Open Veterinary Journal, (2025), Vol. 15(7): 3300-3307

Research Article

10.5455/OVJ.2025.v15.i7.41

Antioxidant and antimicrobial effect of butylated hydroxylanisole and carnosic acid on lamb meat under refrigerated storage

Mohammed Kadhim Wali^1*, Dheyaa Hussein Al-Alwani², Marwa Mohammed Abed³, Mohammed Ibrahim Rahma¹, Ameer Ridha Dirwal¹, Haneen Hazim Ghazi¹, Marwah Hussien Ali Hussein¹ and Walaa Farhan Obied¹

¹Veterinary Public Health, College of Veterinary Medicine, Al-Qasim Green University, Al Qasim, Iraq

²Department of Food Health and Nutrition, College of Food Science, Al- Qasim Green University, Al Qasim, Iraq

³Kufa Technical Institute, Al-Furat Al-Awsat Technical University, Najaf, Iraq

*Corresponding Author: Mohammed Kadhim Wali. Veterinary Public Health, College of Veterinary Medicine, Al-Qasim Green University, Al Qasim, Iraq. Email: mohammed87 [at] vet.uoqasim.edu.iq

Submitted: 05/12/2024 Revised: 13/05/2025 Accepted: 25/06/2025 Published: 31/07/2025

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ABSTRACT

Background: The main causes of quality degradation of meat products are microbial growth and lipid oxidation, which decrease their shelf life. Carnosic acid, also known as CA, is a phenolic compound extracted from rosemary leaves and is considered to be a natural antioxidant. It can remove free radicals. Butylated hydroxyanisole (BHA) is a synthetic antioxidant that is widely used in meat products.

Aim: CA and BHA have antioxidant properties that protect cell membranes from destruction, thus protecting proteins from decomposition, water from escaping, and delaying lipid peroxidation.

Methods: Meat samples were divided into five groups: T1 control treatment (without adding), T2: treatment with 100 ppm CA, T3: treatment with 150 ppm CA, T4: 200 ppm treatment with CA, and T5: treatment with 150 ppm BHA. The samples were kept at 5°C for a period of 7 days.

Result: All sample treatments with BHA and CA for ground lamb meat during the refrigerated storage period led to a reduction in drip loss value, thiobarbituric acid value, and total bacterial count compared with those in the control group. According to the results, there was a significant difference (p < 0.05) increase in the water-holding capacity and pH values in all samples treated compared with the control during the storage period.

Conclusion: The addition of BHA and CA, which are antioxidant and antibacterial agents, can enhance the shelf life of lamb meat. Additionally, the addition of CA was more effective than the addition of BHA in terms of the oxidation and quality characteristics of fresh lamb meat during refrigerated storage at 5°C.

Keywords: Meat, Lipid oxidation, Carnosic acid, BHA.

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Introduction

Meat and meat products are exposed to physical and chemical changes, which make them sensitive and easily affected by oxidation and spoilage (Al-Shibli et al., 2024). These changes start from the slaughtering process, through the preparation of the carcass inside the slaughterhouse, and then to the market. However, external environments, equipment, and workers are sources of meat contamination (García-Díez et al., 2023).

Microbial growth and lipid oxidation are the two leading factors for quality deterioration of meat quality because they decrease the shelf life of meat and meat products (Alam et al., 2017). Additionally, microbial growth causes foodborne illness and economic losses in meat (Al-Salmany and Al-Rubeii, 2020).

Oxidation is also one of the causes of meat spoilage and corruption, as it is one of the important problems facing meat manufacturers, which produce secondary compounds such as aldehydes, ketones, alcohols, and others, which lead to the production of undesirable flavors and odors in meat during the storage period (Al-Shibli et al., 2024). Oxidation can be reduced using different types of antioxidants, either natural or synthetic antioxidant forms such as carnosic acid (CA) and butylated hydroxyanisole (BHA) (Sadakuzzaman et al., 2021).

