Control of microbial growth and lipid oxidation in beef using a Lepidium perfoliatum seed mucilage edible coating incorporated with chicory essential oil

Abstract In this study, chicory essential oil (CEO) was obtained by hydrodistillation‐based extraction method and it was rich in camphor (31.3%) and phenolic compounds with outstanding antioxidant and antimicrobial properties. The CEO was then incorporated into Lepidium perfoliatum seed mucilage (LPSM) based aqueous solution to prepare an active CEO‐loaded LPSM edible coating. The effect of the edible coating was then investigated on the quality and shelf life of beef slices during 7 days storage at 4°C. The results revealed that beef slice coated with CEO‐loaded LPSM edible coating had a significant inhibitory effect on its lipid oxidation and microbial growth. The CEO‐LPSM coating also inhibited the weight and texture losses of beef slices during display more efficiently compared with the control and CEO‐free LPSM coating. Besides, the beef slices coated with CEO‐LPSM were the preferred samples in terms of sensory scores throughout the storage. Thus, using CEO‐rich LPSM edible coating might inhibit decay and significantly improve the shelf life of fresh beef.

packaging for improving the shelf life of various food products (Seyedi et al., 2015). Nonetheless, edible coatings based on polysaccharides have hardly antioxidant and antimicrobial activity themselves; therefore, food-grade ingredients (e.g., plant-based antioxidant and antimicrobial components) are commonly added to edible coatings to improve their biological and functional properties (Barzegar et al., 2020).
In this context, essential oils (EOs) are frequently used in food preservation technologies as natural and are generally recognized as safe agents, owing to antimicrobial and antioxidant compounds they carry (Calo et al., 2015;Rehman et al., 2020). For example, it has been reported that the lemon/thyme EOs-enriched chitosan coating maintained higher hardness and color and retarded lipid/protein oxidation and microbial growth in grass carp filled during cold storage (Cai et al., 2018). Similarly, Cai et al. (2015) demonstrated that the fresh fish fillets treated with clove, cumin, and spearmint oils maintained the hardness, delayed protein and nucleotide degradation, retarded the sensory deterioration, and inhibited the microbial growth and the formation of biogenic amines.
Chicory (Cichorium intybus L.) is a perennial herb and rich in bioactive compounds for human food fortification purposes. It also has many types of biological properties, such as anticancer, antidiabetic, anti-inflammatory, hepatoprotective, hypolipidemic, antioxidant, and antimicrobial effects (Šaponjac et al., 2021). Moreover, the chicory essential oil (CEO) has remarkable antioxidant and antimicrobial properties (Gol et al., 2014). Accordingly, CEO could be used as a potential natural preservative in edible coatings to amend their oxidation-and microbial growth-suppression functions.
To the best of our knowledge, there is no evidence about the role of the oxidative and microbial stability of meat and meat products wrapped by CEO-loaded LPSM edible coating. The objective of this study was therefore to isolate and characterize the CEO and develop a novel CEO-loaded LPSM edible coating to improve the microbial and oxidative stability of beef slices during cold storage.

| CEO extraction
The CEO extraction was performed according to the hydrodistillation method, which is known as one of the famous, routine, and official standard procedures to extract and control the quality of EOs of medicinal herbs (Taherpour et al., 2017). For the extraction of CEO, the chicory was dried, powdered, and added to the Clevenger device (containing 50 g powder in 750 ml distilled water).
The extraction process was performed for 3 hr at 335 power, according to a method reported by Heydari et al. (2020), with some changes. The oil (CEO) was then collected, dehydrated, and stored at 4°C.

| Gas chromatography-mass spectroscopy (GC/ MS)
A gas chromatograph (GC; Agilent 7890A) coupled to a mass spectrometer (MS; Agilent 5975C) was employed for the identification and quantification of the main chemical compounds of the CEO.
Briefly, 0.2 µl of the oil was injected to the DB-5 capillary column (30 m × 0.25 mm × 0.25 µm) and the heating rate, ionization energy, helium gas flow rate was set at 5°C/min, 70 eV, and 1 ml/min, respectively. Finally, the retention profiles were obtained and compared with those of know samples which were analyzed under the same conditions (Alizadeh Behbahani & Shahidi, 2019).

