁴¹ for fresh beef. In all the meat samples evaluated, both color attributes remained stable during the storage period, indicating that none of the solutions used caused negative effects on these color parameters. Only slight differences were observed among treatments and between the dates when the samples were tested, but without a clear trend. This could have been influenced by the great variability observed in the superficial color of the raw meat, which in turn, may mask differences among treatments and evaluation dates.

The initial a* value for the control was of 15.31 ± 1.18, which was similar to the values reported by López-Gajardo ³⁶ and Li ⁴¹. After the immersion in the treatment solutions, a significant decrease to 11.65 ± 0.05, 12.38 ± 0.44 and 10.18 ±0.41 was observed in the PE1, PE2 and PE3-treated meat samples, respectively. This could be caused by the dark brown color of the propolis and the honeydew used to prepare the treatment solutions. During storage, a significant decrease in a*, which could be indicative of oxidation, occurred in all the samples. However, this reduction was more pronounced in the control samples as the solutions containing either propolis or honeydew were effective in mitigating this oxidation process. Several authors ^34,36,41,42 reported a decrease in a* value over time. According to these authors, the changes are related to the redox status of the pigment myoglobin, responsible for color evolution in fresh meat.

Texture                   

Texture is an important quality parameter determining the acceptability of fresh meat ⁴³. The hardness and cohesiveness values determined using the TPA method are listed in Table 3. This method, measures the meat resistance to compression simulating the force used by the molar teeth during the mastication ³⁶. On day 0, no significant differences were observed among treatments for any of the texture parameters, indicating that none of the solutions used caused negative effects on the meat samples. The hardness of the meat ranged from 17.33 ± 2.70 to 20.36 ± 9.44 N, which is similar to the hardness reported by Ruiz-de-Huidobro ²⁸ for raw meat. Regarding cohesiveness, the values ranged from 12.17 ± 3.66 to 15.88 ± 4.54, showing that the harder the meat, the lower was its cohesiveness. During storage, all the samples exhibited a similar behavior, with no clear tendency found for texture evolution. This could be attributed to the great variability observed among samples. According to López-Gajardo ³⁶, this variability in instrumental texture usually found in beef can be attributed to the intrinsic properties of the meat such as the fat content and the fiber distribution in each piece. As it can be seen in Table 3, during storage, there were no significant effects of the solutions on the texture of the meat. Similar results were found in beef meat marinated with fruit vinegar ⁴⁴ and pork meat marinated with vinegar and honey ⁴⁵.

Table 3: Changes in Instrumental Texture of Control and Treated Raw Beef Samples During Refrigerated Storage.

Values are the mean ± standard deviation (n=9). For each evaluation date, different capital letters indicate significant differences (p < 0.05) among treatments; for each treatment, different lowercase letters indicate significant differences (p < 0.05) among evaluation dates.

Sensory Evaluation

Regardless of the treatment, the scores for the color, aroma, tenderness and appearance were above the acceptance limit of 4, with slight, yet insignificant differences among the treated meat (Table 4). Similarly, the color, aroma and appearance of chicken fillets ⁴⁶, minced beef ^47,48 and minced Cyprinus carpio ¹¹ were not negatively affected by the addition of propolis extract solutions. On the other hand, the scores for both, the flavor and the global impression were significantly lower in the PE1-treated samples, being the former below the acceptance limit. The presence of ethanol could be the responsible for the unpleasant taste causing the consumer’s rejection of these samples. The evaporation of the ethanol in the PE2 solution was found to be effective at reducing the negative effects on the sensory quality of the meat observed in PE1. Propolis has been studied for its antimicrobial and antioxidant properties in diverse formulations, with variable results in the sensory quality. In effect, due to its intense flavor, which can impair the organoleptic properties of the foods, it is usually added in concentrations lower than 1% ⁴⁹, microencapsulated ⁵⁰ or combined with other ingredients that are able to mask its taste ⁵¹. In our study, despite the fact the differences against PE2 are not statistically significant, meat treated with the PE3 solution, containing honeydew, received the higher scores for all the sensory parameters studied with no off-flavors or off-odors described by the panelists for these samples. Moreover, when asked about their intention to purchase the meat, 66.7% of the panelists answered they would be willing to buy the PE3-treated meat whilst only 11.1% and 29.6% would buy the meat treated with the PE1 and PE2 solutions, respectively.

