**2. Antimicrobials agents against** *Staphylococcus aureus*

#### **2.1. Spices, herbs and plant extract as antimicrobials against** *Staphylococcus aureus*

The compounds found in herbs and found beneficial as traditional medicine can also be used as antibacterials and natural preservatives. The use of antibacterial synthetic or synthetic preservatives in foods such as the addition of formaldehyde or borax (borax) if taken continuously will cause a disease. The existence of the above phenomenon encourages people to find the best solution for health. An alternative solution is to replace synthetic antibacterial agents with natural antibacterial agents. Preventive methods have been used through the application of indigenous Indonesian herbs, plant extracts, and spices. The antimicrobial activities of plant extracts used for seasoning in foods have been recognized. The most common plant secondary metabolites that have antimicrobial activities occur in the following groups: alkaloids, anthraquinones, coumarins, essential oils (terpenoid and phenylpropanoids), flavonoids, steroids, and triterpenoids [5]. Some of them have antimicrobial activities.

## *2.1.1. Curcuma domestica val*

Turmeric (*Curcuma domestica* val) is one of the plants that is used for traditional medicine by our ancestors long ago. Turmeric has great potential in the pharmacological activity that is, anti-inflammatory, anti-immunodeficiency, anti-virus (bird flu virus), anti-bacterial, and antifungal [6]. The antibacterial properties in turmeric are caused by the chemical content of its main and essential oil curcuminoid.

#### *2.1.2. Ginger*

pathogens that were only seen associated with animals have been presented as illnesscausing agents in humans [1]. *S. aureus* is a Gram-positive bacterium that is an indicator of contamination from the workers and tools. *S. aureus* is a normal flora on the skin and in the respiratory organs in humans, and it is generally found in 20–50% of healthy population [2]. *S. aureus* contamination in food could also occur after the food has been cooked. In relation to cases of food poisoning, *S. aureus* enterotoxin intoxication on consumers occurs through the establishment of contamination on food consumed. This enterotoxin is resistant to heat (heat stable), acid-resistant, and resistant to the effects of proteolytic enzymes such

*S aureus* contamination is found in animal products that are marketed in Indonesia, such as eggs, chickens, and raw beef and also other raw meat products. Poor handling and improper storage methods cause the contamination of *S. aureus,* which survive on kitchen utensils and

Some *S. aureus* contamination that is in animal products marketed in Indonesia is as

Egg contamination can be derived from the environment. *S. aureus* would stick to the eggshell and subsequently on holding it penetrates into the egg through the pores in the

Population of *S. aureus* contamination on chicken breast meats is 2.71 ± 0.02 log CFU/g up to 3.25 ± 0.28 log CFU/g in Java Island, Indonesia (research result). *S. aureus* contamination on breast chicken meat can be derived from the contents of the digestive tract during slaughtering process in the poultry abattoir. Contamination on the carcass also occurs from the air or feces that contaminates skin and carcass [3]. External factors that influence the contamination of *S. aureus* are pH value and *aw* (water activity) on breast chicken meat. *aw* that is optimum in

Contamination of *S. aureus* on fresh beef at traditional market in West Java, Indonesia, has been investigated. The population of *S. aureus* was approximately 2.48 log CFU/g [4] and

**2.1. Spices, herbs and plant extract as antimicrobials against** *Staphylococcus aureus*

The compounds found in herbs and found beneficial as traditional medicine can also be used as antibacterials and natural preservatives. The use of antibacterial synthetic or synthetic preservatives in foods such as the addition of formaldehyde or borax (borax) if taken

increased continuously every hour in the room temperature of storage.

**2. Antimicrobials agents against** *Staphylococcus aureus*

as pepsin and trypsin.

168 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

unwashed hands.

**a.** Chicken eggs

**b.** Chicken meats

food for growth factor of *S. aureus* is 0.8–1.0.

follows:

eggshell.

**c.** Beef

Ginger can grow in the lowlands of the mountainous regions with an altitude of 0–1500 m above sea level. It has been used in food for seasoning in Indonesia. Meat cooked with ginger can have longer storage duration than without ginger. Ginger contains gingerol bioactive compound, which is a major component that can be converted into shogaol or zingerone shogaol formed from gingerol during the heating process [7].

