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PAPER 
 
 
 
 
 

THE EFFECT OF PLANT EXTRACTS 
ON PORK QUALITY DURING STORAGE 

 
 
 

A.M. KACZMAREK*a, M. MUZOLF-PANEKa, M. RUDZIŃSKAb, T. SZABLEWSKIa 
and R. CEGIELSKA-RADZIEJEWSKAa 

aDepartment of Food Quality Management, Faculty of Food Science and Nutrition, Poznań University of Life 
Sciences Wojska Polskiego 31, 60-642 Poznań, Poland 

bDepartment of Food Chemistry and Instrumental Analysis, Faculty of Food Science and Nutrition, Poznań 
University of Life Sciences Wojska Polskiego 31, 60-642 Poznań, Poland 

*Corresponding author. Tel.: +48 8487363 
E-mail address: ak@up.poznan.pl 

 
 
 
 
 
 

ABSTRACT 
 
This study investigated the effects of the natural antioxidants contained in onion, garlic 
and marjoram on the oxidant and microbial stability of raw minced pork during 
refrigerated storage. The most antioxidant active plant extract was marjoram. The highest 
inhibition of secondary oxidation products was observed in the samples with garlic, next 
was onion and finally, marjoram. GC analysis showed that the lowest rates of fatty acids 
degradation were recorded in the samples with garlic. The highest bacteriostatic effect was 
noted in meat samples with fresh onion added. These results suggest that the addition of 
these plants enhances the quality of the raw minced pork meat during refrigerated 
storage. 
 
 
 
 
 

Keywords: antimicrobial, antioxidant, meat, extract, plant, storage 
 



	

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1. INTRODUCTION 
 
Consumer concerns on the quality and safety of meat have greatly increased during the 
past decades. Lipid and microbial stabilities are important parameters that influence the 
quality and acceptance of meat, particularly minced meat. Minced meat has its cell 
structure disrupted, leading to increased exposure of lipids to oxygen, enzymes, heme 
pigments, and metal ions. Oxidative products, which are the results of lipid oxidation 
and/or microbiological changes in the meat, negatively affect the quality and safety of the 
product. For consumers this means the undesirable rancidity of meat. The heme pigment 
content, in conjunction with catalase activity, determines the lipid peroxidation potential 
in raw meat. In cooked meat, the major determinant for lipid peroxidation is the content of 
PUFA (MIN and AHN, 2005). The application of suitable ingredients possessing both 
antimicrobial and antioxidant properties can be useful for extending meat’s shelf life and 
thus preventing economic losses. In recent years, naturally occurring compounds 
exhibiting antioxidant and antimicrobial properties have been preferred for use in meat 
products because of their potential health benefits and safety, especially when compared 
with synthetic derivatives, which were routinely used in processed foods. Studies 
associated with natural antioxidants for the inhibition of microbial spoilage and lipid 
oxidation have been extensively conducted (FALOWO et al., 2014; KARRE et al., 2013; 
SHAH et al., 2014). Onion (Allium cepa L.), garlic (Allium sativum L.) and marjoram 
(Origanum majorana L.) possess both antioxidant and antimicrobial activity (BUSATTA et 
al., 2008; ROBY et al., 2013; SALLAM et al., 2004; YE et al., 2013). Onion and garlic bulbs, as 
well as dried marjoram, are three major meat additives widely used in food preparation. 
Park and Chin (2010) used onion extracts to control the development of lipid oxidation in 
fresh pork patties. EL-ALIM et al. (1999) investigated the use of ground marjoram as an 
antioxidant in raw ground chicken. CAO et al. (2013) proved the antimicrobial and 
antioxidant properties of onion and garlic for extending the shelf life of stewed pork 
during refrigerated storage. Moreover, PARK et al. (2008) demonstrated the antimicrobial 
and antioxidant activity of garlic and onion powder in fresh pork belly and loin during 
storage. Fresh garlic blended with paprika was also successfully used as an antioxidant in 
dry sausages (AGUIRREZÁBAL et al., 2000). However, due to the fact that the rate and 
extent of lipid oxidation are influenced by a number of factors, which include iron content, 
distribution of unsaturated fatty acids, pH, the form of natural additives (fresh, dried or 
extract) and various processing technologies, further research is still needed, particularly 
with reference to the meat system.  
The aim of the present study was to evaluate the effect of natural antioxidants contained in 
onion, garlic and marjoram on the oxidant and microbial stability of refrigerated minced 
pork blade meat during refrigerated storage. 
 