| Total phenolics and flavonoids content
Total phenolic content of the CEO was measured based on the method described by Ahmed et al. (2019). Gallic acid (0-0.5 mg/ml) was used as a standard to obtain a calibration curve and the total phenolic content of the oil was expressed as mg gallic acid equivalent (GAE)/g. The procedure of Saki et al. (2019) was used to determine the total flavonoid content of the CEO. Briefly, the absorbance of the mixture of CEO, NaNO 2 , AlCl 3 , NaOH, and distilled water was recorded at 510 nm, and the content of total flavonoids of the CEO was then expressed as mg quercetin equivalent (QE)/g.

| Antioxidant activity
In this study, DPPH-radical scavenging (DPPH-RS) activity, ABTS radical scavenging (ABTS-RS) activity, and β-carotene-linoleic acid bleaching assays were used to determine the antioxidant activity of the oil, according to the method described by Barzegar et al. (2020).
The resulting solution was stored at 25°C for 30 min, and its absorbance (A) was read at 517 nm. The DPPH-RS activity was then measured as below: For the ABTS-RS activity, ABTS solution and K 2 S 2 O 8 were initially mixed together to generate ABTS radical cation solution. After that, the CEO (0.1 ml) or control (0.1 ml) was mixed with the ABTS radical solution (3.9 ml) and its absorbance was recorded at 734 nm.
The ABTS-RS activity was then measured as follows: The following equation was used to measure the inhibitory effect of the CEO against β-carotene-linoleate solution bleaching: where, A S(120) is the absorbance of the solution at 490 nm after 120 min incubation, A C(0) is the absorbance of control at the time zero, and A C (0) is the absorbance of control at the after 120 min reaction.

| Antimicrobial activity
The method of Noshad et al. (2018)

| Mucilage extraction
The L. perfoliatum seed mucilage (LPSM) was extracted according to the method of Koocheki et al. (2013). The seeds were dispersed in deionized water (1:30 ratio), and the extraction process was performed for 90 min at pH 8 and 48°C. The slurry was oven-dried (45°C), milled, sieved, and stored at 4°C.

| Beef slices coating
The edible coatings were prepared by mixing LPSM (2 g) and Tween-80 (1 ml) in distilled water (the volume was made up to 100 ml). The CEO (0, 0.5, 1, and 1.5% v/v) was then added. The

| Microbial analysis
To determine the microbial load changes of beef slices during display, the slices were firstly mixed and homogenized with 0.1% peptone water in a Stomacher. The slurry was then added to the test tubes containing 0.1% peptone water to prepare subsequent dilutions (10 -1 to 10 -6 ), which were further inoculated into the plates containing culture medium. The following tests were then performed to evaluate the microbial growth during storage (Alizadeh : • Total viable count (TVC) bacterial count in PCA (48 hr incubation at 37°C) • Psychrotrophic count (PTC) in PCA (10 days incubation at 7°C) • Staphylococcus aureus count in MSA (24 hr incubation at 37°C) • Escherichia coli count in EMB (24 hr incubation at 37°C) • Fungi count in SDA (72 hr incubation at 27°C).

| Moisture content
The beef slices were dried at 105°C for 3 hr, and the moisture content was then determined (AOAC, 1995).

| pH measurement
The beef samples (10 g) were mixed with deionized water (90 ml) and homogenized (30 s, 13,000 rpm, 25°C). The pH value of the slurry was then determined by a pH-meter (Barzegar et al., 2020).

| Hardness
A cylindrical probe was used to compress the beef slices (30% of the sample thickness) at a constant pretest speed, test speed, and post-test speed of 3, 1, and 3 mm/s, respectively, by a Stable Micro System Texture Analyzer (TA, XT2i). The highest force (N) was reported as the hardness of the beef slices.

| Sensory evaluation
The odor, color, texture, and overall acceptance of the coated and control beef slices were evaluated by 25-well trained panelists via a nine-point hedonic scale test. The sensory scores ranked from best (9 = like extremely) to worst (1 = dislike extremely).

