Antibacterial inhibitory test of tomi-tomi fruit (Flacourtia iinermis Roxb) extracts against pathogenic bacteria in improving food safety

The tomi-tomi fruit (Flacourtia inermis Roxb) is a rare plant. This fruit has benefits as a flavourings spice and also traditional medicine used by the community, besides it also contains tannins and flavonoid compounds that can inhibit the growth of pathogenic bacteria in food. To support the use of tomi-tomi fruit (F. inermis Roxb) and increase its application in supporting food safety, the purpose of this study is to test and see the antibacterial inhibitory power of tomi-tomi fruit extract against pathogenic bacteria, namely Gram-Negative bacteria (Salmonella typhimurium) and Gram-Positive bacteria (Staphylococcus aureus and Bacillus cereus). This study is an experimental study using the Disc Diffusion method to determine the inhibitory power of bacterial growth using ethanol solvents with concentrations of 25%, 50%, and 75%. The results showed that of the three concentrations of tomi-tomi fruit extract tested against pathogenic bacteria, it turned out that the ethanol extract concentration of tomi-tomi 75% had a very strong inhibitory power category in S. aureus bacteria of 39 mm, S. typhimurium of 32.7 mm and B. cereus of 34.0 mm. Thus, tomi-tomi fruit ethanol extract has an inhibitory power that can be used for decontamination of 3 types of bacteria, especially in Staphylococcus aureus bacteria which have very strong inhibitory power, to maintain quality and improve food safety, among others rice, milk, cheese, fish, and meat that is often contaminated.


Introduction
Indonesia is one of the countries that have problems in health, one of which is the national food poisoning cases reaching 20 million cases per year caused by the use of chemical preservatives that endanger health, the occurrence of diarrhea by pathogenic bacteria, and the issue of the rampant use of chemicals that are carcinogenic or cause chronic toxicity data obtained from [1].Diarrheal disease is caused by the bacteria Escherichia coli, Salmonella typhimurium, Bacillus cereus, Shigella, Vibrio, Clostridia perfringens, and Staphylococcus aureus.Staphylococcus aureus is a commercial and pathogenic bacterium in humans, about 30% of the population is colonized by Staphylococcus aureus bacteria [2].Salmonella typhimurium is one of the gram-negative bacteria that is a pathogenic germ that causes typhoid fever, which is an acute systemic infectious disease, transmitted through the fecal-oral route, especially through contaminated food and water [3,4].Bacillus cereus is a pathogenic bacterium that can cause diarrhea [5,6].So it is necessary to handle the problem by using natural antibacterial as a 1230 (2023) 012171 IOP Publishing doi:10.1088/1755-1315/1230/1/012171 2 food preservative derived from plant extracts that function to improve health, and overcome and prevent bacterial infections.
Various types of plant extracts that produce antibacterial have been widely published for use as an inhibitory force for pathogenic bacteria and food-destroying bacteria such as garlic (Allium sativum L.) [7], young star fruit leaves (Averrhoea carambola L) [8], guava leaves (Psidium guajaya L.) [9], and galangal (Languas galanga Stunz) have proven to have antibacterial properties and food preservatives [10].One of the plant extracts that can be used as an antibacterial is the tomi-tomi fruit (F.inermis Roxb).Tomi-tomi fruit contains proteins, lipids, vitamins, minerals, and carbohydrates and has secondary metabolite compounds such as terpenoids, saponins, alkaloids, and phenolics [11].The existence of these various secondary metabolites is antibacterial in inhibiting various bacteria and as a natural preservative.
Extract F. inermis Roxb with a concentration of 20% have antibacterial activity in inhibiting the growth of Staphylococcus aureus, and Escherichia colli bacteria.Serratia marcescens, Pseudomonas aeruginosa, Klebsiella pneumonia [12].A concentration of 30% water extract of F. inermis Roxb fruit with a long soaking of tiger grouper for 1 hour (60 minutes) gives the best results, namely preserving fish for 18 hours so that it has the potential to be a natural preservative [13].
Thus, antibacterial compounds are chemical or biological compounds that can inhibit the growth of bacteria or mold or also kill bacteria (bactericidal) and mold (fungicidal) and have activities either naturally or intentionally added to food [14].This study aims to determine the best concentration of tomi-tomi fruit ethanol extract for the inhibition of food-pathogenic bacteria.

