The THE POTENCY OF CINNAMON (Cinnamomum burmanni Blume) LEAF EXTRACT AS A BIOPRESERVATIVE AGENT FOR FOOD SAFETY OF SATE LILIT

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ARTICLE HIGLIGHTS
- Cinnamon leaf extract combats E. coli in traditional Balinese food.
- Natural preservative reduces harmful pathogens in sate lilit.
- Cinnamon extract offers a safer alternative to synthetic preservatives.
- Active compounds in cinnamon leaves inhibit bacterial growth.
- Effective biopreservative for enhancing food safety and quality.

ABSTRACT
Escherichia coli O157:H7 contamination of the meat used in preparing sate lilit, a favorite traditional food in Bali, Indonesia, has been a great concern for both local people and foreigners. Although C. burmanni has been included in its spice ingredients, active compounds that play a significant role in this pathogen have limitedly been elucidated. The main objectives of this research were to investigate the potency of this plant to control contaminants and elucidate possible compounds that prevent such contaminants by applying the disk diffusion method and LCMS analysis, respectively. The results showed that the leaf extract of this plant inhibited the in vitro growth of E. coli O157:7, with minimal inhibitory concentration (MIC) and LC50 values of 4% and 2.59%, respectively. The LCMS analysis chromatogram showed that the plant extract's most active fraction produced nine peaks, representing nine possible active compounds. Among those, three compounds (Azoxystrobin, Stigmatellin Y, and 2-arachidonoyl glycerol) were suspected of contributing to control contamination, especially by E. coli O157:H7.

INTRODUCTION

Being one of the tourist islands in Indonesia, Bali is rich in various types of ethnic foods. Such kinds of foods (traditionally served along the streets in small stalls or modern restaurants) can easily be found in areas of tourist destinations in Bali. A specific characteristic of Balinese ethnic foods is the spicy ingredients used in the preparation. Sate lilit is a traditional Balinese Kebab and a favorite food in Bali, enjoyed by both locals and foreigners. Sate lilit has also been part of offerings in the Balinese religious rituals, such as temple festivals, Balinese wedding ceremonies, etc. The meat (beef, pork, chicken, or fish as the primary raw material) of the sate lilit is frequently contaminated by pathogenic microbes(Palupi et al., 2019)that can damage the quality of the sate lilit. These contaminated meats often become a medium for the pathogen to spread and cause food poisoning (foodborne disease outbreak).

Some researchers found that beef (a primary raw material of sate lilit) traded in traditional markets in Bali is frequently contaminated by pathogenic bacteria. For example,(Purni et al., 2020)reported that 39 beef samples collected from some traditional markets located in Denpasar City, Badung, and Klungkung regencies were contaminated by E. coli. Among those samples, 25.6% were infected by E. coli O157:H7, the causative agent of hemorrhagic diarrhea, due to its ability to produce Shiga-like toxins(Patil et al., 2022). Similar results were also reported by(Pinatih et al., 2021), who stated that this serotype of E. coli O157:H7 has been well-known to produce potent toxins identical to those made by Shigella dysentery type 1, and therefore it is often referred to as Shiga like a toxin. As sate lilit is not fully cooked, it has the potential to spread pathogenic microbes that lead to an outbreak.

The application of synthetic chemical-based preservatives has not been encouraged to cope with such pathogenic microbial contamination due to their many harmful side effects on human health. According to the Indonesian Food and Drug Administration (BPOM), food poisoning was considered to be the highest case of poisoning in Indonesia in 2012. Approximately 66,7% of such poisoning cases were due to the consumption of foods containing poisonous preservatives, such as formalin, boric acid, or textile dyes(Utomo & Kholifah, 2018). Based on those food poisoning cases due to the application of inappropriate preservative agents, the possible use of natural preservative agents extracted from plants has been intensively researched so that some alternative and safe food preservations can be invented.

The bark and leaf extracts of C. burmanni Blume have been applied as flavor enhancers in various foods in Indonesia. C. burmanni Blume is a wooden plant. Its height may reach 50 meters(Sujarwo & Keim, 2020). According to(Djarot et al., 2023), this plant's leaves and bark contain high levels of flavonoids, saponin, tannin, and alkaloids.(Mohamed et al., 2020)reported that the bark extract of this plant has the potential as an antibacterial agent. This is due to the antimicrobial effect of its essential oil, containing several active compounds, such as cinnamaldehyde, eugenol, cinnamic acid, and cinnamate(Plumeriastuti et al., 2019). The potential of C. burmanni essential oil to inhibit Staphylococcus aureus, Enterobacter spp., Pseudomonas aeruginosa, and Candida albicans was also reported by(Novita & Sutandhio, 2021). This background has opened the door for its use as an alternative agent in developing food preservatives.

Based on the above rationale, it is urgently needed to elucidate active compounds in the C. burmanni leaf extract that may play an essential role in controlling E. coli O157:H7 contamination before its use as an organic food preservative. This study focused on the leaf extract of the plant, because this part of the plant has been widely used as an important ingredient of sate lilit preparation in Bali. The results of our current research expectedly can be used to develop alternative food preservation methods to control pathogenic microbes (E. coli O157:H7 in particular) in ethnic Balinese food products.

MATERIALS AND METHOD

Extraction of C. burmanni Blume leaves

Mature leaves (located on rows of 4-9 from tips of branches) of C. burmanni Blume were collected from Bedugul village, Tabanan-Bali. These leaf samples were chopped, air dried at ambient temperature (25 – 33^oC), powdered with a warring blender, macerated in 96% ethanol (Sigma Aldrich, Germany) in a ratio of 1:10 w/v, and filtered with 4 layers of Whatman no. 2 filter papers (pore size 8 µm). The filtrate was then evaporated in a vacuum rotary evaporator at 40ºC to obtain crude extract/pellet of the leaf extract and stored at 4^oC before being used in the subsequent experiments. The crude extract obtained from this evaporation was considered to be 100% concentration.

Preparation of C. burmanni Blume crude extract

The crude extract of the plant leaves was diluted with 96% ethanol (Sigma-Aldrich, Germany) to obtain final concentrations of 20% (w/v), 15% (w/v), 10%, and 5% (w/v) by applying the following dilution formula:

V₁. M₁ = V₂. M₂

Where:

V₁ : Volume of the most concentrated extract (mL)

V₂ : Volume of the adjusted extract concentration (mL)

M₁ : Concentration of the most concentrated extract (% w/v)

M₂ : The adjusted extract concentration (% w/v).

Preparation of E. coli O157:H7 cell suspension

The strain of Escherichia coli O157:H7 was obtained from the stock

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