Food Science and Nutrition Studies ISSN 2573-1661 (Print) ISSN 2573-167X (Online) Vol. 1, No. 1, 2017 www.scholink.org/ojs/index.php/fsns 31 Evaluation the Antimicrobial Activity of Artemisia and Portulaca Plant Extracts in Beef Burger Marwa Al-Moghazy1, M. S. Ammar2, Mohamed M. Sief3 & Sherif R. Mohamed3* 1 Dairy Science Department, Food Industries and Nutrition Division, National Research Centre, Dokki, Giza, Egypt 2 Food Science and Technology Department, Faculty of Agriculture, AL-Azhar University, Cairo, Egypt 3 Food Toxicology and Contaminants Department, Food Industries and Nutrition Division, National Research Centre, Dokki, Giza, Egypt * Sherif R. Mohamed, E-mail: sheriframzy4@gmail.com Received: April 9, 2017 Accepted: April 23, 2017 Online Published: May 15, 2017 doi:10.22158/fsns.v1n1p31 URL: http://dx.doi.org/10.22158/fsns.v1n1p31 Abstract Medicinal plants contain substances can alternate the traditional chemical preservatives which used for preserving meat products that have negative effects on consumer health. Several biological activities have been reported for Artemisia and Portulaca as antimicrobial agents, so the current study focused on using Artemisia and Portulaca extracts as antimicrobial agents in beef burger. Phytochemical of Artemisia and Portulaca extracts were analyzed, and both extracts contain alkaloids, flavonoids, phenols, trepenoids and saponin. The results show that Artemisia extract was inhibited all tested microorganisms (Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella Typhimurium, Listeria monocytogenes and E.coli O157:H7) while, Portulaca extract affect Staphylococcus aureus only. The Minimum Cidal Concentration (MCC) and Minimum Inhibitory Concentration (MIC) were carried out for testing microorganisms, since Artemisia extract was very effective against Staphylococcus aureus followed by E.coli, Pseudomonas aeruginosa, Salmonella Typhimurium and L. monocytogenes. Artemisia and Portulaca extracts were separately applied in beef burger as antimicrobials at levels 1% and 1.5%. The sensory evaluation of treated beef burger showed no significant differences between control sample and treatments containing Portulaca extract while, the addition of Artmesia extract had a detrimental effect on taste of beef burger since it causes formation of bitter taste. Keywords antimicrobial, Artemisia, Portulaca, plant extracts, beef burger www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 32 Published by SCHOLINK INC. 1. Introduction Nowadays, new preservation techniques are being developed to extend the storage time with maintaining both the natural appearance and safety of fresh meat (Patsias et al., 2008; Zhou et al., 2010). Synthetic preservatives such as nitrite has been widely used for preserving meat products such as sausages and luncheon meats. Nitrite is efficient for inhibition of Clostridium botulinum (Gibson et al., 1984). Nitrite may react with secondary amines naturally present in the meat to form N-nitrosamines, which are carcinogenic (IARC, 1998). There is an association between consumption of processed meat and increase risk of different cancers (Santarelli et al., 2008; Larsson et al., 2006; Larsson & Wolk, 2012), which cause death (Rohrmann et al., 2013). Unlike synthetic compounds, natural extracts obtained from plants are rich in phenolic compounds which can enhance the overall quality of food by decreasing lipid oxidation and microbial growth (Zhang et al., 2016). Artemisia vulgaris is used in asthma emmenagogue, anthelmintic, and stomachic (Ambasta, 1994). The essential oil of leaves exhibited significant antimicrobial activity (Laxmi & Rao, 1991). The leaves have an antibacterial activity on Staphylococcus aureus, Bacillus typhi, Bacillus dysenteriae, Streptococci, Escherichia coli, Bacillus subtilis and Pseudomonas. Pharmacological studies on Portulaca oleracea showed its activities as antibacterial (Chan et al., 2015), hepatoprotective (Al-Sheddi et al., 2015), anti-inflammatory, analgesia (Zhou et al., 2015), antioxidant (Liu et al., 2015). So the goal of this work is the evaluation of antimicrobial effect for both Artemisia and Portulaca extracts against microorganisms in vitro and as a preservative agent in beef burger. 