Role of quercetin and rutin as natural preservatives in controlling Listeria monocytogenes in minced meat.

Role of quercetin and rutin as natural preservatives in controlling Listeria monocytogenes in minced meat.

Naglaa, A. ELTaib¹. and Shimaa, N. Edris²

¹ Animal Health Research Institute, Tanta Branch, ARC Egypt

² Food Control Department, Fac. Vet. Med., Benha University,

ABSTRACT.

        The important food industry issue nowadays is how to get good quality, healthy and safe food. To increase food safety and shelf life, the food industry has widely used several synthetic preservatives to inhibit the growth of spoilage and pathogenic bacteria. On the other hand, customers' preference pushed food manufacturers to utilize natural-based preservatives. Therefore, the current study aimed to use quercetin and rutin as an example of natural preservatives to study their effects on L. monocytogenes experimentally inoculated into raw minced beef at a concentration of 10⁴ and10⁶ CFU/g. Then, treated minced meat and control groups were analyzed for L. monocytogenes count, aerobic plate count (APC) for determination of shelf life, pH, and sensory properties during cold storage at 4°C. Initial estimation of pH, L. monocytogenes and APC counts (log cfu/g) were (5.92±0.09 and 6.36±0.05), (3.33±0.2 and 5.65±0.08) and (4.66±0.08 and 5.46±0.2) of C-1 and C-2 groups, respectively. The growth of L. monocytogenes in the treated groups was significantly decreased (P<0.05) as compared to the control groups. The degree of inhibition increased with increasing quercetin and rutin concentration however quercetin (0.06 mg/g) recorded the highest effect on L. monocytogenes population by reduction percent (100% and 29.2%) in Q2 and R2 group at 7^th day of storage period. Furthermore, the applied treatments dramatically decreased APC and pH in comparison to the control samples. C-1 and C-2 groups deteriorated after three days while minced beef meat with a natural preservative lasted for nine days under adequate refrigeration (4 ^oC). Additionally, all sensory properties were improved by using natural preservatives. The activity of the applied treatments was according to the following order: quercetin > combination of quercetin and rutin > rutin, for its effect on the growth of L. Monocytogenes in minced meat stored at 4^oC.

Key words: Listeria monocytogenes (L.M.), minced meat, natural additives, Quercetin   and Rutin.