Location and description of the study
The object of study is the tomi-tomi fruit (Flacourtia inermis Roxb).The material used is fruit taken from Kaibobo Village, Western Seram Regency.Province Mollucas.It takes 8-10 hours from the place of collection of the tomi-tomi fruit that has been put in the basket and brought using land and sea transportation.

Preparation sample
Tomi-tomi fruits are sorted, cleaned using water, then chopped.The results of the display are dried in a drying oven for 72 hours.After drying, grinding and grinding are carried out and then sifted, the sieving results are in the form of powder.For maceration an extraction method with a ratio of ethanol material and solvent is used.50 grams of tomi-tomi fruit powder was soaked with 450 ml ethanol in different concentrations (25%, 50%, and 75%) for 3 days and stirred every 1 x 24 hours for 5 minutes.Filtration is carried out using vacuum and filter paper to separate the pulp and its filtrate.The filtrate obtained is then concentrated using a rotary evaporator until the solvent has evaporated so that a thick extract of tomi-tomi fruit is obtained, then dried by freeze drying.The dry extract was used as an antibacterial in testing the inhibitory power of pathogenic bacteria, namely Staphylococcus aureus, Salmonella thypimurium and Bacillus cereus [15].

Extraction treatment
The obtained dry extract is dissolved with aquades and made concentrations of 25%, 50% and 75%.As a comparison of bacterial inhibition, antibiotics are used, namely tetracycline made by means of 10 mg of tetracycline dssolved in 50 ml of aquades (positive control) and 50ml DMSO solution (negative control).After that, qualitative phytochemical tests were carried out including flavonoid tests, tannins/polyphenols and phenol tests.This test was conducted to identify secondary metabolite compounds found in ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) based [16] including flavonoid tests, tannins/polyphenol tests, and phenols test.

Flavonoid test.
Each sample extract as much as 1 ml is put into a test tube, then 2 drops of concentrated HCl are added, homogenized, then magnesium powder is added to produce a color change to orange red and foam, then positive contains flavanoid compounds.

Tannin test.
Each sample extract as much as 1 ml was put into a test tube, then added 1% FeCl 3 as much as 3 drops.The sample will experience a color change to blackish green then positively contain tannin compounds.

Phenol test.
Each sample extract of 1 ml is put into a test tube, then added 10 drops of hot water and 3 drops of 3% FeCl3 reagent.If the sample changes to a bluish-green or dark blue color, then a positive indicates the presence of phenol compounds.

Extract inhibition test against bacteria.
The inhibitory power test uses agar diffusion by the Kirby Bauer method using disc paper.This study used 3 types of concentrations, namely 25%, 50% and 75%, tetracixlyn 10 mg/L antibiotics as positive controls and DMSO 50 ml as negative controls.The inhibitory power test of ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) against bacteria Staphylococcus aureus, Bacillus cereus and Salmonella thyphimurium was carried out as follows: in pure cultures of Staphylococcus aureus, Bacillus cereus and Salmonella thyphimurium that have been rejuvenated then taken and cultured in sterile nutrient broth (NB) media then homogenized in vortex machines.Agar Nutrient Media (NA) in petri dishes are dripping cultures of Staphylococcus aureus, Bacillus cereus and Salmonella thyphimurium bacteria on the surface of agar media that have hardened using sterile cotton sticks.Media that has been smeared with bacteria Staphylococcus aureus, Bacillus cereus and Salmonella thyphimurium is placed with disc paper with a diameter of 6 mm which has been soaked in a solution of ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) with a concentration of 25%, 50%, and 75%, antibiotics tetracycline as a positive control and DMSO as a negative control and each treatment is marked then incubated for 24 hours at a temperature of 37°C.The inhibitory zone or clear zone formed from each media using disc paper is measured using a caliper in mm [17].