2. Materials and Methods 2.1 Materials Herbs: Artemisia vulgaris and Portulaca oleracea were obtained from Egyptian herbal market, Dokki, Giza. Microbial strains: Three Gram negative pathogen bacteria; Salmonella Typhimurium (ATCC 14028), E. coli O157:H7 and Pseudomonas aeruginosa and 2 Gram positive pathogens; Staphylococcus aureus (25923), and Listeria monocytogenes (ATCC 7644), Microbial strains are generously given by Microbiology Department, Faculty of Agriculture, Cairo University. Different microbial strains were preserved at -20oC. Ingredients of beef burger: a. Beef meat: Frozen beef, lean meat was obtained from the local butcher shop in the day before the experiment. The meat was cold stored at 5±1ºC overnight. b. Soybean flour: Soybean flour was obtained from food Technology Research Institute, Agriculture Research Center, Giza, Egypt. c. Other ingredients: Spices, Fresh eggs, onion and salt were obtained from the local market. While, sodium tripolyphosphate, and sodium ascorbate were obtained from the Adwic Laboratory Chemicals Co., Cairo, Egypt. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 33 Published by SCHOLINK INC. 2.2 Methods 2.2.1 Technological Methods Preparation of Artemisia vulgaris and Portulaca oleracea extract: One hundred grams of Artemisia vulgaris or Portulaca oleracea leaves were added to excessive distilled water: ethanol (2:8 v/v) and incubated at room temperature for 24 h, then the slurry was filtered through filter paper. The water extract was concentrated using a rotary evaporator under reduced pressure and the residues were dissolved in 50 ml of distilled water. Beef burger formulation: Beef burger patties were processed by the method described by (Oroszvári et al., 2005; Ou & Mittal, 2006) according to the formula showed in Table 1: Table 1. Beef Burger Formulation Main ingredients (%) Treatments Control Artemisia extract Portulaca extract 1% 1.5% 1% 1.5% Beef meat 62 62 62 62 62 Soy flour 12 12 12 12 12 Iced water 10 10 10 10 10 Fresh eggs 7 7 7 7 7 Fresh onion 7 7 7 7 7 Salt 1.5 1.5 1.5 1.5 1.5 Spices 0.5 0.5 0.5 0.5 0.5 Total 100 100 100 100 100 Additives (%) Sodium tripolyphosphate 0.3 0.3 0.3 0.3 0.3 Sodium ascorbate 0.03 0.03 0.03 0.03 0.03 Artemisia extract 1 1.5 1 1.5 Portulaca extract 1 1.5 1 1.5 2.2.2 Analytical Methods DPPH radical scavenging method: The antioxidant activity of the plant extracts was evaluated using the stable 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) according to a modified method of Bandoniene et al. (2002). Qualitative Phytochemical Analysis: phytochemical of plant extracts was carried out by standard methods described by (Brain & Turner, 1975; Evans, 1996) as follows: Detection of Alkaloids: Extracts were dissolved separately in dilute hydrochloric acid and filtered. The filtrates were used to detect the alkaloids by using Mayer’s test, since filtrates were treated with Mayer’s reagent. Formation of a yellow, cream precipitate indicates the presence of alkaloids. Detection of Saponins: About 0.5 mg of the extract was mixed by agitation with five ml of distilled water. Formation of frothing (appearance of creamy miss of small bubbles) shows that the presence of www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 34 Published by SCHOLINK INC. saponins. Detection of Tannins: A small quantity of extract was mixed with water and heated using water bath. Then the mixture was filtered and ferric chloride was added to the filtrate. The formation of dark green colour indicates the presence of tannins. Detection of Flavonoids: Detection of flavonoids was carried out as follows: -Lead acetate test: Extracts were treated with a few drops of lead acetate solution. The formation of yellow color indicates the presence of flavonoids. -H2SO4 test: Extracts were treated with a few drops of H2SO4. Formation of orange color indicates the presence of flavonoids. Precipitate indicates that the presence of flavonoids. Detection of Phenols: Detection of phenols was carried out as follows: -Ferric chloride test: 10 mg extracts were treated with a few drops of ferric chloride solution. Formation of bluish black color indicates the presence of phenol. -Lead acetate test: 10 mg extract was treated with a few drops of lead acetate solution. Formation of a yellow color precipitate indicates the presence of phenol. 2.2.3 Quantitative Phytochemical Analysis Estimation of Alkaloids was carried out by using the method of (Harborne, 1973) as follows: One gram of extract was weighed into a 250 ml beaker and 200 ml of 10% acetic acid in ethanol was added and the beaker was covered and allowed to stand for 4 h, then the mixture is filtered and concentrated on a water bath to one quarter of the original volume. Concentrated NH4OH was added by drop wise to the extract until the precipitation was complete. The whole solution was allowed to settle and the precipitate was collected and washed with dilute NH4OH, then filtered. The residue is the alkaloid, which was dried and weighed. Estimation of Flavonoids was carried out as follows: One gram of plant sample was repeatedly extracted with 100ml of 80% aqueous methanol at room temperature. The