 
2. MATERIALS AND METHODS 
 
2.1. Samples preparation 
 
Samples of dried marjoram leaves, fresh garlic and onion bulbs were purchased in a local 
market. All the products were produced in Poland. Three pork blades (each from different 
animals) were obtained from a local market (where the process of cutting and deboning is 
conducted locally) and transported to the laboratory under chilled conditions in iceboxes. 
Blade muscles containing approximately 4% intramuscular fat were used. The blades were 
chopped and minced separately in a meat grinder, after the removal of excessive fat and 
connective tissue. Dried marjoram leaves and freshly crushed garlic and onion bulbs were 



	

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mixed with the meat. The following four samples were prepared from each blade: 1 
control (meat without any addition) and 3 treatments, namely, with garlic 0.1% (m/m), 
onion 0.5% (m/m) and marjoram 0.5% (m/m). The mixing process for each sample was 
conducted separately for each blade. The concentrations of the additives were established 
during preliminary studies and were accepted by a sensory panel (data not presented). 
Meat samples were placed in plastic foam meat trays, wrapped with polyethylene and 
kept at 4°C for 9 days. A strict sanitation procedure was followed during the preparation 
of the meat samples to avoid microbial contamination. 
 
2.2. Antioxidant activity of plant extract  
 
The antioxidant activities of marjoram leaves, garlic and onion were determined by 
methods, which reflected the various mechanisms of antioxidant action, such as the 
scavenging of free radicals or chelation of transition metal ions (methods such as DPPH 
and TEAC or FRAP, respectively). Each method has its advantages and limitations, thus a 
combination of these methods was implemented for assessing antioxidant activity to get 
more insight into the antioxidative potential of the extracts studied.  
To investigate the antioxidant activity of the plant material, extracts were prepared in 
methanol. For the extraction, 2 g of dried marjoram leaves, onion or garlic were mixed 
with 30 mL of pure ethanol. The procedure was conducted in the dark at room 
temperature. After 30 min of extraction, each extract was put through a filter (type 388) 
and the solutions of the corresponding concentrations were prepared in methanol for 
antioxidant activity measurements. 
 
2.3. DPPH method 
 
The antioxidant activity of marjoram leaves, garlic and onion were evaluated by the DPPH 
method according to the procedure described by Sánchez-Moreno et al. (1998) with some 
modifications. Briefly, 10 µL of the plant extract samples was added to 990 µL DPPH in 
methanol (giving a final concentration 0.1 mM) and mixed in a vortex. The reaction 
mixtures were incubated in the dark at room temperature for 30 min, and the decrease in 
absorbance caused by the plant extract was measured at 515 nm using a Cary 1E 
spectrophotometer (Varian, Berlose, Australia). For each sample, three separate 
determinations were conducted. The corresponding solvent blank readings were also 
recorded (methanol). The DPPH• radical scavenging activities of the plant extracts were 
expressed in TEAC(DPPH) values - Trolox Equivalents Antioxidant Capacity (mM of 
Trolox/g of dry weight). TEAC(DPPH) values were calculated as the ratio of the slope of 
the linear plot for the scavenging of DPPH• radicals by the plant extract tested to the slope 
of the plot for DPPH• radicals scavenging by the water-soluble vitamin E analogue Trolox, 
used as an antioxidant standard. 
 
2.4. TEAC assay 

 
The TEAC (Trolox Equivalent Antioxidant Capacity) assay is based on the inhibition of the 
absorbance of the blue-green coloured ABTS•+ radical cation by antioxidants (Miller et al. 
1993). In the study, the ABTS•+ radical cation was generated with potassium disulphate 
according to the modifications of Re et al. (1999). Briefly, ABTS was dissolved in water to a 
7 mM concentration and mixed with 2.45 mM potassium disulphate at a ratio of 2:1 
(stoichiometrical reaction). To produce ABTS•+ radical cation, the mixture was allowed to 
stand in the dark at room temperature for 12-16 h. The radical was stable in this form for 
more than two days (i.e. stored in the dark at room temperature). The ABTS•+ solution was 