Rosemary is one of the few herbs used in the food industry due to its antioxidant properties, whether raw or extracted, whereas some other spices are used only to add the desired flavor. A large group of phenolic compounds has been isolated from the rosemary plant, including CA, carnosol, and rosmarinic acid (Yao et al., 2023).

CA is a phenolic compound extracted from rosemary leaves and considered one of the most potent antioxidants among terpene compounds, as it has the ability to remove free radicals (Al-Alwani, 2017). Pure CA is also interesting, as it has begun to be commercially available in its pure form and has good antioxidant activity compared to other compounds (Jordán et al., 2012). It also does not contain any odor, so it can be added to raw or cooked meat. CA has antimicrobial and antioxidant activity and contributes to improving the quality characteristics and undesirable flavors of minced lamb stored in the refrigerator (Al-Alwani, 2017).

BHA is a synthetic antioxidants that is widely used in meat products and used to retard or minimize the oxidative deterioration of foods (Zhang et al, 2023). It may be added to meat and meat products to prevent oxidation and extend their storage life (Kumar et al., 2015). The aim of this study was to investigate the effect of the addition of BHA and CA on the shelf life and quality of fresh lamb meat during refrigerated storage at 4°C.

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Materials and Methods

Samples preparation

Lamb meat without bones was used after removing the external fat on the surface, and meat samples were cut into medium-sized pieces using a knife. The tools used were clean and sterilized, in addition to gloves, to prevent contamination of the meat as much as possible. The meat was minced using an electric mincer with a 0.7 cm sieve diameter, and the meat parts were homogenized to ensure an equal distribution of the thigh muscle components. The meat was divided into five parts of 250 g; each part was allocated to one of the study treatments, where the additives were used on the minced meat. The samples were then divided into five groups and treated with different concentrations of CA and BHA (100, 150, and 200 ppm) of CA and (150 ppm) of BHA. Then, the samples were stored for 7 days at 5°C. Finally, sample analysis was performed on days 0 hours, 3 and 7 days of refrigerated storage at 50°C.

Physicochemical traits

Drip loss measurement

Five grams of meat samples from each treatment were placed in polyethylene bags and stored in a refrigerator at 4°C for 24 hours according to the method described by Young and Lyon (1997). The samples were then redrying, after drying, and the liquids were removed from the surface of the meat samples via filter paper.

Water-holding capacity

Fifty grams of each treatment mixture were weighed and homogenized with 50 ml of distilled water for 1 minute, according to methods of Sadakuzzaman et al. (2021), after which the mixture was placed in a centrifuge at a temperature of 4°C and a speed of 5,000 rpm for 10 minutes, after which the percentage of the meat’s ability to retain water was calculated according to the following equation:

The PH value is expressed as follows

The pH of the minced meat was measured according to the method described by Verma et al. (2008) by homogenizing a 10 g minced meat sample with 100 ml of distilled water via a German Gerhardt homogenizer. The sample solutions were subsequently centrifuged for 15 minutes at 2,000 gx. The mixture was filtered through filter paper No. 1, 1 and the pH was measured using a pH meter (Sadakuzzaman et al., 2021).

Determination of thiobarbituric acid

Approximately 1 g of the meat sample was homogenized with 25 ml of a cold solution containing 20% trichloro acetic acid (TCA) dissolved in phosphoric acid with a concentration of 2 M in a homogenizer for 2 minutes. The mixture was transferred to a 50-ml volumetric flask, and the volume was filled to the mark with distilled water. The mixture was mixed, and 25 ml was collected and centrifuged at a speed of 3,000 rpm for 30 minutes and filtered through filter paper No. (1). Then, transferred 5 ml of the filtrate to a test tube and 5 ml of thiobarbituric acid reagent solution to it [dissolve a 1 g powder of TBA reagent with 75 ml of double distilled water (90°C–100°C) and mix thoroughly]. Then, 30 ml of N 0.6 of glacial acetic acid was added mixed fully with 50 ml of 25% TCA solution, and cooled to room temperature. Prepared 0.005 M. Blanc’s solution was prepared by mixing 5 ml of distilled water with 5 ml of the reagent solution. The contents of the test tubes were mixed well, closed tightly, and kept in a dark place for 15–16 hours at room temperature. The contents were heated in a water bath for 30 minutes (Tarladgis et al., 1964). The absorbance (A) of the resulting color was measured at a wavelength of 530 nm using a spectrophotometer. The thiobarbituric acid value was calculated by multiplying the absorbance value by the factor 5.2, and the value was expressed on the basis of mg malondialdehyde/kg meat according to the following equation: thiobarbituric acid value (mg MDA/kg meat)=5.2 × A530.