#### *2.1.3. Garlic*

Raw garlic can be minced, pressed, sautéed, pickled, boiled, and juiced. Garlic sulphur compound(s) is(are) the primary bioactive agent(s). The major thiosulfanates, allicin, account for approximately half of the total of thiosulfanates from the *Allium sativum* genus [8]. Allicin was described as colorless oil, extremely pungent for the principal odor and taste of garlic. It was reported that allicin in concentrations of 1: 85,000 in broth was bactericidal to a wide variety of Gram-negative and Gram-positive organisms. A 5% garlic extract concentration has a germicidal effect on *S. aureus* [9]. Garlic extract used for seasoning in Indonesian food has strong antibacterial activities against *S. aureus* (**Figure 1**).

#### *2.1.4. Clove oil*

Clove oil has a potential as a preservative for food products and is known as Generally Recognized As Safe (GRAS) as a food ingredient. In addition, various studies have shown that clove oil has antimicrobial properties against *Salmonella* sp., *Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli,* and *S. aureus*. An amount of 0.25/100 ml of clove oil could inhibit *S. aureus*. The application of clove oil in processed meat products showed that at a concentration of 1 ml/l, it reduces the bacterial population significantly (*P* < 0.05), as much as 0.88 log CFU/g.

**Figure 1.** Inhibition zone of antimicrobial activities of garlic extract against *S. aureus.*

#### *2.1.5. Roselle flower*

An essential ingredient contained in rosella flower petals is the pigment anthocyanin that forms flavonoids and acts as antioxidants. Anthocyanin that causes the red color of this plant contains delfinidin-3-siloglukosida, delfinidin-3-glucoside, and sianidin-3-siloglukosida, while flavonoids contain gossypetin and mucilage (rhamnogalacturonan, arabinogalactan, and arabinan). *Hibiscus sabdariffa* Linn (Roselle flower) also contains phenol compounds that can be chemically defined by the presence of the aromatic ring carrying one (phenol) or more (polyphenols) substitution of hydroxyls [10]. The working of phenol in killing microorganisms is by cell protein denaturation. Phenol derivatives interact with bacterial cells through adsorption process involving hydrogen bond. At low levels, protein complex forms phenol by weak bonds and immediately occurs as decomposition, followed by phenol penetration into cells, causing precipitation and protein denaturation. Roselle flower extracts were proven for their antibacterial activities against *S. aureus* (**Figure 2**) and are used in yoghurt products in Indonesia as flavoring and preservatives.

#### *2.1.6. Red dragon fruit extract*

Flavonoids, phenols, hydroquinones, and saponins are the phytochemical compounds found in red dragon fruit peel extract. Steroids and triterpenoids compounds are also found in the red dragon fruit peel. The phytochemical substances of red dragon fruit extract have antibacterial activity that reacts with the bacterial cell wall proteins.

Red dragon fruit peels were extracted by modification maceration. Dragon fruits were cleaned and peeled manually before being cut into small sizes (2 mm). Red dragon fruit peels were dried at 50°C with an oven and ground to a powder. Peel powder was added with a solvent (1:50) for 60 min and filtered. The solution was evaporated at a vacuum evaporator temperature of 60°C. The extract was stored at −20°C and continued to be used in the Prevention of *Staphylococcus aureus* Contamination on Animal Products Using Indonesian Natural Products http://dx.doi.org/10.5772/66045 171

**Figure 2.** Inhibition zone of antimicrobial activities of Roselle flower extract against *S. aureus.*

antimicrobial analysis. Analysis of antimicrobial activity was performed by the well diffusion method. Bacterial culture was inoculated in NaCl 0.85% to obtain the bacteria concentration of 108 CFU mL−1. The dilution of the bacterial culture was done to obtain a culture concentration of 106 CFU mL−1. The other culture was grown in Mueller-Hinton Agar medium (DifcoTM, USA) and provided with holes as well with a predetermined diameter. Extracts were inserted into the well and covered with filter paper. Grail was stored in a refrigerator for 2–3 h, followed by incubation at 37°C for 24 and 48 h. The antimicrobial activity was characterized by the formation of clear zones around wells and measured for its diameter (mm). The inhibition zone produced by red dragon fruit peel extracts showed strong antibacterial activity (**Figure 3**).