	

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diluted with PBS (phosphate buffer saline), pH 7.4 to an absorbance of 0.7 (±0.02). 10 µL of 
plant extract was added to 990 µL ABTS•+ solution in PBS, pH 7.4 and the decrease in the 
absorbance of the mixture after 6 min of incubation in the dark was monitored 
spectrophotometrically at 734 nm. The corresponding solvent blank readings were run 
(ABTS in PBS, pH 7.4 with methanol). All determinations were conducted in triplicate, and 
the results were expressed as TEAC values. The TEAC value represents the ratio of the 
angle of the plot for the scavenging of ABTS•+ by the particular extract under investigation 
to the slope of the plot for ABTS•+ scavenging by Trolox, used as an antioxidant standard 
(MILLER et al., 1993). The TEAC value is expressed in milimolar concentrations (mM), 
according to the definition of the TEAC value introduced by MILLER et al. (1993). The 
TEAC value is defined as the concentration of Trolox solution with an equivalent 
antioxidant potential to a 1 mM concentration of the compound under investigation 
(MILLER et al., 1993). 
 
2.5. FRAP assay 
 
The FRAP (Ferric Reducing Antioxidant Power) assay directly measures the ability of 
antioxidants to reduce a ferric tripyridyltriazine complex (Fe+3-TPTZ) to a ferrous 
complex (Fe+2-TPTZ) at a low pH, with an intense blue color and an absorption of 593 nm 
(BENZIE and STRAIN 1996). The FRAP solution was prepared by mixing an acetate buffer 
(300 mM, pH 3.6), 10mM TPTZ (2,4,6-tripyridyl-s-triazine) and FeCl3•6H2O (20 mM in 40 
mM HCl) at a ratio of 10:1:1. The mixture was allowed to stand in the dark at 30°C for 30 
min directly before use. Then, 50 µL of an increasing concentration of plant extract was 
added to the FRAP solution in a 1mL total volume and allowed to react for 10 min in the 
dark. Readings were taken at 593 nm and results were expressed as mM Trolox equivalent 
(TE) per g of dry weight. All determinations were conducted in triplicate. 
 
2.6. Total polyphenol content of plant extracts 
 
The Total Polyphenol Content (TPC) of the plant materials was determined using the 
Folin- Ciocalteu reagent (FCR) as described by Singleton and Rossi (1995). An aliquot of 20 
µl of plant extract (onion, garlic or marjoram), prepared in the same way as for antioxidant 
activity measurements, was added to 100 µl of FCR, mixed and incubated for 3 min at 
room temperature in a dark place. Then, 300 µl of sodium carbonate (20% m/v) was added 
and filled to 2 mL with distilled water. After 2h of incubation in the dark at room 
temperature the absorbance was read at 765 nm against the blank sample (solvent instead 
of extract). The results were presented as mg Gallic Acid Equivalent (GAE) per g of dry 
weight (DW). All determinations were conducted in triplicate. 
 
2.7. Total flavonoid content 
 
The total flavonoid content was determined by using the aluminum chloride colorimetric 
method as described by MEDAA et al. (2005). The basis of this assay is that aluminium 
chloride forms acid stable complexes with the C-4 keto group and either the C-3 or C-5 
hydroxyl group of flavones and flavonols. In addition, it also forms acid labile complexes 
with the ortho-dihydroxyl groups in the A or B ring of flavonoids. 100 µL of plant extracts 
were mixed with aluminium chloride in methanol (2% m/v) to a final volume of 1 mL. 
The mixtures were incubated for 15 min in the dark at room temperature. Then, the 
absorbance was monitored at 410 nm. The results were expressed in mg of quercetin per g 
of dry weight. 
 



