Effect of Essential Oils and Vacuum Packaging on Spoilage-Causing Microorganisms of Marinated Camel Meat during Storage

The use of essential oils (EOs) and/or vacuum packaging (VP) with meats could increase product shelf-life. However, no studies investigating the effect of EOs and VP on camel meat background microbiota have been conducted previously. The study aimed to analyze the antimicrobial effect of essential oils (EOs) carvacrol (CA), cinnamaldehyde (CI), and thymol (TH) at 1 or 2% plus vacuum packaging (VP) on the growth of spoilage-causing microorganisms in marinated camel meat chunks during storage at 4 and 10 °C. VP is an effective means to control spoilage in unmarinated camel meat (CM) and marinated camel meat (MCM) compared to aerobic packaging (AP). However, after EO addition to MCM, maximum decreases in spoilage-causing microorganisms were observed under AP on day 7. Increasing the temperature from 4 to 10 °C under AP increased the rate of spoilage-causing bacterial growth in CM and MCM; however, EOs were more effective at 10 °C. At 10 °C the maximum reductions in total mesophilic plate counts, yeast and molds, mesophilic lactic Acid bacteria, Enterobacteriaceae, and Pseudomonas spp. were 1.2, 1.4, 2.1, 3.1, and 4.8 log CFU/g, respectively. Incorporating EOs at 2% in MCM, held aerobically under temperature abuse conditions, delayed spoilage.


Introduction
The world production of meat is estimated to be around 340 million tonnes per year. Average annual consumption is estimated at 43 kg meat/person, with the level being even higher in developed countries [1]. Camelids are traditionally reared in the Middle East

Camel Meat Samples
On the same day of experiments, fresh camel leg meat was obtained from a local butcher shop. Multiple samples were taken on different days. The meat was transported to the Nutrition and Food Research laboratory, University of Sharjah in an icebox to maintain refrigeration. Using a knife sanitized with 70% (v/v) alcohol and wiped with disposable tissue, the camel meat was portioned into cubes weighing 10.0 ± 0.1 g. Then the meat cubes were stored at 4 • C in a polystyrene tray covered with plastic cling wrap.

Marinade Preparation
A marinade was prepared as per a previously published protocol [15]. It consisted of (g/100 g meat): 20.0 full-fat yogurt, 4.0 crushed tomato, 14.0 crushed onion, 4.0 olive oil, 4.0 vinegar, 1.9 salt, 1.6 kiwi fruit, 1.5 paprika powder, and 0.7 black pepper. For every 100 g of meat, 52 g marinade was used. The fruit and vegetables were washed, and all marinade ingredients were aseptically weighed and mixed in a sterilized stainless-steel bowl.

Preparation of Treatment and Packaging of the Samples
The camel meat samples were randomly divided into eight individual treatments. All analyses were conducted in triplicate. Meat samples without marination (CM) and marinated camel meat (MCM) were considered as control 1 and 2, respectively. To the MCM, 1 or 2% each of CA, CI, or TH was individually added. Care was taken to mix the meat thoroughly. All work was performed under sterile conditions in a biosafety cabinet (5' Purifier logic+ class II, Labconco, MO, USA). The treated products were stored under aerobic (AP) or anaerobic (VP) conditions. Vacuum packaging was performed using sous-vide vacuum pouches 20 cm wide by 15 cm long, which were evacuated and closed using a vacuum-sealing machine (Henkelman, 's-Hertogenboschm, The Netherlands). Both the AP and VP products were kept at 4 and 10 • C for 0, 1, 4 and 7 days.

Microbial Enumeration
After storage, individual samples were aseptically transferred into sterile stomacher bags containing 90 mL peptone water (Himedia, Mumbai, India). Homogenization of the samples was conducted using a stomacher (Interscience, Saint Nom la Brétèche, France) for 1 min. After that, 0.1 or 1 mL of appropriate decimal dilutions was plated in duplicate for Mesophilic total plate count (TPC), yeast and molds (Y&M), mesophilic LAB, and Enterobacteriaceae (EN) via the pour plate method using Plate Count Agar (30 • C for 3 day), Sabaroud Dextrose Agar (SDA, 25 • C for 5 day), De Man Rogosa Sharpe Agar (MRS, with an overlay, anaerobically, 25 • C for 5 day), and Violet Red Bile Glucose Agar (VRBGA, with an overlay at 32 • C for 24-48 h), respectively. A spread plate method was used to enumerate pseudomonads using Pseudomonas Agar supplemented with Pseudomonas CFC, and incubated at 25 • C for 48 h. Subsequently, plates containing 25-250 colony-forming units (CFU) were counted manually using a Stuart ® Colony Counter (Cole-Parmer, Eaton Socon, UK) and the resulting data were transformed into log CFU/g [17].