| Statistical analysis
Data were analyzed by Minitab software (version 16) via one-way ANOVA. The Tukey test, at confidence level of 95% (p < .05), was applied to determine the differences between the data means. It is also necessary to note that the experiments were done at three replications.
However, the variation found in the concentration and type of the major constituents is due to the fact that the composition of volatile organic compounds is greatly dependent on the genetic factors, harvest time, growing location, storage conditions, nutrients, light, water, and extraction methods (Šaponjac et al., 2021). the CEO is mainly due to the redox properties of its phenolic compounds, thereby acting as hydrogen/electron donors, singlet oxygen quenchers, and metal ions chelation (Indrianingsih et al., 2015).
As can be seen from Figure 1 and  Barzegar et al., 2020). It could be also noteworthy that low levels of CEO were generally enough to possess growth-suppression or killing effect against gram-positive microorganisms, in comparison with the gram-negative ones ( Table 1)

| Application of CEO-loaded LPSM edible coating on beef slices
The CEO with considerable antimicrobial and antioxidant properties was therefore used as a natural preservative to develop novel active edible coatings (i.e., CEO-loaded LPSM coating) to ameliorate quality and shelf life of beef slices. hardness loss (p < .05). The samples coated by CEO-loaded LPSM had significantly higher hardness value compared with the control sample, at the 7th day of storage. This could probably due to microbial growth-suppression and meat enzymes activity-inhibitory effects of the CEO and edible coating, thereby preventing meat protein degradation and subsequent pH increase and texture loss (Mohan et al., 2012;Omidi-Mirzaei et al., 2020).
The edible coating was also able to efficiently inhibit weight loss of the beef slices over time ( Figure 5). Despite the fact that all the samples underwent a significant decrease in moisture content (i.e., higher weight loss) during cold storage, the beef slices It is also necessary to point out that lipid oxidation is considered as one of the main factors of quality deterioration in beef.
The PV and TBA value were then measured as lipid oxidation indices ( Figure 6). As can be observed in Figure 6a, the PV increased significantly during refrigeration storage. The uncoated beef sample experienced the highest PV increase during storage (6.69-fold).
While the PV of the coated samples increased remarkably slowly, and the LPSM + 0%CEO, LPSM + 0.5%CEO, LPSM + 1%CEO, and LPSM + 1.5%CEO showed approximately 5. 85-, 5.95-, 5.71-, and 4.31-fold PV increase as the storage time rose from 1 to 7 days. It is noteworthy that the permitted PV limit for meat is 7 meq O 2 / kg (Alizadeh . Accordingly, the uncoated beef sample exceeded the permitted limit on the 7th day and its shelf life is therefore predicted to be 5  Oxidation and microbial growth in foods are also associated with consumer rejection. As shown in Figure 7, all sensory properties of beef samples (odor, color, texture, and overall acceptance), whether coated or not, decreased progressively as a function of storage time; however, the beef slices with CEO were the preferred beef and all treatments with edible coating received higher scores in comparison with the control. From the panelist points of view, the meat samples could be only accepted when the sensory properties received high scores, greater than 4 (Heydari et al., 2020). In this context, the control sample was unacceptable in terms of all sensory features after 7 days storage, whereas the CEO-loaded LPSM coated beef slices were acceptable throughout the display. Indeed, the beef slices coated by CEO-rich LPSM, which had the highest oxidative and microbial stability, ranked the highest sensory scores, as well. This is mainly due to the antimicrobial and antioxidant activity of the oil and oxygen/water barrier properties of the edible coating, inhibiting oxidation and microbial growth and subsequent quality loss of meat (Alizadeh Barzegar et al., 2020;Kiarsi et al., 2020;Rezaeifar et al., 2020). Moreover, the flavor and odor conferred on the beef by the CEO could also have influenced its consumer acceptance.

| CON CLUS IONS
The chicory essential oil showed a large number of bioactive compounds with superb antioxidant and antimicrobial properties. The incorporation of chicory essential oil into the L. perfoliatum seed mucilage-based edible coating reduced the beef lipid oxidation and microbial growth more efficiently compared with the oil-free coating. The bioactive-loaded edible coating also decreased weight and texture losses during display and improved beef acceptability.
Therefore, edible coatings rich in natural compounds with superb antimicrobial and antioxidant effects could be used in animal meat products to ameliorate their shelf life.

ACK N OWLED G M ENT
The authors would like to express their sincere gratitude to the Vice-

chancellor for Research and Technology of Ferdowsi University of
Mashhad for supporting this study as the project No. 52734.

CO N FLI C T O F I NTE R E S T
The authors have declared no conflict of interest.

E TH I C A L A PPROVA L
This article does not contain any studies with human or animal subjects.