Gram-positive bacterium, *S. aureus* ATCC 25923, was more sensitive to the antibacterial activity of red dragon fruit peel extract. The Gram-positive bacteria are more susceptible to antibacterial activity due to the absence of a lipoprotein wall that is capable of preventing antimicrobial compounds. Red dragon fruit peel extract due to its antibacterial compounds such as phenolic compounds could inhibit the growth of bacteria [13]. Application of red dragon fruit extract on beef sausages showed that *S. aureus* was not detected during 20 days of cold storage.

#### *2.1.7. Teak leaf extract*

*2.1.5. Roselle flower*

170 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

Indonesia as flavoring and preservatives.

terial activity that reacts with the bacterial cell wall proteins.

*2.1.6. Red dragon fruit extract*

An essential ingredient contained in rosella flower petals is the pigment anthocyanin that forms flavonoids and acts as antioxidants. Anthocyanin that causes the red color of this plant contains delfinidin-3-siloglukosida, delfinidin-3-glucoside, and sianidin-3-siloglukosida, while flavonoids contain gossypetin and mucilage (rhamnogalacturonan, arabinogalactan, and arabinan). *Hibiscus sabdariffa* Linn (Roselle flower) also contains phenol compounds that can be chemically defined by the presence of the aromatic ring carrying one (phenol) or more (polyphenols) substitution of hydroxyls [10]. The working of phenol in killing microorganisms is by cell protein denaturation. Phenol derivatives interact with bacterial cells through adsorption process involving hydrogen bond. At low levels, protein complex forms phenol by weak bonds and immediately occurs as decomposition, followed by phenol penetration into cells, causing precipitation and protein denaturation. Roselle flower extracts were proven for their antibacterial activities against *S. aureus* (**Figure 2**) and are used in yoghurt products in

**Figure 1.** Inhibition zone of antimicrobial activities of garlic extract against *S. aureus.*

Flavonoids, phenols, hydroquinones, and saponins are the phytochemical compounds found in red dragon fruit peel extract. Steroids and triterpenoids compounds are also found in the red dragon fruit peel. The phytochemical substances of red dragon fruit extract have antibac-

Red dragon fruit peels were extracted by modification maceration. Dragon fruits were cleaned and peeled manually before being cut into small sizes (2 mm). Red dragon fruit peels were dried at 50°C with an oven and ground to a powder. Peel powder was added with a solvent (1:50) for 60 min and filtered. The solution was evaporated at a vacuum evaporator temperature of 60°C. The extract was stored at −20°C and continued to be used in the Teak leaf extracts have a composition of flavonoids, alkaloids, tannins, anthraquinones, and naphthoquinones as antimicrobial substances that inhibit the growth of bacteria [11]. Addition of teak leaf extracts effectively inhibited *S. aureus* in the sausages. The 0.5 and 1% concentrations of teak leaf extracts addition on sausage formula in the processing could effectively inhibit *S. aureus* [12]. The method of teak leaf extraction is as follows: The extraction of teak

**Figure 3.** Inhibition zone of antimicrobial activities of red dragon fruit extract against *S. aureus.*

leaf was performed using ethanol extraction. Fresh teak leaf was oven-dried at 60°C for 24 h, chopped, and blended. Two hundred milliliters of 96% ethanol was then added into 20 g of teak leaf powder (10:1 ratio) and was boiled using waterbath at 70°C for 2 h. The mixture was centrifuged at 6000 rpm for 15 min. Ethanol was then removed by air-dry evaporation. The inhibition zone of antimicrobial activities was performed using diffusion methods [12]. The result of the antibacterial activities of teak leaf extract against *S. aureus* is shown in **Figure 4**.