Table 4: Effect of Propolis Extract Solutions. (PE1: Propolis Extract Solution with Ethanol; PE2: Propolis Extract Solution without Ethanol; PE3: Propolis Extract Solution with Honeydew) on the Sensory Quality of Beef (on an Intensity Scale from 1: Worst to 7: Best Quality)

Values are the mean ± standard deviation (n=27). Different letters indicate significant differences (p < 0.05) among treatments.

Multivariate Analysis

In the discriminant analysis, two functions together, (F1 and F2): (1) and (2), explained  99.7% of the variance. F1 was associated with a positive value of the Enterobacteria counts and a negative value of the color coordinate a*, and explained 97.2% of the variance. F2 explained 2.5% of the variance and was associated with a positive value of the total aerobic mesophiles’ counts. Meat samples could be classified according to both, the treatment applied and the storage time (Figure 2). In effect, in figure 2 it is possible to identify five groups: on the top left part (quadrant II) are the samples of day 0 of all the treatments. From day 7 onwards, the antimicrobial and antioxidant effects of the applied solutions became evident, with the PE1, PE2 and PE3-treated samples conforming the second group in the bottom left part (quadrant III) as the control moved to the bottom right (quadrant IV), together with the treated samples of day 10. The fourth group, located in the top right (quadrant I), comprised the samples from day 14 of all the treated meat together with the control of day 10. This confirms that all propolis and honey solutions were effective in retarding microbial growth and preserving the meat from oxidation. Finally, in the last group only the control samples of day 14 were included. The microbial counts of these samples were above the limits of acceptance established in the European normative ³².

F1=1.080E-0.471a*                                                    (1)

F2=0.928A                                                                (2)

where; E: Enterobacteria; a*: color parameter (red) and A: total aerobic mesophiles.

Figure 2: Discriminant Functions for the Instrumental Parameters and Microbiological Analysis in Treatments: Untreated Meat Samples (C) and Meat Treated with Propolis Extract Solutions: PE1, PE2. PE3, According to Storage Time (From 0 To 14 Days). Click here to view Figure

Conclusion

In this study, the combination of propolis with honeydew and the evaporation of the ethanol from the propolis ethanolic solution, resulted in promising strategies to mitigate the strong flavor associated to propolis. The solution containing propolis extract (8%) in mixture with honeydew (10%) was well accepted by the sensorial panelists, who expressed their willingness to buy the product. Moreover, this treatment demonstrated antioxidant and antibacterial properties when applied to beef. In effect, the oxidation process along with the Enterobacteria and total aerobic mesophiles growth were delayed with no negative effects on the physicochemical quality of the meat. From these results, further studies including the combination of propolis with different natural products (e.g. essential oils) are suggested with the aim of broadening propolis use as a natural preservative in the food industry.

Acknowledgments

The authors thank the Beekeepers Association of Navarre for providing the propolis and honeydew used in this study. Carmenza López-Patiño acknowledges to the University of Cauca (Colombia) for the financial support for her Ph.D. studies.

Funding Sources

The authors received no financial support for the research of this article.

Author Contributions

C. López-Patiño, C. Arroqui and P. Vírseda designed the experiment. C. López-Patiño, collected test data. C. López-Patiño and S. Horvitz interpreted the results and drafted the original paper. C. Arroqui, S. Horvitz and P. Vírseda, reviewed and revised the paper. C. Arroqui and P. Vírseda were both responsible for project administration.

Conflicts of Interest

The authors declare no conflict of interest.