	

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2.8. Measurement of lipid oxidation 
 
The extent of lipid oxidation was monitored by the formation of Thiobarbituric Acid-
Reactive Substances (TBARS). The TBARS index was determined, in triplicate samples, by 
the extraction method of SØRENSEN and JØRGENSEN (1996) with some modification. 
For extraction, 10 g meat was homogenized (15,000 rpm, 30 s, 20°C) together with 30 mL of 
a 7.5% aqueous solution of trichloroacetic acid (TCA). After filtration and centrifugation, 
(5 min, 5000 rpm), a 5.0 mL extract was mixed with 5.0 mL of 0.02 mol l-1 aqueous 
thiobarbituric acid (TBA) in a stoppered test tube. The samples were incubated at 100C for 
35 min in a water-bath and subsequently cooled for 10 min in cold water. Absorbance was 
measured at 532nm by a Carry 1E UV/VIS spectrophotometer against a blank containing 
5mL distilled water and 5mL TBA reagent. The results, expressed as milligrams 
malondialdehyde per kg meat, were calculated from the standard curve of TEP (1,1,3,3-
tetraethyoxypropane) standards. The antioxidant potential, which is expressed in terms of 
the percentage of antioxidant activity (%AOA), were calculated using the following 
equation (WIJEWICKREME and KITTS 1998):  
 

%AOA= ((X value of the control – X value of the test sample) × 100)/ 
X value of the control 

 
where X is the TBARS value. 
 
2.9. Fatty acids composition analysis 
 
The meat samples were minced in a blender. Lipids were extracted from the samples with 
chloroform:methanol (2:1 v/v) according to FOLCH et al. (1957). The extracts were dried 
in a vacuum on a rotary evaporator and under a nitrogen flow. The fatty acid content was 
determined as Fatty Acid Methyl Ester (FAME) derivatives in a HP 5980 series II gas 
chromatographer (Hewlett Packard, Oalo Alto, USA) using the AOCS Official Method Ce 
1k-07. The GC system consisted of an Innowax capillary column (30 m x 0.2 mm x 0.2 mm) 
and FID detector. Hydrogen was used as the carrier gas, at a flow rate of 1.5 mL/min. The 
temperature of both the injector and detector was set at 240°C. The column temperature 
was maintained at 220°C isotherm. Peak identification was based on the fatty acid ester 
standards, and fatty acid compositions were estimated from the chromatogram peak areas 
and were expressed as mg/1 g meat sample. 
 
2.10. Microbial analysis 
 
An amount of 10 g of meat was collected from prepared samples and introduced, under 
sterile conditions, to a flask containing 90 mL of 0.1% sterile peptone diluents 
(Bacteriological Peptone, Oxoid, England). The samples were homogenized using an 
Ultra-Turrax T25 homogeniser (IKA, Germany), producing an initial suspension of 1:10. 
The microbiological analyses were conducted in line with ISO reference methods 
(according to the Polish Standard of PN-ISO). Bacterial counts were given in CFU g-1. The 
total bacterial count was determined on a Standard Plate Count Agar (CM 463, Oxoid, 
England). Incubation was run at 30°C for 72 h. For counts of the Enterobacteriaceae rods, a 1 
mL sample was inoculated into 15 mL of a molten selective VRBG medium (P-0256, BTL, 
Poland). After setting, a 10 mL overlay of molten medium was added and incubation was 
conducted at 37°C for 24-48 h. The counts of Pseudomonas were determined on a solid 
Pseudomonas Agar medium (CM 0559, Oxoid, England) supplemented with 
Pseudomonas CFC Selective Agar Suplement (SR 0103, Oxoid, England) after incubation 



	

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at 30°C for 48 h. De Man, Rogosa and Sharpe (MRS) Agar (CM 0361, Oxoid, England) was 
used for determining lactic acid bacteria counts. MRS agar was also overlayed with a 
molten medium and incubation was conducted at 30°C for 48-72 h. An oxidase test was 
used to confirm lactic acid bacteria (MBO 266, Oxoid, England). Dichloran Rose Bengal 
Chloramphenicol DRBC Agar (CM 0727B, Oxoid, England) was used in the determination 
of moulds and yeasts. Incubation was conducted at 20°C for 5 days. Total Viable aerobic 
bacteria Counts (TVC) were conducted as an indicator of microbial spoilage in the pork 
meat samples. The samples (10 g) were homogenized with 90 mL with sterile peptone 
water (1g/l) using an Ultra-Turrax T25 homogenizer (IKA, Germany). Serial decimal 
dilution was performed and plated onto a Standard Plate Count Agar (CM 463, Oxoid, 
Basingstoke, England). Incubation was performed at 30°C for 72 h. Bacterial counts were 
enumerated and expressed as log10 cfu/g. 
 