#### **2.2. Bacteriocins as antimicrobials and their application as meat product biopreservatives**

Bacteriocins produced by Indonesian lactic acid bacteria *Lactobacillus plantarum* IIA-1A5 was purified and characterized. Plantaricin IIA-1A5 has been previously isolated from Indonesian lactic acid bacteria of *L. plantarum* IIA-1A5. This plantaricin has been shown

**Figure 4.** Inhibition zone of antimicrobial activities of teak leaf extract against *S. aureus.*

to inhibit the growth of *S. aureus* [13], making it a promising preservative substance to replace the use of chemical preservatives. Plantaricin could be digested by trypsin enzyme. It was heat stable at 80°C for 30 min and 121°C at 15 min, also active in a broad pH range of 4.0–9.0. Plantaricin IIA-1A5 could inhibit the growth of pathogenic bacteria, such as *E. coli, Salmonella Typhimurium, Bacillus cereus,* and *S. aureus.* Plantaricin IIA-1A5 showed good characteristics as an antimicrobial [14]. Plantaricin IIA-1A5 employs bactericidal activity since it disrupts the cell membrane and promotes the release of ions, proteinaceous, and genetic materials [13]. The cell wall of Gram positive has a thicker peptidoglycan layer, which is dominantly composed of lipoteichoic acid (LTA). The LTA is the target recognition of bacteriocin, facilitating the absorption of bacteriocin in the cell wall of Gram-positive bacteria [15].

#### *2.2.1. Genes involved in the production of plantaricin*

leaf was performed using ethanol extraction. Fresh teak leaf was oven-dried at 60°C for 24 h, chopped, and blended. Two hundred milliliters of 96% ethanol was then added into 20 g of teak leaf powder (10:1 ratio) and was boiled using waterbath at 70°C for 2 h. The mixture was centrifuged at 6000 rpm for 15 min. Ethanol was then removed by air-dry evaporation. The inhibition zone of antimicrobial activities was performed using diffusion methods [12]. The result of the antibacterial activities of teak leaf extract against *S. aureus* is shown in **Figure 4**.

**Figure 3.** Inhibition zone of antimicrobial activities of red dragon fruit extract against *S. aureus.*

172 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

**Figure 4.** Inhibition zone of antimicrobial activities of teak leaf extract against *S. aureus.*

**2.2. Bacteriocins as antimicrobials and their application as meat product biopreservatives** Bacteriocins produced by Indonesian lactic acid bacteria *Lactobacillus plantarum* IIA-1A5 was purified and characterized. Plantaricin IIA-1A5 has been previously isolated from Indonesian lactic acid bacteria of *L. plantarum* IIA-1A5. This plantaricin has been shown The genes responsible for bacteriocin production in *L. plantarum* IIA-1A5 are at least organized in two different operons: *plnABCD* and *plnEFI* [13]. The genes have been sequenced. PlnB (representative of operon plnABCD) amino acid sequence is derived from translation of partial DNA sequences using the software APE plasmid editor (http://biologylabs.utah.edu/ jorgensen/wayned/ape/). Regardless of its open-frame reading, partial sequence of plnB is shown in **Figure 5**.

To identify what kind of protein is encoded by plnB gene, we performed protein BLAST. BLAST results showed that the DNA sequence has 100% similarity with the histidine kinase genes for plantaricin on *L. plantarum* (**Figure 6**). The histidine kinase has been reported as one of the genes responsible for bacteriocin production. It is located in the locus responsible for plantaricin production in some of the plantarum strain. Histidine kinase is a quorum sensor to monitor the cell density of a bacterial population. At a certain concentration threshold, histidine kinase will be activated through a certain mechanism and induced with the production of bacteriocin [13].

Sequencing of plnEF gene and translation plnEF gene to amino acid has been conducted. PlnEF amino acid sequences were also obtained from the translation of DNA sequences using the software ApE plasmid editor (http://biologylabs.utah.edu/jorgensen/wayned/ape/). PlnB translation of DNA sequences to amino acid sequences is presented in **Figure 7**.

**Figure 5.** Amino acid sequences translation of plnB derived from its DNA sequences. The number on the left and right side shows the numbering sequence of the DNA sequence (top row) and amino acids (the second row). Bases in DNA are written in capital letters, while the amino acids are written in the format of three letters.


**Figure 6.** Multiple amino acid sequence alignment PlnB (35555) with homologous proteins. Each homologous proteins used in the alignment presented in the access code in the database. The red sequence shows the location of homology in the alignment.


**Figure 7.** PlnE translation of DNA sequences into amino acids. The number on the left and right side shows the numbering sequence of the DNA sequence (top row) and amino acids (the second row). Bases in DNA are written in capital letters, while the amino acids are written in one letter.