References

1. Pearce K. L., Rosenvold K., Andersen H.J., Hopkins D.L. Water distribution and mobility in meat during the conversion of muscle to meat and ageing and the impacts on fresh meat quality attributes―A review. Meat Sci. 2011; 89(2): 111-124. doi:10.1016/j.meatsci.2011.04.007.
   CrossRef
2. Aguilar-Morales J. Métodos de Conservacion de alimentos. Red Tercer Milenio; 2012.
3. European Commission. Commission regulation No 853/2004 of 30 April 2004 laying down specific hygiene rules for food of animal origin. Official Journal of the European Union. 2004; L 226/22-8.
4. Recio M.C., Rios J.L., Villar A. Antimicrobial activity of selected plants employed in the Spanish Mediterranean area. Part II. Phyther Res. 1989; 3(3): 77-80. doi:10.1002/ptr.2650030302.
   CrossRef
5. Vargas-Sánchez R., Torrescano-Urrutia G., Torres-Martínez B., Pateiro M., Lorenzo J., Sánchez-Escalante A. Propolis extract as antioxidant to improve oxidative stability of fresh patties during refrigerated storage. Foods. 2019; 8(12): 614-629. doi:10.3390/foods8120614.
   CrossRef
6. Ali F.H., Kassem G.M., Atta-Alla O.A. Propolis as a natural decontaminant and antioxidant in fresh oriental sausage. Vet Ital. 2010; 46(2): 167-172.
7. Silici S., Karaman K. Inhibitory effect of propolis on patulin production of penicillium expansum in apple juice. J Food Process Preserv. 2014; 38(3): 1129-1134. doi:10.1111/jfpp.12072.
   CrossRef
8. Viera V.B., Piovesan N., Moro K.I.B., Rodrigues A.S., Scapin G., da Rosa C.S., Kubota E.H. Preparation and microbiological analysis of Tuscan sausage with added propolis extract. Food Sci Technol. 2016; 36: 37-41. doi:10.1590/1678-457X.0045.
   CrossRef
9. Mehdizadeh T., Langroodi A.M. Chitosan coatings incorporated with propolis extract and Zataria multiflora  Boiss oil for active packaging of chicken breast meat. Int J Biol Macromol. 2019; 141: 401-409. doi:10.1016/j.ijbiomac.2019.08.267.
   CrossRef
10. El-Mossalami H.H.A., Abdel-Hakeim Y.A. Using of propolis extract as a trial to extend the shelf-life and improving the quality criteria of fresh Egyptian sausage. Assiut Vet Med J. 2013; 59(139): 23-33.
    CrossRef
11. Payandan E., Sayyed-Alangi S.Z., Shamloofar M., Koohsari H. Study of chemical composition and efficacy of different extracts of Iranian propolis on the microbiological and sensory parameters of minced cyprinus carpio meat at 4°C storage. J Aquat Food Prod Technol. 2017; 26(5): 593-603. doi:10.1080/10498850.2016.1240281.
    CrossRef
12. Casquete R., Castro S.M., Jácome S., Teixeira P. Antimicrobial activity of ethanolic extract of propolis in “Alheira”, a fermented meat sausage. Cogent Food Agric. 2016; 2(1). doi:10.1080/23311932.2015.1125774.
    CrossRef
13. Gutiérrez-Cortés C., Suarez-Mahecha H. Antimicrobial activity of propolis and its effect on the physicochemical and sensoral characteristics in sausages. Vitae. 2014; 21(2):90-96.
14. Bankova V., Galabov A.S., Antonova D., Vilhelmova N., Di Perri B. Chemical composition of propolis extract ACF® and activity against herpes simplex virus. Phytomedicine. 2014; 21(11): 1432-1438. doi:10.1016/j.phymed.2014.04.026.
    CrossRef
15. Marques-das Neves M.V., Sarmento-da Silva T.M., de Oliveira-Lima E., Leitão-da Cunha EV, Oliveira E. de J. Isoflavone formononetin from red propolis acts as a fungicide against Candida sp. Brazilian J Microbiol. 2016; 47(1): 159-166. doi:10.1016/j.bjm.2015.11.009.
    CrossRef
16. Nolkemper S., Reichling J., Sensch K.H., Schnitzler P. Mechanism of herpes simplex virus type 2 suppression by propolis extracts. Phytomedicine. 2010; 17(2): 132-138. doi:10.1016/j.phymed.2009.07.006.
    CrossRef
17. Santana-Andrade J.K., Denadai M., Santos-de- Oliveira C., Nunes M.L., Narain N. Evaluation of bioactive compounds potential and antioxidant activity of brown, green and red propolis from Brazilian northeast region. Food Res Int. 2017; 101(August):129-138. doi:10.1016/j.foodres.2017.08.066.