2.11. Statistical analysis 
 
All tests were run in triplicate. The statistical package Statistic 13.1 was used for analysis 
of covariance (ANCOVA). The influence of various plant additions on oxidative and 
microbial raw meat stability was assessed using analysis of covariance. This analysis 
procedure is applied when looking at group effects on a continuous outcome (TBARS 
value, fatty acid content and microbial count) when another continuous explanatory 
variable (storage time) may also have an effect on the outcome. Comparison of the 
treatment means was based on Tukey’s Honest Significant Difference (HSD) test. This test 
is the most useful for multiple comparisons and avoids Type II errors. In addition, 
Dunnett’s T-test was performed for a comparison of the treatments to the control. To 
compare the rates (slope of regression equation) of fatty acids' degradation, a linear 
regression analysis between the storage time and the fatty acid content was calculated. To 
compare the regression coefficients (slope) of the control with the other treatments, the 
null hypothesis H0: Bc=Bt, (where Bc is the regression coefficient for the control and Bt is the 
regression coefficient of the sample with additives) was tested. The comparisons between 
the coefficients were performed introducing two dummy variables as predictors to 
regression analysis. The first dummy variable was coded “0” for the control and “1” for 
the meat sample with plant additive, and the second, which was the product of the first 
dummy variable and storage time. The significant differences between the regression 
coefficients were based on the result of the T-test (P ≤ 0.05) for the second dummy 
variable. Bacterial counts were converted to their equivalent log cfu for data uniformity. 
Differences were considered significant at the P ≤ 0.05 level.  
 
 
3. RESULTS AND DISCUSSIONS 
 
3.1. Antioxidant activity and total polyphenol content of the plant extracts 

 
All results of the antioxidant activity and phenolic content (total polyphenol content TPC 
and total flavonoid content TFC) measurements are presented in Table 1. According to 
radical scavenging methods like DPPH and ABTS and ion chelating methods such as 
FRAP, the most antioxidant active plant extract was marjoram. There were no statistically 
significant differences between the antioxidant activities of garlic and onion. 
GORINSTEIN et al. (2008) showed higher antioxidant activity of garlic in comparison to 
onion in the DPPH method but significantly lower antioxidant activity of garlic than onion 
in the ABTS and FRAP methods. MARIUTTI et al. (2008) reported that marjoram is the 
most antioxidant active among the tested extracts in the DPPH and ABTS methods, which 



	

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is in accordance with the results presented in this paper. However, the authors showed 
that garlic is more antioxidant active than onion as a DPPH• and ABTS•+ radical 
scavenger 
 
 
Table 1. Phenolic content and the antioxidant activities of marjoram, garlic and onion. 
 
 Plant extract  

 Marjoram Garlic Onion 
Phenolic content Mean±SD Mean±SD Mean±SD 
TPC (mg GAE/g) 24.48 ± 2.66 a 0.79 ± 0.23 b 2.74 ± 0.65 b 
TFC(mg QE/g) 15.72 ± 1.26 a 0.22 ± 0.00 b 0.27 ± 0.01 b 

Antioxidant activity    
TEAC(DPPH)(mM TE/g) 175.11 ± 11.43 a 3.05 ± 0.46b 3.77 ± 0.51b 
TEAC(ABTS)(mM TE/g) 261.84 ± 15.04 a 7.68 ± 0.25b 19.09 ± 1.15b 

FRAP(mM TE/g) 186.9 ± 10.60a 4.16 ± 0.06b 3.86 ± 0.19b 
 
(a-b)Means with the same superscript within the same row are not different (P > 0.05). 
 
 
In this study, the TPC and TFC values correspond to the antioxidant activity (Table 1). r-
Pearson coefficients for the linear correlations between the antioxidant activity and TPC or 
TFC values are equal to 0.99. The higher the TPC (and TFC) value, the higher the 
antioxidant activity is. In literature, data on the TPC values of onion, garlic and marjoram 
vary. Generally, it was shown that onion possesses a higher TPC value compared to garlic 
(GORINSTEIN et al. 2008; NUUTILA, et al. 2002). It is also hard to compare the TPC values 
of marjoram to reference results since various modifications to the methods and units 
were implemented by the authors. 
 