Statistical analysis
The data obtained for phytochemical tests were carried out by description in the table while for the resistance diameter test using Complete Randomized Design (RAL) analysis as many as 3 treatments with 3 repetitions by Steel and Torie adapted by [18].

Results and discussion
Ethanol is used as a solvent for maceration because ethanol solvents have a level of safety and ease of evaporation so that it is easy in the separation process.Ethanol is polar and has the property of attracting secondary metabolites in simplicia with optimal amount.The extraction process uses prisnsip like disolves like, namely solutions that are polar will be dissolved in polar and nonpolar solutions will dissolve in nonpolar compounds, active compounds in a plant will be easily dissolved or bound by solvents according to the nature of polarity.So that polar ethanol solutions will more easily extract flavanoid compounds, tannins and phenols in plant tissues [19].

Phytochemical test
Qualitative phytochemical testing is carried out to identify the content of active compounds contained in plants.In this study, the test was carried out by taking a small sample of macerated extract, and then adding reagents according to the compounds to be identified.The results of phytochemical tests on ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) showed that there were bioactive compounds, namely flavonoids, tannins and phenols (Table 1, 2 and 3 From the results of the study, it is known that each concentration of ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) contains flavanoid compounds, tannins and phenols.This is in line with previous research that states that Flacourtia inermis Roxb contains flavanoids, tannins and phenols.The types of flavanoids contained in Flacourtia inermis Roxb are catechins malic acid and quinic acid [20].
Qualitative tests of flavanoids (Table 1) showed that all three concentrations of ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) were positive for flavanoids.This is due to a reduction reaction with concentrated magnesium and HCl.The presence of a hydoksicil group (-OH) on flavanoids causes the formation of hydrogen bonds so that they are polar) [21].The occurrence of this reaction is characterized by the formation of yellow or yellow gradients such as dark yellow or orange.Qualitative tannin tests at all three concentrations of ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) positively produced tannins characterized by the formation of blackish-green color (Table 2).This is due to the reaction of tannins with FeCl3 which produces aromatic compounds.The reaction between tannins and FeCl3 forms complex compounds because the presence of Fe 3+ ions binds 2 donor atoms, namely at position 4' and dihydroxy in tannins to form complex compounds [22].Tannins are a group of phenols that can be distinguished from other enols because of their ability to precipitate proteins, this can be proven by reacting tannins with gelatin will produce precipitates.The precipitate is due to the presence of hydrogen bonds [23].
Phenol qualitative test at all three concentrations of ethanol extract of tomi-tomi fruit (Roxb) was positively characterized by a change in red color to blackish green.Phenol is acidic, because it has OHgroup properties that easily break away.The characteristic of phenol is its ability to form chelate compounds into metals, easily oxidized and form polymers that cause a dark color [24].