mixture was filtered through a Whatman No.1 filter paper into a pre-weighed 250 ml beaker. The filtrate was transferred into a water bath and allowed to evaporate to dryness and weighed (Krishnaiah et al., 2009). Estimation of Total Phenols was carried out as follows: The fat free sample was boiled with 50 ml of ether for extraction of phenolic compounds for 15 min. Five ml of the extract was pipetted into a 50 ml flask, then 10 ml of distilled water was added. Two ml of NH4OH solution and 5 ml of concentrated amyl alcohol were also added. The samples were made up to mark and left to react for 30 min for color development. This was read spectrophotometry at 505 nm. Screening of antimicrobial activity: Screening of antimicrobial activity was performed using the agar diffusion method. The Nutrient agar plate was over layered with approximately 2 mL soft agar inoculated with 105-106 cfu/mL of overnight activated microbial cultures, then wells of 8 mm diameter were holed by cork borer, 60 µL of each tested compounds were injected in every well. Negative control was performed using sterile distilled water. Plates were incubated for 24 h at 37oC. Diameters of inhibition clear zones were measured using graded ruler. www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 35 Published by SCHOLINK INC. 2.2.4 Microbiological Methods Determination of Minimum Inhibitory Concentrations (MIC) and Minimum Cidal Concentrations (MCC): Determination of Minimum Cidal Concentrations (MCC) of extracts were performed using microplate methods, 100 µL of sterile nutrient broth were loaded in each well of 96 wells plate, then defferent volumes of extract were added to achieve final concentrations ranged from1 to 15% (v/v), then the inoculums of different pathogens were added to have 106 CFU/mL final concentration. After incubation for 24 h at 37oC, bacterial count of each sample was determined using the drop plate method (Naghili et al., 2013). Drops of 10 µL from each well were pipetted onto surface of solidified nutrient agar, and then the plates were incubated for 24 h at 37oC. Minimum inhibitory concentration defined as the concentration which shows no increase or decrease of the initial counting after 24 h of incubation, while Minimum Cidal Concentration (MCC) defined as the less concentration showed no bacterial viability. Microbiological assessment of frozen beef burger: Microbiological assay of the different beef burger samples was carried out at zero time and after 3 months of frozen storage. Total count of bacteria was determined to evaluate the antimicrobial activity of plant extract in a real food system such as beef burger and the ability of this extract to prolong the storage period of beef burger. Ten g of each sample was added to 90 ml of sterile pepton water and mixed well for 1 min to homogenize. Decimal dilutions in sterilized pepton water were prepared and 1 ml was poured in Plate count agar (PCA, CM0325, Oxoid) to enumerate total bacterial count after incubation for 24 h at 37oC. 2.2.5 Statistical Analysis One-way analysis of variance (ANOVA) was performed to test for differences between the groups mean. Significant differences between the means were determined by Duncan’s multiple range test and p<0.05 were regarded as significant (Sokal & Rohlf, 1995). 3. Results Table 2 shows the qualitative phytochemical of Artemisia and Portulaca Extracts. From the obtained results it cleared that both extracts contain alkaloids, flavonoids, phenols, trepenoids, saponin while the tannins was positive in Artemisia and negative in Portulaca extract. Table 2. Qualitative Phytochemical Analysis of Artemisia and Portulaca Extracts Phytochemicals Artemisia extract Portulaca extract Alkaloids Mayer s test + + Flavonoids Lead acetate test H2SO4 test + + + + Phenols Ferric chloride test + + www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 36 Published by SCHOLINK INC. Lead acetate test + + Terpenoids + + Saponin + + Tannin + - Note. (+) Present; (-) Not detected. Table 3 shows the quantitative phytochemical of Artemisia and Portulaca Extracts. From the results it cleared that both extracts contained Alkaloids, Flavonoids, phenol and antioxidant activity at levels 4.25-5.34, 14.34-9.46, 9.47-3.85 W/w, 74%-68% respectively. Table 3. Quantitative Phytochemical Analysis of Artemisia and Portulaca Extracts Phytochemicals Artemisia extract (W/w) Portulaca extract (W/w) Alkaloids 4.25 5.34 Flavonoids 14.34 9.46 Phenol 9.47 3.85 Antioxidant Activity 74% 68% Table 4 shows the Artemisia and Portulaca water extract against pathogenic bacteria. The obtained results showed that Artemisia extract has a potent antimicrobial effect against all tested