3.2. Measurement of lipid oxidation 
 
The lipid oxidation in the fresh minced pork meat subjected to refrigerated storage was 
determined using the TBARS method. This method is widely used to estimate the degree 
of lipid oxidation because of its relatively simple measurement and good correlation with 
the sensory quality of foods (MOTTRAM 1998). TBARS are formed through the second 
stage of lipid oxidation, during which peroxides are oxidized to aldehydes and ketones 
such as malondialdehyde (MDA). Fig. 1 presents the TBARS values of the samples under 
study. The results showed that both the storage period and the type of additive affected 
the TBARS values (with P < 0.05). The TBARS values of all the tested samples (control and 
treatment) increase constantly with the time of storage (Fig. 1). This increase was the most 
pronounced for the control sample, whose TBARS values rose from 0.11 mg MDA/kg 
meat on the first day of analysis to 0.352 mg MDA/kg meat at the end of storage (9th day). 
The TBARS values in the minced meat were influenced by onion, garlic and marjoram, 
resulting in significantly lower values than those without treatment. As shown in Fig. 1, 
the most effective treatments in inhibiting the lipid oxidation were the onion and garlic 
ones. There were no statistically significant differences between these two samples (P 
>0.05). Samples treated with onion and garlic reached TBARS values of 0.22 and 0.186 mg 
MDA/kg respectively on the last day of storage, compared to the control samples which 
exhibited a TBARS value equal to 3.5 MDA/kg. Significant differences in the TBARS were 
found between the treated samples and the control from day 3 to the end of storage. YIN 
and CHENG (1998) found that garlic had a stronger antioxidant effect than onion in their 



	

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liposome model system. In this study, due to sensory acceptability, different levels of 
additives were tested (0.15 m/m for garlic and 0.5% m/m for onion), which may have 
caused the lack of difference between the onion and garlic inhibition of lipid oxidation. A 
statistically significant protective effect for lipid oxidation in meat was also noted for 
samples with the addition of marjoram. The TBARS value for the treatment with marjoram 
at the end of storage was 0.28 mg MDA/kg and was significantly lower than the control 
one. However, this effect was less pronounced when compared to the samples with garlic 
and onion. In conclusion, the highest inhibition of secondary oxidation products was 
observed in the samples with garlic (47%). In the samples with onion and marjoram, the 
percentage of inhibition was 37% and 25% respectively. These results were consistent with 
those of previous studies in which fresh onion and garlic bulbs or their extracts showed 
antioxidant effects in the meat matrix (CAO et al., 2013; KIM et al., 2010; PARK et al., 2008; 
SALLAM et al., 2004).  
 
 

 
 
Figure 1. Changes in the TBARS values of fresh minced pork with different treatments during refrigerated 
storage at 4°C. 
 
 
3.3. Fatty acid composition analysis 

 
The fatty acid composition of minced pork muscle fat is shown in Table 2. Both the type of 
additive and storage time influenced the fatty acid composition. The predominant fatty 
acids in all meat samples were oleic (C18:1n-9), palmitic (C16:0), stearic (18:0) and linoleic 
(C18:2n-6) acids, in order of the increasing concentration in the samples. The amount of 
these acids accounted for more than 91% of the total fatty acid contents in all samples. 
PARK et al. (2008) obtained similar results in fresh pork belly and loin. MUFAs were the 
major part followed by SFAs. Generally, a decrease in the fatty acid content in the 



	

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intramuscular lipid was noted during storage. This effect was expected because, due to 
cellular structure damage and exposure to the oxygen, minced muscle is very prone to 
lipid and protein oxidation (MIN and AHN, 2005). Therefore, to evaluate whether the fatty 
acid contents were equal in the groups being studied (type of additive) analysis of 
covariance was performed. This analysis allows for comparisons of the effects of additives 
and control the influence of storage time simultaneously. Comparison of the additives 
showed that a stronger inhibition of lipid oxidation was exhibited by garlic (Table 2). 
Statistically significantly higher contents of stearic (56.82 mg/g) palmitileic (15.28 mg/g), 
linoleic (41.1 mg/g), cis-10-heptadanoic (1.62 mg/g) and arachidonic acids (5.14 mg/g) 
were observed in this treatment when compared to the control. The lowest content of fatty 
acids was noted in the samples with the addition of fresh onion, which makes it the least 
effective in the retardation of lipid oxidation. The marjoram additive was the most 
effective in inhibiting the decomposition of linoleic (but only until the 7th day of storage) 
and cis-10-heptadanoic acids. Furthermore, to compare the effect of the added natural 
ingredients on the change rates of fatty acids, linear regression analysis was performed.  
 