    CrossRef
18. Pobiega K., Kraśniewska K., Gniewosz M. Application of propolis in antimicrobial and antioxidative protection of food quality―A review. Trends Food Sci Technol. 2019; (83): 53-62. doi:10.1016/j.tifs.2018.11.007.
    CrossRef
19. Osés S.M., Pascual-Maté A., Fernández-Muiño M.A., López-Díaz T.M., Sancho M.T. Bioactive properties of honey with propolis. Food Chem. 2016; 196: 1215-1223. doi:10.1016/j.foodchem.2015.10.050.
    CrossRef
20. Petruzzi L, Corbo M.R., Campaniello D., Speranza B., Sinigaglia M., Bevilacqua A. Antifungal and antibacterial effect of propolis: a comparative hit for food-borne Pseudomonas, Enterobacteriaceae and fungi. Foods. 2020; 9(5). doi:10.3390/foods9050559.
    CrossRef
21. Keskin Ş. Orange peel volatile oil: a green solvent for propolis extraction, enhanced α-amylase inhibition activity. Flavour Fragr J. 2020; 35(4): 411-416. doi:10.1002/ffj.3576.
    CrossRef
22. Presidencia del Gobierno de España. Orden, de 12 de junio de 1986, por la que se aprueban los métodos oficiales de análisis para la miel. Boletín Oficial del Estado. 1986; (145): 22195-22202.
23. Bobo-García G., Davidov-Pardo G., Arroqui C., Vírseda P., Marín-Arroyo M.R., Navarro M. Intra-laboratory validation of microplate methods for total phenolic content and antioxidant activity on polyphenolic extracts, and comparison with conventional spectrophotometric methods. J Sci Food Agric. 2015; 95(1): 204-209. doi:10.1002/jsfa.6706.
    CrossRef
24. Meda A., Lamien C.E., Romito M., Millogo J., Nacoulma O.G. Determination of the total phenolic, flavonoid and proline contents in Burkina Fasan honey, as well as their radical scavenging activity. Food Chem. 2005; 91(3): 571-577. doi:10.1016/j.foodchem.2004.10.006.
    CrossRef
25. S. Food & Drug Administration. Bacteriological analytical manual for food, 8th Edition. 8th edition. Published 1995. https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam
26. Mossel D.A.A., Mengerink W.H.J., Scholts HH. Use of a modified MacConkey agar medium for the selective growth and enumeration of Enterobacteriaceae. J Bacteriol. 1962; 84(2): 381.
    CrossRef
27. Commission Internationale de l’Eclairage. Recomendations of Uniform Color Spaces-Color Difference Equations Psychometric Color Terms. Commission Internationale de l’Eclairage; 1978. doi:10.25039/TR.015.2018.
    CrossRef
28. Ruiz-De-Huidobro F., Miguel E., Blázquez B., Onega E. A comparison between two methods (Warner-Bratzler and Texture Profile Analysis) for testing either raw meat or cooked meat. Meat Sci. 2005; 69(3): 527-536. doi:10.1016/j.meatsci.2004.09.008.
    CrossRef
29. Palomino L.R., García C.M., Gil J.H., Rojano B.A., Durango D.L. Determination of phenolic content and evaluation of antioxidant activity of propolis from Antioquia (Colombia). VITAE, Rev la Fac Química Farm. 2009; 16(3): 388-395.
30. Vargas-Sánchez R.D., Torrescano-Urrutia G.R., Acedo-Félix E., Carvajal-Millán E., Gónzález-Códova A.F., Vallejo-Galland B., Torres-Llanez M.J., Sánchez-Escalante A. Antioxidant and antimicrobial activity of commercial propolis extract in beef patties. J Food Sci. 2014; 79(8): 1499-1504. doi:10.1111/1750-3841.12533.
    CrossRef
31. Vit P., Gutiérrez M.G., Titěra D., Bednař M., Rodríguez-Malaver A.J. Czech honey categorized according to their antioxidant activity. Acta Bioquim Clin Latinoam. 2008; 42(2): 237-244.
32. European Commission. Commission regulation No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Off J Eur Union. 2005; (2073): L 338/1-26.
33. Istrati D., Constantin O., Ionescu A., Vizireanu C., Dinica R. Study of the combined effect of spices and matination on beef meat vacuum packaged. Food Technol. 2011; 35(2): 75-85.
34. Rojo M. Evaluación del empleo de miel artesanal en la conservación de carne picada de ternera. Doctoral thesis. Universidad Politécnica de Cartagena, Spain. Published online 2013.