INTRODUCTION

           Meat and meat products are essential nutrient sources for humans due to their excellent protein content, essential amino acids, vitamin B groups, and minerals (Bohrer,2017). Minced meat is particularly sensitive to microbiological spoilage and chemical changes due to the possible contamination with various microorganisms and elevated contact with oxygen. (Lorenzo et al. 2018). High water activity and nutritional variables make meat and meat products ideal for spoiling microbes and food-borne diseases (Zhou et al. 2010). Foodborne outbreaks have severe adverse effects on public health on a global scale, leading to rising rates of illness and mortality. Listeria monocytogenes, a prevalent zoonotic food-borne pathogen that is motile and Gram-positive, non-spores former, found in an extensive variety of habitats (Park et al. 2021). Listeriosis, a zoonotic illness, where infection occurs through the consumption of contaminated food. Non-invasive listeriosis is a mild, gastrointestinal-only infection that may cause flu-like symptoms. Asymptomatic shedding or gastrointestinal colonization has been documented in both humans and animals (Schoder et al. 2022). According to the Center for Disease Control and Prevention (CDC, 2022), approximate estimates of the number of cases per year are 1600, with 260 fatalities. Pregnant women and their unborn children, seniors 65 years of age and older, and those with compromised immune systems are the groups most prone to contract the L. monocytogenes. Furthermore, listeriosis accounts as fewer than 2% of food-borne infections in the US, with 40% of mortalities. Among food-borne infections, listeriosis has the greatest hospitalization rate (92%) as illustrated by Tack et al. (2020). Without applying any antibacterial strategies, L. monocytogenes can persist in the food processing environment, resulting in potential bacterial cross-contamination among the foods (Jordan & McAuliffe, 2018). The APC is often used in food safety studies to assess its microbiological quality within the determined time. Because of its heightened susceptibility to chemical and microbiological changes, minced beef requires strict controls to overcome the growth of spoilage and harmful bacteria to guarantee consumer safety. Thus, preservatives, heat treatment, drying, freezing, packaging, and beneficial microorganisms have an antagonistic effect on the pathogenic bacteria, are all part of the preservation techniques used to protect food from harmful bacteria and increase its shelf-life (Matthews et al. 2017). In general, chilling is the most preservation method utilized for fresh meat and meat products to increase the duration of refrigerated storage, foods are supplemented with antibacterial and antioxidant chemicals, particularly those derived from synthetic sources (Solomakos et al. 2008). Consumers are more concerned about the worldwide problem of the multi-drug resistance of microorganisms. This has led to a growing trend to seek natural food additives that can be used for the inhibition of chemical and microbiological spoilage of foods (Raudsepp et al. 2018). Increasing consumer awareness about the possible health implications of using synthetic additives has encouraged consumers to ask some requirements for their foods, which should be nutritious, of high quality, and free from any chemical preservative for achieving the safety demands and decreasing the health hazards. This has encouraged meat industries to exploit natural plant derived additives in meat production as an alternative to the use of synthetic ones. Therefore, fruits, vegetables, herbs, and other plant extracts or powders are commonly used as natural preservatives to improve the quality of meat products and extend their shelf-life (Rotimi et al. 2023). As a natural alternative, phenolic compounds possess antioxidant, anti-carcinogenic, anti-microbial, anti-mutagenic, anti-inflammatory, and cardiovascular disease prevention properties (Yang et al. 2017). Flavonoids possess antibacterial properties, and their action is enhanced by hydroxyl groups located at specific places on their aromatic rings. Hydrophobic substituents, including nitrogen or oxygen-containing heterocyclic moieties, prenyl groups, alkylamino chains, and alkyl chains, typically augment the activity of flavonoids (Xie et al. 2014). Quercetin, a significant phytochemical, is classified as a flavonoid polyphenol. It is found in an extensive variety of fruits, vegetables, and beverages, in addition to flowers, leaves, and seeds (Osonga et al. 2019).  Quercetin has also achieved GRAS (Generally Recognized as Safe) status by the United States Food and Drug Organization (Magar and Sohng, 2020). The antimicrobial properties of quercetin make it effective in combating a variety of bacterial infections, including those of the respiratory, integumentary, gastrointestinal, and urinary tract infection (Oliveira et al. 2016). Rutin is a highly prevalent polyphenolic compound that is present in over 70 % of wild plant species. Buckwheat (Fagopyrum esculentum Moench), a member of the Polygonaceae family, is reportedly the most significant natural source of rutin (Kim et al. 2005). Pure rutin takes the form of a yellow powder with a structure of fine crystals. Due to its lipophilic nature, retinoid is soluble in organic solvents, but very poorly soluble in water (Rodríguez-Valdovinos, et al. 2021). The structure of rutin determines its biological activity. The main advantage of rutin is represented by its antioxidant properties. Due to the presence of free hydroxyl groups, rutin could neutralize reactive oxygen species. Rutin protects body cells through reducing free oxygen radicals and subsequently prevents the damage-causing mutations in the cell genetic material. In addition, quercetin-3-O-rutoside as an antioxidant prevents the peroxidation of phospholipids that form the cell membrane and consequently rutin, have antibacterial properties in vitro on bacterial cells. Escherichia coli reduces the damage caused by free radicals. (Jhanji et al.2020). Therefore, the current study was aimed to compare the effects of quercetin and rutin as examples of natural preservatives on the growth potential of experimentally inoculated L. monocytogenes, APC, pH and sensory properties in raw minced meat samples during refrigeration storage at 4^oC.

MATERIAL and METHODS:

1- Preparation of bacterial strain:

Listeria monocytogenes (NCTC 10885) pure strain was obtained from Reference Laboratory for Food Safety, Animal Health Research Institute (AHRI), Dokki, Egypt. The strain approximately 10⁵ CFU/ml was targeted, and cell counts were confirmed by using the Eddy Jet spiral plater (Neutec Group Inc., Farmingdale, NY, USA) with Plate Count Agar (PCA).

2 – Preparation of natural preservatives compounds:

Quercetin and rutin were purchased from I.C.N. Pharmaceuticals (Bryan, OH, USA). All of the compounds are commercially available (powder form) and they were ≥95% pure.