Statistical Analysis
Two-factor ANOVA including post-hoc analysis (Tukey HSD) was performed to analyze the effects of EO treatments (fixed factor, 8 levels), storage period (fixed factor, 4 levels,) and their interactive effects on the survival of spoilage-causing microorganisms in the yogurt-based marinade [15]. Data were checked for normality via the Shapiro-Wilk test before ANOVA analysis. In addition, an independent T-test was performed to compare aerobic and vacuum packaging. Statistical difference was tested at (p < 0.05). Statistical analysis was carried out using IBM SPSS Statistics (version 26) software. Graphical representations of the analyzed data were prepared using GraphPad Prism Version 7.0 (Graph Pad Software, Inc., La Jolla, CA, USA) [15]. However, the data here are presented in tabular form only.

Results
The effects of marinades with the added active EOs on the indigenous microbiota of CM during 4 and 10 • C storage for up to 7 d are presented in Tables 1-10. Table 1. Population changes of mesophilic total plate count (log (N0/N) ± SD CFU/g) in marinated camel meat with essential oils under aerobic (AC) and vacuum (VC) conditions after storage at 4 • C for 0, 1, 4 and 7 days.
On day 7 under AP, the microbial numbers in CM for TPC, Y&M, LAB, and EN at 10 • C were higher than at 4 • C by 0.7, 0.3, 1.6, and 0.7 log CFU/g, and in MCM they were also higher by 1.7, 3.0, 1.5, and 1.0 log CFU/g, respectively. In contrast, PS numbers were 1.1 log CFU/g higher in CM at 4 • C compared to 10 • C.
When AP and VP treatments were compared after the addition of EOs, the decrease in microbial populations recovered with Y&M, LAB, EN, and PS media towards the end of the 7-d storage period was the highest under AP at 10 • C, in contrast to the control.