35. Piedrahíta-Márquez D.G., Fuenmayor C.A., Suarez-Mahecha H. Effect of chitosan-propolis edible coatings on stability of refrigerated cachama (Piaractus brachypomus) vacuum-packed fish fillets. Packag Technol Sci. 2019; 32(3): 143-153. doi:10.1002/pts.2422.
    CrossRef
36. López-Gajardo A. Parámetros de calidad y características sensoriales de la carne de terneros de raza retinta criados en dos modelos de producción ecológica. Doctoral thesis. Universidad de Sevilla, Spain. Published online 2018.
37. Naveena B.M., Sen A.R., Vaithiyanathan S., Muthukumar M., Babji Y. Effect of honey and vitamin C on quality of microwave cooked chicken patties. J Food Sci Technol. 2007; 44(5): 505-508.
38. Alabdulkarim B., Abdel-Nabi-Bakeet Z., Arzoo S. Effect of frying oils on quality characteristics of frozen chicken patties incorporated with honey. African J Biotechnol. 2012; 11(12): 2985-2992. doi:10.5897/ajb11.3389.
    CrossRef
39. Suman S.P., Joseph P. Myoglobin chemistry and meat color. Annu Rev Food Sci Technol. 2013; 4(1): 79-99. doi:10.1146/annurev-food-030212-182623.
    CrossRef
40. García-Esteban M., Ansorena D., Astiasarán I. Comparison of modified atmosphere packaging and vacuum packaging for long period storage of dry-cured ham: effects on colour, texture and microbiological quality. Meat Sci. 2004; 67(1):57-63. doi:10.1016/j.meatsci.2003.09.005.
    CrossRef
41. Li X., Zhang D., Ijaz M., Tian G., Chen J., Du M. Colour characteristics of beef longissimus thoracis during early 72 h postmortem. Meat Sci. 2020; 170(February): 108245. doi:10.1016/j.meatsci.2020.108245.
    CrossRef
42. Hernández-Salueña B., Sáenz-Gamasa C., Diñeiro-Rubial J.M., Alberdi-Odriozola C. CIELAB color paths during meat shelf life. Meat Sci. 2019; 157(July): 107889. doi:10.1016/j.meatsci.2019.107889.
    CrossRef
43. Mittal G.S., Nadulski R., Barbut S., Negi S.C. Textural profile analysis test conditions for meat products. Food Res Int. 1992; 25(6): 411-417. doi:10.1016/0963-9969(92)90165-2.
    CrossRef
44. Sengun I.Y., Turp G.Y., Cicek S.N., Avci T., Ozturk B., Kilic G. Assessment of the effect of marination with organic fruit vinegars on safety and quality of beef. Int J Food Microbiol. Published online 2020. doi:10.1016/j.ijfoodmicro.2020.108904.
    CrossRef
45. Tänavots A., Põldvere A., Kerner K., Veri K., Kaart T., Torp J. Effects of mustard-honey, apple vinegar, white wine vinegar and kefir acidic marinades on the properties of pork. Vet ir Zootech. 2018; 76(98): 76-84.
46. Jonaidi-Jafari N., Kargozari M., Ranjbar R., Rostami H., Hamedi H. The effect of chitosan coating incorporated with ethanolic extract of propolis on the quality of refrigerated chicken fillet. J Food Process Preserv. 2018; 42(1): 1-8. doi:10.1111/jfpp.13336.
    CrossRef
47. Shavisi N., Khanjari A., Basti A.A., Misaghi A., Shahbazi Y. Effect of PLA films containing propolis ethanolic extract, cellulose nanoparticle and Ziziphora clinopodioides essential oil on chemical, microbial and sensory properties of minced beef. Meat Sci. 2017; 124(July 2014): 95-104. doi:10.1016/j.meatsci.2016.10.015.
    CrossRef
48. Shahbazi Y., Shavisi N. A novel active food packaging film for shelf-life extension of minced beef meat. J Food Saf. 2018; 38(6). doi:10.1111/jfs.12569.
    CrossRef
49. Habryka C., Socha R., Juszczak L. The effect of enriching honey with propolis on the antioxidant activity, sensory characteristics, and quality parameters. Molecules. 2020; 25(5). doi:10.3390/molecules25051176.
    CrossRef
50. Bernardi S., Favaro-Trindade C.S., Trindade M.A., Balieiro J.C.C., Cavenaghi A.D., Contreras-Castilo C.J. Italian -type salami with propolis as antioxidant. Ital J Food Sci. 2013; 25(4): 433-440.
51. Spinelli S., Conte A., Lecce L., Incoronato A.L., Del Nobile M.A. Microencapsulated propolis to enhance the antioxidant properties of fresh fish burgers. J Food Process Eng. 2015; 38(6): 527-535. doi:10.1111/jfpe.12183.
    CrossRef