3- Preparation of meat Samples:

 Fresh beef minced meat 1300 gm was used for each trial was purchased from butcher shop at Tanta city, Egypt then transferred directly to the Tanta laboratory in ice box under complete aseptic condition without delay. The sample was divided for two equal batches (Q and R) then sub-divided to 13 sub-group each (100 gm) and immediately, each batch was contaminated with 10⁴cfu/g L. monocytogenes strain suspension (Group Q) and 10⁶ cfu/g (Group R) respectively, except control negative group(C-ve) which remained without strain contamination. The minced meat mixed homogeneously for 3 min to ensure proper distribution of the pathogen and were left for 30 min at 25^οC to allow attachment and absorption of the inoculated bacteria at room temperature (Dubal et al. 2004), then quercetin and rutin were added to the samples in the amount of 0.03, 0.06 and mix of 0.03 mg/g to form 10 treatments groups: C-control negative free from L. monocytogenes and not treated with either quercetin or  rutin ; C-1: 10⁴  CFU/g L. Monocytogenes; Q1: 10⁴ CFU/g L. Monocytogenes in addition to quercetin 0.03 mg/g; Q2: 10⁴ CFU/g L. Monocytogenes + quercetin 0.06 mg/g; Q3: 10⁴ CFU/g L. Monocytogenes + rutin 0.03 mg/g; Q4: 10⁴ CFU/g L. Monocytogenes+ rutin 0.06 mg/g and Q5: 10⁴ CFU/g L. Monocytogenes+ Mix quercetin and rutin0.03 mg/g; C-2: 10 CFU/g L. monocytogenes  R1: 10⁶ CFU/g L. monocytogenes+ quercetin 0.03 mg/g; R2: 10⁶ CFU/g L. monocytogenes + quercetin 0.06 mg/g; R3: 10⁶ CFU/g L. monocytogenes + rutin 0.03 mg/g; R4: 10⁶ CFU/g L. monocytogenes+ rutin 0.06 mg/g and R5: 10⁶ CFU/g L. monocytogenes + Mix quercetin and rutin 0.03 mg/g. The procedures were performed under the laminar-flow cabinet Kojair KR-130 (Kojair Tech Oy, Mänttä-Vilppula, Finland). Finally, each sample was packed in a polyethylene bag, labeled and stored at 4 °C, where examined at days 0, 3, 5, 7, 9 of storage for the following examination: enumeration of L. monocytogenes, Aerobic plate count (APC), pH and sensory evaluation. Each experiment was performed in triplicate.

3.1. Microbiological Analysis: FDA (2001)

       Ten grams of each sample was placed in a stomacher containing sterilized peptone water (90 mL) 0.1% (Merck, Germany) and homogenized. Decimal dilutions of the samples were then prepared then each sample (100 μL) was applied to the surface of Palcam agar media (Oxoid) and then incubated at 37 °C for 48 h for the enumeration of L. monocytogenes in minced meat (Hitchins and Jinneman 2011). Aerobic plate count (APC) was determined by plating appropriate dilution on plate count agar (Difco, USA). The colonies were counted, and the results are expressed as CFU/g of the minced meat sample.

3.2. Determination of pH according to Ben Hsouna et al. (2017).

The pH was determined for the homogeneous mixtures of meat with distilled water in the ratio 1:10, w/v. A minced meat sample (5 g) was homogenized in distilled water (50 mL). The suspension was filtered, and the pH of the filtrate was measured using Consort C833 digital pH meter (Consort, Turnhout, Belgium).

3.3. Sensory Analysis:

      Different sensory attributes (color, odor, texture, and overall acceptability) were determined over the experimental period. A trained 9 members who were regular consumers of minced meat and were selected depending on their availability and willingness to participate in the study, from staff of Department of Food Hygiene Animal Health Research Institute, Tanta Branch using a structured-point hedonic scale with numerical scores ranging from 5 (like very much) to 1 (dislike very much) (Deda et al 2007). Sensory evaluation was carried out individually under controlled conditions of light, temperature, and humidity.

4. Statistical analysis:

The data was statistically analyzed using one-way ANOVA of SPSS program for windows (Version 20) (SPSS Inc. Chicago, IL and USA) and Duncan’s post hoc test with p<0.05 considered to be statistically significant.

Results:

Table (1): Effect of various concentrations of Quercetin and Rutin on APC count and survival of L. Monocytogenes (10⁴cfu/g) experimentally contaminating minced beef samples during storage at 4^oC.

• The values represented by Mean ± SE of three experiments. Means within a column followed by different letters are significantly different (P < 0.05).
• Unaccepted = the recorded APC value exceeded the permissible limit mentioned in ES of minced meat No. (1694/2005).
• Xx = Samples does not examine any more

Table (2): The effect of Quercetin and Rutin at various concentrations on APC count and survival of L. Monocytogenes (10⁶cfu/g) experimentally contaminating minced beef samples during storage at 4^oC.

The values represent Mean ± SD of three experiments. Means within a column followed by different letters are significantly different (P < 0.05).