microorganisms, Staph aureus, Pseudomonas aeruginosa, Salmonella Typhimurium, Listeria monocytogenes and E.coli O157:H7. The maximum effect of the Artemisia extract as an antibacterial was against Pseudomonas aeruginosa and gave 28 mm Inhibition zone diameter followed by Listeria monocytogenes, Staph aureus, Salmonella typhimurium and E.coli O157:H7 since the extract could inhibit the microbial growth and the inhibition zones were 26.25, 26, 24 and 15.4 mm respectively. The Portulaca extract was also tested as an antimicrobial against the same microorganisms, but it had a potent effect only against Staph aureus while, did not have any effect against Pseudomonas aeruginosa, Salmonella Typhimurium, Listeria monocytogenes and E.coli O157:H7. Table 4. Screening of Antimicrobial Activity (Inhibition Zone Diameter in mm) of Artemisia and Portulaca against Tested Pathogenic Bacteria Microorganism Artemisia extract Portulaca extract Staph aureus 26 20 Pseudomonas aeruginosa 28 Nil Salmonella typhimurium 24 Nil Listeria monocytogenes 26.25 Nil E.coli O157:H7 15.5 Nil Table 5 shows the Minimum Inhibitory Concentration (MIC) and Minimum Cidal Concentrations www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 37 Published by SCHOLINK INC. (MCC) of Artemisia extract against pathogenic bacteria (Portulaca extract was excluded because that it had not antimicrobial activity except against Staph aureus only). The Minimum Cidal Concentration (MCC) is the lowest concentration of an antibacterial agent required to kill a particular bacterium. It can be determined from broth dilution Minimum Inhibitory Concentration (MIC) tests by subculturing to agar plates that do not contain the test agent. The Minimum inhibitory concentration MIC and Minimum Cidal Concentration (MCC) were carried out for testing pathogenic bacteria are E. coli, Pseudomonas aeruginosa, Staph aureus, Salmonella Typhimurium, L.monocytogenes. The MIC was regarded as the lowest concentration of the extracts that prevent the growth of any tested bacterial colony on the medium. From the obtained results in Table 4 the Artemisia extract was very effective on Staph aureus and the MIC was appeared at 5 µL/100 µL, while MIC for both E.coli and Pseudomonas aeruginosa were medium and detected at 7 µL/100 µL, moreover Salmonella Typhimurium and L.monocytogenes possess the lowest MIC which recorded at 9 µL/100 µL. Also the minimum cidal concentration MCC was carried out and found that the results related and agreed with the results of MIC and the MCC at 6 µL/100 µL was for Staph aureus followed by E.coli, Pseudomonas aeruginosa and Salmonella Typhimurium, L.monocytogenes at 8, 10 µL/100 µL respectively. Table 5. Minimum Inhibitory Concentration (MIC) and Minimum Cidal Concentrations (MCC) of Artemisia Extract against Pathogenic Bacteria Microorganism MIC (µL/100 µL) MCC (µL/100 µL ) E.coli 7 8 Staph aureus 7 8 Pseudomonas aeruginosa 5 6 Salmonella typhimurium 9 10 Listeria monocytogenes 9 10 Staph aureus pseudomonas aeruginosa www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 38 Published by SCHOLINK INC. Salmonella typhimurium Listeria monocytogenes E.coli O157:H7 Figure 1. The Minimum Inhibitory Concentration (MIC) and Minimum Cidal Concentrations (MCC) of Artemisia Extract against Pathogenic Bacteria Data in Table 6 show changes in the total bacterial count of beef burger during freezing, storage which was determined at zero time and after three months of frozen storage. The results show that Portulaca extract had a slight effect on beef burger microbial quality, but the rate of growth was slow which may be due to the polyphenols content of Artemisia extract which cause reducing the growth rate and maximum growth population and/or extending the lag-phase of the target microorganism (Zhou et al., 2010). The Artemisia extract possesses a potent effect as an antimicrobial agent against all tested bacteria opposite the Portulaca extract that had only effect against Staph aureus, but unfortunately based on the sensory evaluation of beef burger results the Artemisia extract has a detrimental effect on the taste of burger patties which impart bitter taste and were unpalatable. So, more researchers are required to remove the bitterness of Artemisia extract until we can be used it in beef burger preservation without its affecting the sensory properties. Table 6. Microbiological Changes in Total Bacterial Count (cfu/g) Analysis of Beef Burger Samples during Frozen Storage Period Storage time Treatments Control Artemisia extract Portulaca extract 1% 1.5% 1% 1.5% Zero time 2.40 E+06 3.70 E+06 2.10 E+06 2.66 E+06 2.68 E+06 3 months 3.07 E+06 3.85 E+06 2.86 E+06 3.00 E+06 3.40 E+06 