Microbiological counts

A total of 10 g of lamb meat sample was homogenized with 90 ml of peptone buffer solution for microbial assessment of the total bacteria count and coliform count. The dilutions were prepared from the 10¹–10⁹ dilution and then cultured via the pour plate method using the diluted 10⁻⁶ and enumerated after incubation at 32°C for 24 hours. The cultured media consisted of plate count agar and violet red bile agar (Wali and Abed, 2019; Terrones-Fernandez et al., 2023).

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Static Analysis

All data were estimated as the mean ± SD of the experimental data. The data were analyzed using SPSS for analysis of variance (p < 0.05) using (ANOVA) on a two-way basis (SPSS, 2008). The number of repetitions for the analyses (n value) was five.

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Result and Discussion

Physical characterized

Drip loss value of lamb meat

As shown in Figure 1, there were significant differences (p < 0.05) lower on drip loss value in all groups: 0, 3, and 7 days of storage at 4°C. Notably, the percentage of drip loss in the T4 treatment group (200 ppm) was 1.39% ± 0.50%, 2.22% ± 0.32%, and 2.70% ± 0.38%, respectively, whereas the lower drip loss value was recorded in T5: 1.49% ± 0.16%, 2.30% ± 0.48%, and 2.76% ± 1.33%, respectively, in all treatment groups and storage periods: 0, 3, and 7 days. The highest percentage of drip loss was 1.64% ± 0.30%, 2.76% ± 0.24%, and 2.96% ± 0.27%, respectively, in control treatment T1 (without treatment) for all storage periods.

Water-hold capacity of lamb meat

The results (Fig. 2) revealed a significant difference (p < 0.05) increase in water-holding capacity between the fresh lamb meat of the treatment and control group throughout the period and during refrigerator storage. In the sample treated with T4 (200 ppm) and T3 (150 ppm) CA, the increase in water-holding capacity recorded was 30.69% ± 2.75%, 36.24% ± 5.16%, and 42.08% ± 5.83%, respectively, in treatment T4 and 29.68% ± 2.89%, 35.44% ± 4.83%, and 38.88% ± 7.22%, respectively, in treatment T3. The lowest values for the ability of meat to retain water were 27.59% ± 2.34%, 24.34% ± 1.85%, and 19.68% ± 1.43%, respectively, in the control treatment (T1) in all treatments and all storage periods (0, 3, and 7 days).

PH value of lamb meat

The present study (Fig. 3) revealed a significant (p < 0.05) difference in the pH value of fresh lamb meat between different concentrations and storage periods in all treated meat samples that were stored at 4°C. The results reveal a significantly different increase (p < 0.05) in the pH value for lamb meat samples treated with (200 and 150 ppm) of CA and BHA, respectively, and control samples during the storage period at 4°C. The control group recorded lower pH values of 5.75 ± 0.037, 5.82 ± 0.031, and 5.95 ± 0.033, while the groups treated (200 ppm) with CA recorded high pH values of 0.020 ± 5.80, 5.96 ± 0.032, and 5.99% ± 0.036%, respectively, during the storage period. In addition, the sample treated with T5 (150 ppm) the pH values were 5.72 ± 5.72 ± 0.022, 0.067, and 5.92 ± 0.038, respectively.