• Unaccepted = the recorded APC value exceeded the permissible limit mentioned in ES of minced meat No. (1694/2005).
• Xx = Samples does not examine any more

Fig.1: Sensory analysis for (GQ) minced beef during storage at 4^oC.  Values are the means of three individual replicates), Groups (C-ve: control necative,C-1: control pos.10⁴ CFU/g L. monocytogenes; Q1: 0.03mg/g quercetin+10⁴ CFU/g L. monocytogenes., Q2: 0.06mg/g qurecetin+10³  CFU/g L. monocytogenes, Q3: 0.03 mg/g rutin+10⁴  CFU/g L. monocytogenes, Q4: 0.06mg/g rutin+10⁴  CFU/g L monocytogenes., Q5:Mix 0.03mg-g quercetin and rutin+10⁴  CFU/g L. monocytogenes.

  Fig.2: Sensory analysis for (GR) minced beef meat during storage at 4^oC. Values are the means of three individual replicates), Groups (C-ve: control necative,C-2: control pos.10⁶  CFU/g L.M; R1: 0.03mg/g qurecetin+10⁶  CFU/g L. monocytogenes + R2 0.06mg/g qurecetin+10⁶ CFU/g L. monocytogenes, R3: 0.03 mg/g rutin+10⁶CFU/g L. monocytogenes, R4 0.06mg/g rutin+10⁶CFU/g L monocytogenes.,  R5:Mix0.03mg-g quercetin and rutin+10⁶  CFU/g L. monocytogenes.

DISCUSSION:

       Because of safety and health concerns, consumers should impose strict criteria for their food, which must be healthy, high-quality, and synthetic preservative-free. This has prompted meat industries to employ natural additives instead of synthetic ones (Rodríguez-Valdovinos et al.2021). Firstly, Table (1 and 2) discussed the effect of Rutin and Quercetin on the survival of L. monocytogenes Inoculated by (4 & 6) log cfu/g in minced meat during storage time at 4^oC where, the initial count of L. monocytogenes in minced beef samples after inoculation was 3.33±0.2 and 5.65±0.08 log CFU/g, respectively.

Table (1) showed that in the C-1 group, L. monocytogenes count increased from 3.33±0.2 to 3.83±0.05, while the pH was in the range of 5.92±0.09 to 6.73±0.01 at the 3^rd day of storage at which the samples were unaccepted as the APC exceeded the permissible limit according to ES No (1964/2005). In contrast, L. monocytogenes count decreased significantly in minced meat groups treated with quercetin and rutin, or their combinations during refrigerated storage (p < 0.05), where quercetin at 0.06 mg/g (Q2 group) consistently displayed the strongest inhibitory effect on L. monocytogenes count across all storage days and showed completely inhibition of its population at 7^th and 9^th days of storage. While Q4 and Q5 groups maintained lower L. monocytogenes population throughout the storage period till the 7^th day than Q1and Q3 groups.