 
Table 2. Effect of onion, garlic and marjoram on the fatty acid profile of fresh minced pork during storage at 
4°C (mg/g). 
 

Fatty acid 
Treatment  

Control Onion Garlic Marjoram SE 
C14:0 4.90a 4.62a 6.08a 5.01a 0.39 
C16:0 84.31ab 79.95a 106.26b 89.22ab 6.86 
C18:0 41.41a 39.89a 56.82b* 47.02ab 3.40 
C16:1 12.42a 11.56a 15.28b* 12.64a 0.62 
C17:1 1.26a 1.18a 1.62b* 1.85c* 0.05 
C18:1 176.59ab 166.92a 198.84b 159.50a 12.06 
C20:1 3.35a 3.33a 3.20a 2.74a 0.15 
C18:2 35.53ab 33.27a 41.10bc* 42.75c* 1.79 
C20:2 1.52a 1.52a 1.47a 1.52a 0.08 
C18:3 4.91a 4.56a 2.38b 2.52b 0.21 
C20:4 3.33a 3.31a 5.14b* 3.92a* 0.17 
SFA 130.63ab 124.46a 169.15b* 141.26ab 10.62 

MUFA 193.62ab 182.99a 218.95b 176.73a 8.94 
PUFA 45.29ab 42.66a 50.08ab 50.70b 3.19 

 
 a-c)Means with the same superscript within the same row are not different (P > 0.05). (*) Paired comparison 
(control compared with other natural additives) significantly greater than control at the P < 0.05 level using 
Dunnett’s-T test. 
 
 
Results showing the regression coefficients from the linear models of the decay of fatty 
acids are presented in Table 3. The linear model was statistically significant in almost all 
cases (P ≤ 0.05). Only for saturated fatty acids in the meat samples with garlic and 
marjoram, was the linear model not significant (P > 0.05). In the samples with garlic, the 
level of saturated fatty acids was stable during storage. However, in the samples with 
marjoram the level of saturated fatty acids increased till the 7th day of storage and then 
rapidly decreased, reaching the lowest values of all the meat samples. The values of the 
determination coefficients ranged from 74% to 96%. 
 



	

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Table 3. Effect of onion, garlic and marjoram on the fatty acids content changes of fresh minced pork during 
storage at 4°C (expressed as slope (mg/g/day)). 
 

Fatty acid 
Treatment  

Control Onion Garlic Marjoram 
C14:0 -0.568 -0.472 -0.057N -0.340N 
C16:0 -0.500 -7.801 -1.282N -6.130N 
C18:0 -4.357 -3.713 -0.831N -3.040N 
C16:1 -1.391 -1.298 -0.762* -1.865 
C17:1 -0.123 -0.098 -0.076* -0.173 
C18:1 -19.89 -18.97 -9.381* -23.94 
C20:1 -0.401 -0.422 -0.144* -0.346 
C18:2 -3.299 -3.169 -1.613* -6.089* 
C20:2 -0.162 -0.148 -0.068* -0.133 
C18:3 -0.652 -0531 -0.214* -0.256 
C20:4 -0.282 -0.200 -0.165* -0.621* 
SFA -14.42 -11.99 -2.165* -9.512 

MUFA -21.81 -20.79 -10.36* -26.32 
PUFA -4.396 -4.051 -2.060* -7.101* 

 
(N)Regression coefficients are not significant (P > 0.05). (*) Paired comparison (control compared with other 
natural additives) were significantly different to the control at P < 0.05. 
 