Chemical characterized

Thiobarbituric acid value of lamb meat

The present study (Fig. 4) revealed a significant (p < 0.05) difference between the treatments and the different storage periods in terms of the thiobarbituric acid value of fresh lamb meat. Treatment T4 (200 ppm) resulted in lower values of thiobarbituric acid during the entire storage period with values of 0.10 ± 0.10 ± 0.008, 0.007, and 1.21% ± 0.09%, respectively, than the 0.12 0.12 ± 0.01, ± 0.008, and 1.36Z ± 0.06% for T3 (150 ppm) CA, respectively. Compared with those of the control groups, T1 (without added) had high values of 0.19 ± 0.01, 0.25 ± 0.02, and 2.3 ± 0.23, respectively, during the entire storage period. There was a significant difference between all the different treatments during the storage period.

Fig. 1. Drip loss percentage of fresh lamb meat treated with different concentrations of BHA and CA during refrigerator storage.

Fig. 2. WHC percentage of fresh lamb meat treated with different concentrations of BHA and CA during refrigerator storage.

Fig. 3. PH value of fresh lamb meat treated with different concentration of BHA and CA during refrigerator storage.

Microbial characterized

Total bacterial count of lamb meat

The present study (Fig. 5) shows a significant (p < 0.05) difference reduction in the total bacterial count of the treated samples compared with the control during storage. The treated sample with T4 (200 ppm) reduced the total bacterial count during the entire storage period to 7.70 ± 0.02, 6.08 ± 0.49, and 5.96 ± 0.46 (log cfu/g), respectively, whereas the total bacterial count was 7.73 ± 0.04, 6.83 ± 0.02, and 6.80 ± 0.09 (log cfu/g) for T3 (150 ppm) in all storage periods compared with the control group; T1 (without added) increased the total bacterial count during all storage periods to 7.88 ± 0.02, 8.07 ± 0.06, and 8.08 ± 0.42 (log cfu/g), respectively. Significant differences were detected between the groups during the storage period.

Fig. 4. TBAR value of fresh lamb meat treated with different concentration of BHA and CA during refrigerator storage.

Fig. 5. Total bacterial counts (log cfug) of fresh lamb meat treated with different concentrations of BHA and CA during refrigerator storage.

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Discussion

Drip loss value of lamb meat

These studies reported that there was a significant difference (p < 0.05) in the lower drip loss value of the lamb meat sample between all groups during the storage period. This is due to the addition of CA, which has a strong antioxidant effect and the ability to protect the cell wall from damage caused by the oxidation process, increase its stability, and prevent the release of exudate from inside the cell (Al-Alwani, 2017).

Water-holding capacity value of lamb meat

There was a significant difference increase in the water-holding capacity between the fresh lamb meat of the treatment and the control group throughout the period and during refrigerator storage. These results mention agree with Al-Alwani (2017), who found that the addition of CA and BHA to meat increases the water-holding capacity of all treated samples and storage periods compared with the control group. However, it shows that increase in water-holding capacity in treated samples due to the CA, which has the ability to protect cell membranes from destruction and thus protect proteins from decomposition and prevent water from escaping and remaining linked to the protein by protein-water bonds. The reason may be that these compounds contributed to raising the pH of the treated meat, which increased the meat’s ability to retain water. These results agree with de Lima et al. (2024) found that the chitosan coatings with 4% or 8% rosemary extract efficiently preserved water in meat during cooking. Water is essential for muscle structure and its subsequent effects on muscle quality. Proteins become less elastic and more rigid as water is lost from muscle structures during heating and cooking. The rate and extent of pH decline associated with anaerobic glycolysis in postmortem muscle tissue are the primary factors determining the water content of raw, processed, and cooked meat products (Warner, 2017).