Discussion
Camel meat demand is growing in the marketplace because it is perceived to be a healthy alternative based on its greater content of vitamins, minerals and amino acids compared to other red meats [4]. A previous study indicated that EOs are an effective means to destroy pathogenic bacteria in camel meat [15]. The EOs and their concentrations used in this study were evaluated in a previous published study, where it was observed that the EOs did not cause any significant changes in color and texture [15]. Amongst the three EOs used in the study (namely, Ca, Ci a nd Th), the greatest scores in terms of color, taste, texture, flavor and overall acceptability were found when Ci (1 or 2%) was added. The lowest scores in terms of taste, flavor and overall acceptance were observed with a Ca substitution. Overall, the study reported a fair overall acceptability of the EO marinated meat when compared to the control. Thereby, the current study has attempted to characterize any interactive inhibitory effects of vacuum packaging and EO treatment on the spoilage-causing microorganisms of marinated camel meat, in order to improve its shelf-life.
In the present study, storing camel meat without (CM) or with marinade (MCM) up to 7 days aerobically at both 4 and 10 • C increased the numbers of spoilage-causing microorganisms. The increase in microbial populations at 4 and 10 • C in response to AP has been recorded previously; the total viable count (TVC)/TPC in beef and sausages under AP were reported to increase by 1.2-5.3 log CFU/g at 4 • C and 4.1 log CFU/g at 10 • C upon storage for 6-7 day, respectively [18][19][20]. The populations of Y&M increased by 2.1-2.4 log CFU/g at 4 • C when stored for about a week in beef and pork, respectively [18,21]. Meanwhile, LAB under AP was reported to increase by 1.2-4.3 log CFU/g at 4 • C, and by 3.9 log CFU/g at 10 • C, upon storage for about a week [18][19][20][21]. Furthermore, the populations of EN increased by 2.2-4.1 log CFU/g at 4 • C in beef [18,20], while the population of PS under AP increased by 3.8 log CFU/g at 4 • C by 7 days [18].
It was observed in the present study that the microbial population increase towards the end of aerobic storage was higher at 10 • C than at 4 • C (except for PS in MCM). The improved microbial growth at the higher of the two temperatures used here has been reported previously. TPC, Y&M, LAB, EN and PS were higher in ground camel meat stored at 10 • C for 7 days compared to 4 • C by 2.4, 1.9, 0.4, 1.1, and 1.8 log CFU/g, respectively [17]. Similarly, in Greek taverna sausages stored up to 6 d, the TVC and LAB populations at 10 • C were higher by 2.9 and 2.7 log CFU/g in the AP product, while they were higher by 2.4 and 2.6 log CFU/g in the VP product, respectively [19]. Another study reported that the total aerobic numbers and the LAB populations in sausages stored at 10 • C were higher by 3 log CFU/g compared to 4 • C after 14 d storage [22].
In the current study, VP was comparatively more effective than AP in retarding bacterial growth in CM and MCM samples. This is understandable as VP reduces oxygen availability, which is vital to the growth of strictly aerobic microorganisms, and its absence slows the growth of facultative organisms. It has been shown that TVC increased in red meat by 1.4-5.3 log CFU/g when held near 4 • C, and by 4.3 log CFU/g at 10 • C, when stored for about a week under vacuum [18][19][20]23]. Y&M in VP beef was observed to increase by 0.6 log CFU/g at 4 • C by day 7 [18], and in pork it reached 2.9 log CFU/g at 8 d [24]. Similarly, the LAB in red meat increased by 1.6-3.4 log CFU/g at 4 • C and by 4.3 log CFU/g at 10 • C when stored up to a week [18][19][20]23,24]. The EN increased by 0.4-3.3 log CFU/g when meat was stored at 3-4 • C under VP for a week [18,20,23], while the PS increased by 0.9 log CFU/g after storage for 7 days in VP beef [18].
The antimicrobial effects of EOs are ascribed to their ability to disrupt the cell wall/ membrane, inhibit adenosine triphosphate (ATP) production, interrupt protein synthesis and unbalance intracellular pH [25]. It is the phenolic compounds in EOs that mainly exert an antimicrobial effect [26].
The antimicrobial effect observed in the current study is similar to observations made under aerobic conditions, where the addition of 0.3-0.5% CA and TH was seen to reduce the aerobic plate count in marinated beef stored at 4 • C for 7 days [27]. Likewise, adding 1% CI, oregano and thyme EOs to pork stored for 6 d at 4 • C decreased Y&M by 2.6, 1.1, and 1.1 log CFU/g, respectively [21]. A similar observation was made in another study [28]. Thyme EO at 1% in pork stored for 6 d at 4 • C decreased LAB numbers by 0.1 log CFU/g [21]. Decreases in PS in beef upon the addition of 0.4 and 0.8% TH and CA have also been reported [28].
Similarly, when the addition of EOs to MCM was followed by VP, there was an additional antimicrobial effect in the current study. A previous study found that when VP pork had been treated with 0.9% TH and stored for 6 d at 3 • C, the TVC numbers were maintained [23]. Similarly, when ham slices were sprayed with mixed rosemary/liquorice extract, vacuum-packaged, and stored at 4 • C for 28 days, the mesophilic aerobic bacteria (MAB) numbers decreased by 3.2 log [29]. Adding oregano decreased LAB by 2.5 log CFU/g, and when vacuum-packed, decreased PS by 6 log CFU/g in beef stored at 5 • C [30]. Likewise, adding 0.9% thyme EO to pork stored at 3 • C for 6 d maintained EB numbers [23].
When the AP and VP results from the present study are compared, the effectiveness of the EOs is seen to be higher under AP conditions in almost all treatments examined. A similar observation has been made previously [28]. This is contrary to the expectation of an additive antimicrobial interaction of VP with EOs. VP has been reported to result in higher drip losses compared to an AP system with beef [31]. It is thought that the increased moisture developing from the meat exudate would have diluted the antimicrobial action of EOs.
Additionally, it was anticipated that there would be an additive or synergistic inhibitory interaction between the use of 4 • C and the EOs. The maximum decreases in Y&M, LAB, EN, and PS were observed when the meat containing EOs was stored at 10 • C. This can be explained by the higher membrane fluidity observed at higher temperatures [16], thereby facilitating greater microbial disruption by the more volatile EOs. Overall, the maximum decreases in TPC, Y&M, LAB, EN, and PS were 1.2, 1.4, 2.1, 3.1 and 4.8 log CFU/g, respectively, based on the treatment conditions.
Meat is sometimes kept briefly at room temperature to accelerate thawing. Further, the retail refrigerators where marinated meat is stored might not always be able to maintain the mandated refrigeration temperatures due to repeated employee entry/exit. Adding EOs would aid in protecting meat under such temperature abuse from spoilage. Additionally, since EOs would be relatively easy to apply at a commercial level and would not require significant equipment investment, transition to their adoption could be rapid.

Conclusions
These findings illustrate that a greater antimicrobial effectiveness of EOs on marinated camel meat may be anticipated during mild temperature abuse, which can periodically be observed on a practical basis. Antimicrobial effects were enhanced in the presence of EOs if the meat was stored aerobically, rather than under vacuum. Storing MCM with added EOs aerobically at 10 • C was observed to deter spoilage-causing microorganisms to the greatest extent.