BLAST results showed that the DNA sequence of plasmid Ape editors has a high homology with the pln locus from several strains of plantaricin from *L. plantarum*. This means plnE correct encoding plantaricin [13]. Alignment results either in whole or in part show that the homology of plnE is more than 90% with various strains of the plantaricin (**Figure 8**).

Prevention of *Staphylococcus aureus* Contamination on Animal Products Using Indonesian Natural Products http://dx.doi.org/10.5772/66045 175


**Figure 8.** Multiple sequence alignment of amino acid sequencing of plantaricin EF.

#### *2.2.2. Application of plantaricin IIA-1A5 as a biopreservative*

Plantaricin IIA-1A5 could be used as biopreservatives for raw beef after slaughtering from abattoir. The initial contamination of *S. aureus* on raw beef is 2 log CFU/g. The addition of 0.2% plantaricin IIA-1A5 by spraying it onto raw beef surface could enhance the safety of beef from *S. aureus* contamination. Population of *S. aureus* on beef with 0.2% plantaricin is lower than maximum standard allowed by Indonesian standard of fresh beef (2 log CFU/g). In control (without plantaricin addition), population of *S. aureus* increased continuously every hour (3 log CFU/g). Plantaricin IIA-1A5 is able to extend the shelf life of meat stored at room temperature, according to physicochemical and microbiology quality [4].

Another application of plantaricin is as a biopreservative in meat products. *S. aureus* has been observed in meatballs without preservatives after 5 h of storage at room temperature. The 0.3% plantaricin IIA-1A5 addition displayed inhibition of *S. aureus* to be as strong as 0.3% nitrite. Until 20 h storage at room temperature, meatballs with nitrite or plantaricin IIA-1A5 were considerably safe to be consumed, which is a proven and promising potential use of plantaricin as a nitrite replacer for meatballs preservative [16].

## **3. Bacteriocin produced by** *S. aureus*

BLAST results showed that the DNA sequence of plasmid Ape editors has a high homology with the pln locus from several strains of plantaricin from *L. plantarum*. This means plnE correct encoding plantaricin [13]. Alignment results either in whole or in part show that the

**Figure 7.** PlnE translation of DNA sequences into amino acids. The number on the left and right side shows the numbering sequence of the DNA sequence (top row) and amino acids (the second row). Bases in DNA are written in

**Figure 6.** Multiple amino acid sequence alignment PlnB (35555) with homologous proteins. Each homologous proteins used in the alignment presented in the access code in the database. The red sequence shows the location of homology

in the alignment.

174 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

homology of plnE is more than 90% with various strains of the plantaricin (**Figure 8**).

capital letters, while the amino acids are written in one letter.

*S. aureus* produced bacteriocins and bacteriocin-like substances that were correlated with the presence of a plasmid usually involved in type B exfoliative toxin production. The bacteriocinogenic plasmids carried by the *S. aureus* strains are identified as plasmids larger than 40 kb that code for a high-M bacteriocin and that do not confer immunity [17]. *S. aureus* was isolated from bovine mastitis cases in 56 different Brazilian dairy herds and has been successfully investigated to produce antimicrobial substance (AMS). The bacteriocins may possess potential practical applications since they were able to inhibit important pathogens such as *B. cereus* and *L. monocytogenes* isolated from nosocomial infections [18] and show a potential application in food preservation [19]; meanwhile, the pathogenicity of *S. aureus* should be discussed for safety of bacteriocin. The antimicrobial activity of the bacteriocin produced by *S. aureus* is detected to be resistant to heat treatment at 65°C; however, treatment at 80°C completely abolished its antimicrobial properties [19].

Although *S. aureus* also produced bacteriocin, it could not kill and inhibit the cell itself because of immunity system. Bacteriocin-producing bacteria protect themselves from similar bacteriocin by immunity proteins. When these proteins are expressed in sensitive cells, they strongly protect against externally added similar bacteriocin. The immune system can work synergistically to protect the producing cells from their own bacteriocin [17]. Plasmid carried by the *S. aureus* strains confers immunity identified as small plasmids (8.0–10.4 kb), which code for bacteriocins or bacteriocin-like substances with a low M [18].