PH value of lamb meat

The results reveal an increase in the pH value for lamb meat samples treated with different concentrations of CA and BHA compared with the control samples during the storage period at 4°C. These results are similar to those Falowo et al. (2017). However, this may be due to the presence of compounds present in the addition treatments, including CA, in maintaining the stability of meat proteins, thus increasing the ability of meat to retain water, which in turn leads to an increase in the pH. The PH value increases during protein deterioration in raw meat due to the metabolism of microorganisms, release of alkaline compounds, and amines compound by proteolytic bacteria (Zahid et al., 2019). During the early storage period, meat has a low PH because of meat water retention or the rapid activity of cathepsins and calpain-µ (Li et al., 2014).

TBARS value of lamb meat

Previous studies have reported a significant difference lower thiobarbituric acid value of fresh lamb meat between the different concentrations and all storage periods. This result is in agreement with those of Mokhtar and Youssef (2014), who reported that some plant extracts and BHA lead to decreased TBARS values compared with those controlled during storage at 4°C in beef burgers. The main reason for the reduction in thiobarbituric acid values in treatments containing CA may be that it is considered an antioxidant (Jordan et al., 2014). However, the phenolic compounds in rosemary extracts, particularly carnosolic and carnosol acids, are responsible for 90% of rosemary’s antioxidant properties. These compounds convert lipids and hydroxyl radicals into stable products. These compounds reduce the formation of reactive oxygen species as reported by de Lima et al. (2024). It has high effectiveness in removing free radicals by has two orthophenolic hydroxyl groups located on carbon atoms C11 and C12 (Richhelmer et al., 1999). This helps break the oxidation chain reaction because of the ability of these substances to donate a hydrogen atom to fatty acid (Naveena et al., 2013). The increase in TBA value during periods of refrigerators’ storage of meat is normal due to the production of oxidation processes, ROS, free radicals, and so on (Amaral et al., 2018).

Total bacterial count of lamb meat

The results reported in (Fig. 5) mentions agree with Baker et al. (2013), who detected that rosemary extract reduced the total bacterial count to 11.50 ± 0.28 log. cfu/g in the treated group, whereas the control recorded 39.00 ± 0.57 log. cfu/g. This may be due to the presence of CA, which has shown antibacterial activity (Romano et al., 2009). The mechanism of action of CA as an antibacterial agent is that it changes genetic material, electron transport, nutrients, and fatty acid production, and leads to leakage of cellular components by interacting with the cell membrane, which leads to its death Ojeda-Sana et al. (2013) and Nieto et al. (2018). The use of rosemary oil, grape seed extract, pine bark extract, and BHA/butylated hydroxytoluene (BHT) in ground meat leads to a reduction in lipid oxidation, microbial growth, and color change Ahn et al. (2007). Because biologically active substances such as phenolic compounds in natural extracts may participate in inhibiting lipid peroxidation, by inhibiting the formation of free radicals, and preventing the propagation of free radical chain reactions through chelating transition metal ions, especially iron and copper ions (Youssef, 2014).

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Conclusion

The addition of CA to lamb meat lead to in a decrease in the drip loss and total bacterial count with a clear improvement in the ability of the meat to water holding-capacity and pH values, compared to the synthetic antioxidant butylated hydroxyanisole, in addition to its effectiveness as a natural antioxidant through the decrease in the oxidation indicators thiobarbituric acid. The results revealed that the addition of BHA and CA, both as antioxidants and antibacterial agents, effectively extended the shelf life of lamb meat. The results further indicated that CA exhibited greater efficacy than BHA in preserving the oxidation stability and overall quality of fresh lamb meat during refrigerated storage at 5°C. 2. We recommend using CA and BHA as an antioxidant and antirancid to increase the shelf life of meat and meat products.

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Acknowledgments

I would like to thank the Department of Vets. Public HealthCollege of Veterinary MedicineAl-Qasim Green University.

Conflict of interest

No conflicts of interest.

Funding

This study did not receive any external fund.

Author’s contribution

All authors contributed equally.

Data availability

All data are provided in the manuscript.

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