www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 39 Published by SCHOLINK INC. Table 7 shows that there is no significant differences (P≤0.05) between control and treatments containing Portulaca extract in all sensory properties of beef burger, furthermore addition of Portulaca extract with 1.5% was enhanced the taste, tenderness, juiciness and overall acceptability of beef burger. On the other hand the addition of Artmesia extract had a detrimental effect on taste of beef burger since it impart it bitter taste. Also addition of Artmesia extract was slightly lower the sensory score of beef burger for tenderness and juiciness but did not affect color and appearance. Table 7. Effect of Addition Plant Extracts on Sensory Characteristics of Beef Burger Sensory characteristics Treatments Control Artemisia extract Portulaca extract 1% 1.5% 1% 1.5% Color 8a 8a 8a 8a 8a Taste 8a 1b 1b 8a 9a Flavor 8a 1b 1b 8a 8 a Tenderness 8ab 7b 7b 8ab 9a Juiciness 8ab 7b 7b 8ab 9a Appearance 8a 8a 8a 8a 8a Overall acceptability 8a 1b 1b 8a 9a * Scores ranging from 0-3 = very poor, 4 = poor, 5 = fair, 6-7 = good and 8-10 = very good. ** In rows means have the same superscript are not significantly different. 4. Discussion Our obtained phytochemical analysis of Artemisia and Portulaca extracts in agreement with (Alireza et al., 2013) who showed that tannins, saponins, alkaloids, amino acids, phenolic compounds, quinines and terpenoids are present in Artemisia extract using mass gas-chromatograph. Also, Okafor (2014) studied the water extract of aerial parts of Portulaca and showed that it contain steroids, protein, and alkaloids. The Artemisia extract had a potent effect as an antimicrobial against all tested microorganisms while, Portulaca extract had a potent effect against Staph aureus only. In our respect study (Abdul & Waheeta, 2010) showed that Artemisia nilagirica extracts had a broad spectrum of antibacterial activity against phytopathogens and clinical pathogens except S. aureus, E. faecalis and K. pneumoniae. Our results showed that plant extracts contain effective biological compounds such as alkaloids, flavonoids, phenols, tannins and terpenoids, which could be alternate the traditional chemicals to inhibit phytopathogenic bacteria and decrease the negative effects of synthetic drugs. The MIC of Artemisia extract that gave a positive effect against all tested microorganisms was analyzed and the results was in agreement agreed with (Abdul & Waheeta, 2010) who studied the effects of various Artemisia extracts against bacteria and reported that hexane extract was effective www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 1, 2017 40 Published by SCHOLINK INC. against all phytopathogens with low MIC of 32 μg/ml, while the methanol extract showed higher inhibition activity against Escherichia coli, Yersinia enterocolitica, Salmonella typhi, Enterobacter aerogenes, Proteus vulgaris, Pseudomonas aeruginosa (32 μg/ml), Bacillus subtilis (64 μg/ml) and Shigella flaxneri (128 μg/ml). The MIC analyses of clinical pathogens showed an activity against Gram-positive and Gram-negative bacteria, which indicate that plant extracts contains several antimicrobials. The phytochemical screening of plant menthol extracts showed that it contain flavonoids, terpenoids, phenols, amino acids, alkaloids and tannins, which have antimicrobial properties (Fernandez et al., 1996; Mendoza et al., 1997; Amaral et al., 1998; Cowan, 1999 ; Shaheen et al., 2003; Amarowicz et al., 2008; Chowdhury et al., 2008). Artemisia and Portulaca extracts were incorporated into the beef burger formula as an antimicrobial agent and both extracts gave good results as antibacterial, but Artemisia extract was had superior antimicrobial activity than Portulaca extract. The sensory quality of the formulated beef burger was carried out, beef burgers incorporated with Portulaca extract were having good sensory quality, while the beef burgers containing Artemisia extract were having bad sensory quality (bitter taste), although it showed good microbiological quality, thus further researches are needed to remove the compounds which induce the bitter taste until we can use the Artemisia extract as an antimicrobial in meat products. 5. Conclusion Finally, Beef burger can be formulated with incorporation of Portulaca extract as antimicrobial without any detrimental effect on its sensory properties, whereas more researches are needed to remove the compounds of Artemisia extract which cause the bitter taste to make it suitable for using in persevering meat products without detrimental effect on its sensory properties. References Abdul, R. A., & Waheeta, H. (2010). 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