The obtained results in Table (2) showed that L. monocytogenes level in C-2 group increased from 5.65±0.08 to 6.73±0.1 log cfu/g at which the samples were unaccepted as the APC exceeded the permissible limit according to ES No (1964/2005) and pH was in the range of 6.36±0.05 to 6.5±0.06 at the 3^rd day of storage. However, L. monocytogenes count significantly decreased (p<0.05) from 5.22±0.05 to 4.0±0.05 log cfu/g in R2 from the beginning of the experiment till the 7^th day of storage while, the count elevated once again at the 9^th day to record 4.36±0.05. The mean cunt of L. monocytogenes continued to decrease until reached 4.59±0.08, 4.8±0.02, 4.44±0.03 and 4.37±0.05 log cfu/g in R1, R3, R4 and R5 groups, respectively (p< 0.05) at the 5^th day of storage and begin to increase at the 7the day. From Table (2) the highest reduction levels of L. monocytogenes were in R2 & R5 (4. ±0.05 & 4.41±0.09) both at the 7^th day of storage. This is consistent with the findings of Djenane et al. (2011), who observed a significant reduction in the number of L. monocytogenes in minced beef treated with essential oils (EOs). Nearly like the findings of Vaquero et al. (2011), where quercetin and rutin decrease L. monocytogenes count to 1.22 and 1.12 log CFU/ml when compared to control by 2.18 log CFU/ml, respectively. Also, these findings align with Amin et al. (2015), who identified Quercetin as the most effective flavonoid against methicillin-resistant Staphylococcus aureus with a MIC of 260 μg/ml (MRSA), the authors mentioned that growth inhibition percentage was higher at 5^th day of storage, for both concentration of natural additives investigated. The activity of natural additives towards L. monocytogenes was in dose dependent manner, this means that with increase the concentration of quercetin and rutin, they are more effective where, the groups Q1, Q2, R2, Q4 and  Q5 exhibited the highest reduction percent by 28.83, 100%, 29.2%, 33.63%, and 32.43% , in comparison to  Q3, R1, , R3, R4 and R5 (25.83%, 18.76%, 15.04%,  and 21.42%, and 22.56), respectively. Quercetin alone as well as in combined from with rutin were effective in decreasing the population of L. monocytogenes in minced meat, which is consistent with Vaquero et al. (2011), who reported that the reduction in growth of inoculated L. monocytogenes by mixture of quercetin and rutin at two measured doses (100 and 200 mg/L) in culture medium at 4 °C was 0.14 and 1.54 log cycle, respectively. Quercetin's antibacterial methods involve modifying bacterial cell permeability, breaking cell walls, blocking nucleic acid synthesis, influencing proprotein production, and lowering enzyme activity (Qi et al. 2022). In contrast, the findings of Jhanji et al. 2021, who investigated the antimicrobial capacity of rutin against four specific reference strains, revealed that the growth of B. subtilis, P. aeruginosa, E. coli, and S. aureus was inhibited by approximately 75%, 90%, 70% and 70%, respectively. Also, the effect of natural additives depends on the dose of inoculum where, Q2 group resulted in listeriocidal effect in compared to R2 group which resulted only in bacteriostatic effect, and this agreed with Soriano et al. (2004) who stated that the bacteriostatic effect was observed against Listeria in the batches inoculated with 5 log cfu/g. So, it has been reported that the inhibitory activity of natural additives against L. monocytogenes in minced beef was found to depend on concentration of the additives and the strain of L. monocytogenes used, and its supplementation level at the same storage temperature. Finally, this agreed with Zhang et al (2020) who stated that plant extracts with high flavonoid contents can effectively inhibit the growth of Gram-positive bacteria by damaging the integrity of the cell wall and cell membrane, inhibiting the activity of the intracellular enzymes, changing the expression of associated genes, and inducing bacterial apoptosis.

      In general, Egyptian Standards and Quality Authority Established some criteria for accepting or rejecting the product based on some microbiological parameters including APC count as its permissible limit should not exceed (10⁶ log cfu/g) in minced meat (ES-1694/2005). Table (1and 2) indicates the effect of  natural preservatives treatment in groups Q and R on APC in minced beef samples during storage at 4°C. Initial APC for control samples (C-ve, C1 and C2) was 2.31±0.04, 4.66±0.08 and 5.46±0.2 log cfu/g and then sharply increased with advanced storage to 4.7±0.09, 7.1±0.1 and 7.31±0.3 log CFU/g, at 3 day of storage respectively. In addition, C-ve group was unaccepted on the 5^th day vas 3^rd day for C-1 and C-2. APC at the end of the shelf life was higher than 7 log cfu/g, which is considered a spoilage level for this product and the samples became unaccepted (Fernandez-Lopez et al. 2006). The refrigerated shelf life was seven days for treated groups (Q2, Q4, Q5, R2, R4 & R5), compared to Five days for Q1& Q3, R1 & R3), while C –ve, remained sound till the 3^rd day of storage. In addition, Q2and R2 group at concentration 0.06 mg/ml quercetin appeared to be the most effective inhibitor, where APC decreased from 4.26±0.1 and 4.93±0.2 to 3.28±0.08 and 3.83±0.3 cfu/g at 3^rd respectively. This growth inhibitory effect can be explained by the biggest drop in pH that made the environment unfavorable to microbial growth (Table 1and 2). In the present study, the plant materials were incorporated into minced meat in the form of a powder, as it was found that the natural additives as powder were especially well suited for minced meat, because they are easily and uniformly mixed and all treatments due to its addition to meat helped prolong the shelf life of the products by keeping APC of low level, when compared to controls group.