 
All the slopes have a negative value indicating the inverse relationship between the 
storage time and fatty acid content. The lowest rate of fatty acid degradation was recorded 
in the samples with the fresh garlic addition. This dependency was noted for all fatty 
acids, which confirms the highest inhibiting effect of garlic on lipid degradation. In 
addition, discriminant analysis corroborates these outcomes. As shown in Fig. 2, the first 
discriminant function extracts the sample of meat with garlic, while the second extracts the 
meat samples with marjoram. The meat samples with onion exhibit the same fatty acid 
composition during storage as the meat samples without any addition (the points are 
located in the same place of the graph). The LDA model allows meat samples enriched 
with garlic and marjoram to be distinguished with 100% accuracy. The highest 
decomposition rate was noted in the meat samples with marjoram, namely, arachidonic 
and linoleic acid. The contents of these acids decreased from 6.24 to 1.63 and from 60.2 to 
9.2 mg/g respectively. The level of linoleic acid in the samples with marjoram was 
significantly the lowest among the tested samples at the end of storage. 
 
3.4. Microbial changes  

 
The bacteria selected in this study such as Pseudomonas and Enterobacteriaceae belong to the 
genera of bacteria constituting microflora typical for spoilage processes in meat during 
storage. To assess the effect of the addition of the plants on bacterial growth in meat 
samples covariance analysis was performed.  
Throughout the storage period, the inhibition effect of the plant additives on bacterial 
growth was observed. Only for lactic acid bacteria was this effect not statistically 
significant. 
 



	

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Figure 2. Discrimination of minced meat samples based on the fatty acid composition during cold storage 
(4°C); C-control, G-meat with garlic (0,1%), M- meat with marjoram (0.5%) and O- meat with onion (0.5%). 
 
 
As shown in Table 4, the highest numbers of all the tested bacteria were noted in the 
control samples. The results showed that the addition of onion and marjoram to fresh 
meat exerted a statistically significant bacteriostatic effect against TVC, Enterobacteriaceae 
and Pseudomonas bacteria. The highest inhibitory effect against Enterobacteriaceae and 
Pseudomonas bacteria was noted in the meat samples with the addition of fresh onion. The 
results of our study supported the findings of other authors. GROHS et al., (2000) reported 
that spice mixtures might delay bacteria growth in fresh pork and beef. PARK et al., (2008) 
showed that the addition of garlic and onion powder to pork loin and belly inhibited the 
growth of the total amount of bacteria and Enterobacteriaceae. SALLAM et al., (2004) noted 
that fresh garlic and garlic powder, through their antioxidant and antimicrobial effects, are 
potentially useful in preserving meat products. However, MURRAY (1997) claimed that 
only fresh garlic preparations provide the full range of beneficial compounds. 
 
 
Table 4. Effect of onion, garlic and marjoram additions on the microbial changes (cfu/g) of fresh minced 
pork stored at 4°C. 
 

Type of bacteria 
Treatment  

Control Onion Garlic Marjoram 
TVC 8.02a 7.54c* 7.92ab 7.8b* 
Enterobacteriaceae 6.16a 5.65b* 6.09a 5.70b* 
Pseudomonas 8.00a 7.52c* 7.84ba 7.74bc* 
Lactic acid bacteria 2.84 2.64 2.71 2.65 

 
(a-c)Means with the same superscript within the same row are not different (P > 0.05). 
(*) Paired comparison (control compared with other natural additives) is significant at the P < 0.05 level 
using Dunnett’s T- test. 



	

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4. CONCLUSIONS 
 
Due to the natural potential health benefits and safety of the plant species when compared 
to synthetic derivatives, natural species have gained appreciable interest among the 
research and industry community. The highest antioxidant activity is demonstrated by 
fresh garlic, whereas fresh onion in minced pork meat reveals the highest antibacterial 
activity. The synergistic behaviour of these plants would require further research. 
Although marjoram was characterised by the highest antioxidant activity, its protective 
effect on lipids in meat was less significant than other treatments. This result confirms the 
necessity to examine the plant antioxidant effect in particular food products. Our data 
indicated that the addition of fresh garlic and onion, as well as dried marjoram, enhances 
raw minced meat safety and shelf life. In addition to their economical and health 
promoting benefits, the application of plants, as natural and safe bio-preservatives, could 
be highly recommended for the improvement in the quality of ground pork. 
 
 
ACKNOWLEDGEMENTS 
 
This research project was partly supported by the Polish State Committee for Scientific Research (No. 
2014/15/D/NZ9/04261). 
 
 
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Paper Received March 24, 2017  Accepted July 10, 2017