          Meat pH affects color, tenderness, and microbial growth, all of which are indicators of meat quality and freshness (Bojorges et al. 2020). During refrigerated storage, quercetin and rutin treated meat groups had significantly lower pH values than the control groups (P<0.05), but there were no significant changes in pH values within the treated meat groups (P>0.05) immediately after treatment in some groups. Table 1 and 2 recorded that all samples nearly displayed similar starting pH values ranged from 5.65±0.05 to 5.67 ± 0.04 (Table 1) and from 6.2±0.05, to 6.36±0.05 (Table 2). On the 3^rd day of storage, pH value strongly increased in C-1 and C-2 group, reaching 6.73±0.01 and 6.5±0.05 (table1 and2). Also pH showed strong increase in C-ve group, reaching 6.81±0.11, 6.3±0.05 in compared to Q3 and Q4 groups which recorded lower pH value by 5.45±0.05 and 5.15±0.01 on 5^th day of storage (table 1). While, on 7^th day of storage, samples of Q2 and R2 showed the significantly (p<0.05) lowest values of pH 4.38±0.03 and 6.06±0.03 followed by Q5and R5 4.79 ± 0.01and 6.16 ± 0.03(Table 1and 2).The pH levels increased at the end of the storage period in all treatments, except R2 group recorded 5.9±0.05 on 9^th day of storage (table 2) and this agreed with Biswas et al. (2004) who reported that during storage, the pH values of control samples showed an upward trend, probably due to the formation of basic microbial metabolites or metabolites derived from the deamination of beef protein. Also, this is in line with Pawars et al. (2000), revealed that raw minced meat inoculated with 10³ CFU/g of L. monocytogenes and stored at 4°C had an initial pH of 5.74. Listeria monocytogenes grew well on those meat products with a pH value near or above 6.0, while meats near or below pH 5.0, the organism grew poorly or not at all, for that reason, decreasing the pH with natural additives treatment could contribute to controlling the growth of L. monocytogenes (Glass and Doyle 1989).The increase in pH levels observed in meat samples could potentially be attributed to protein degradation during the final phase of storage. This degradation process facilitates the formation of volatile alkaline nitrogen molecules, such as amines and ammonia, via the action of meat endogenous proteases and microorganisms (Ruan et al. 2019).

       Consumer acceptability is largely affected by meat's sensory properties. The results of sensory evaluation are shown in Fig (1 & 2). Considering overall acceptance of minced meat by panelist, it is evident that panelist liked minced meat alone and they gave the same judgement to the groups with addition of quercetin and rutin powder. In C -ve, C-1 and C-2 groups, then at 5 and 3 days, the panelist mostly disliked color and flavor and would refuse such meat of these groups which, become unaccepted. Due to oxidation processes the color and flavor of the meat have changed. Changes in meat color are due to oxidation of red oxymyoglobin to metmyoglobin, which gives meat an unattractive brown color (Velasco and Williams, 2011). The color of meat is at first glance what is acceptable to the customer since people usually relate color to freshness (Carpenter et al. 2007). As for the minced meat treated with investigated natural preservatives, change in color, odor and texture were recorded by panelists. At zero-day significance differences did not exist (P > 0.05) in color, odor, texture and overall acceptability between control samples and treated minced beef samples with quercetin and rutin (0.03, 0.06 and mix of 0.03 mg/g). Generally, overall acceptability values were higher in samples treated with quercetin and rutin (0.06 and mix of 0.03 mg/g) than other treatment, when compared to control samples and the acceptability scores of panelists were improved till the end of storage period in most of groups. The best accepted samples for color, odor and texture to panelist were arranged as Q2 and R2 groups, followed by Q5, R5, Q4 and R4 groups, finally Q1, R1, Q3and R3. Similar judgements were observed when Salvia essential oils at higher concentration as natural additives significantly improved the sensory characteristics of the minced meat during the investigated storage period than lower one (Ben Akacha et al. 2023). Consumers pay great attention to minced meat color before they decide whether to buy a product. In the present study, all added natural preservative powders changed the color of minced meat depending on what pigments they contained. The color of both control groups was pale pink and was regarded as characteristic of minced meat. The quercetin and rutin powder had a golden-yellow color and simultaneously reduced the normal redness color of meat and which seemed pleasant to the panelists especially with Q2, Q5, R2 and R5 groups. The same effect was reported by Hayes et al. (2013), as natural lipophilic compound, colored the fat in minced meat to a pleasant golden orange.

Conclusion:

       In the present study, Treatments with quercetin and Rutin were effective in reducing populations of L. monocytogenes, APC, improved sensory proprieties and increase the shelf life of minced beef in comparison to controls. The application of Quercetin and Rutin cannot replace the rules of strict hygiene and good manufacturing practice, but it may be used as an additional hurdle contributing to extending the shelf life of raw minced meat. They can be used in many biotechnological fields as a natural preservative’s ingredient of food.