Bacterial inhibitory power
The inhibitory power of the tested extract is indicated by the presence of a clear zone around the paper disc.The clear zone around the paper disc is the diffusion area of the extract that affects the growth of bacteria.The large diameter of the inhibitory zone formed may indicate the antibacterial strength of the extract used.The classification of antibacterial forces makes it easier to classify the ability of the diameter obtained [25].Extracts with a resistance diameter of more than 20 mm belong to the category of very strong, the resistance diameter ranges from 10-20 mm belongs to the strong category, the resistance diameter ranges from 5-10 mm belongs to the medium category and the resistance diameter of less than 5 mm belongs to the weak category.
The results of testing the inhibitory power of tomi-tomi fruit ethanol extract (Flacourtia.inermis at concentrations of 25%, 50% and 75% formed clear zones around the paper disc in Staphylococcus aureus, Salmonella typhimurium, and Bacillus cereus bacteria can be addressed in Table 4. Table 4. Inhibitory power of pathogenic bacteria.The results of research on the inhibitory power of bacteria by ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) against Staphylococcus aureus with a concentration of 75% had a significant effect on concentrations of 50% and 25%.The diameter of the inhibitory zone for 75% concentration is 39 mm, 50% is 30 mm and 25% concentration is 23.8 mm.The higher the concentration of the extract on the growth of Staphylococcus aureus, the greater the inhibitory zone formed.The large diameter of the inhibitory zone formed is due to the greater content of antibacterial substances at higher concentrations.The formation of a bacterial growth inhibition zone indicates that the extract has antibacterial active compounds.This is in line with [26] who had previously conducted research on the inhibitory power of Staphylococcus aureus bacteria using papaya leaf extract has active compounds with a growth inhibition zone of Staphylococcus aureus bacteria of 15 mm at a concentration of 1.5%.The inhibitory power of ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) against Staphylococcus aureus bacteria can be seen in Figure 1.The results of testing the diameter of the inhibitory power of Bacillus cereus bacteria with a concentration of 75% ethanol of tomi-tomi fruit (Flacourtia inermis Roxb) greatly affect the concentration of 50% and 25%.For 75% ethanol concentration, the inhibitory power is 34 mm, the 50% inhibitory power concentration is 33.9 mm, and the 25% inhibitory power concentration is 22.2 mm.The cell wall of Bacillus cereus consists of a very thin lipid layer and peptidoglycan as much as 50%, the higher the concentration used the larger the size of the clear zone formed around the soaked paper disc with various concentrations determined (Figure 2).This is because the higher the concentration used, the higher the active ingredient compounds found in different concentrations of ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb).The results of testing the diameter of the inhibitory power of Salmonella typhimurium bacteria with a concentration of 75% ethanol tomi-tomi fruit (Flacourtia inermis Roxb) are very influential on concentrations of 50% and 25%.75% concentration inhibitory power of 32 mm; 50% concentration is 26.7 mm and 25% concentration is 21.1 mm.The size or size of the inhibitory zone formed can indicate that an antibacterial is weak, medium, strong and very strong against the growth of a bacterium.Based on the diameter of the inhibitory zone, it can be seen in Table 4.The difference in the diameter of the inhibitory zone obtained at each concentration occurs because it is influenced by the difference in ethanol concentration of tomi-tomi fruit (Flacourtia inermis Roxb) used.The higher the ethanol concentration of tomi-tomi fruit (Flacourtia inermis Roxb), the more active compounds contained in the tomi-tomi fruit (Flacourtia inermis Roxb).This is evidenced by the difference in inhibitory zones formed at each concentration.The results showed that there was a proportional relationship between the ethanol concentration of tomi-tomi fruit (Flacourtia inermis Roxb), and the diameter of the resulting inhibitory zone.The higher the ethanol concentration of the tomi-tomi fruit (Flacourtia inermis Roxb), the wider the inhibitory zone formed (Figure 3).The higher the concentration, the greater the clear zone formed due to the antibacterial contained in the ethanol extract of tomi-tomi fruit (Flacourtia Inermis Roxb) the larger.Staphylococcus aureus, Bacillus cerues and Salmonella typhimurium bacteria are sensitive to tetracycline it is seen from the clear zone formed.The clear zones formed indicate that the bacteria Staphylococcus aureus, Bacillus cerues and Salmonella typhimurium are sensitive to tetracycline Thus, the ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) contains flavonoids, phenols and tannins which are included in secondary metabolite compounds so that they have potential as antibacterials.This is the main factor in the antibacterial activity of ethanol extract of tomi-tomi fruit (Flacourtia inermis Roxb) in inhibiting food pathogenic bacteria, namely Staphylococcus aureus, Bacillus cereus and Salmonella typhimurium

Conclusion
Based on the results of the study, it can be concluded that the best concentration of 75% ethanol extract f tomi-tomi fruit (Flacourtia inermis Roxb) has secondary metabolite compounds, namely flavonoids, phenols and tannins; has inhibitory power against bacteria Staphylococcus aureus, Bacillus cereus and Salmonella typhimurium has a large diameter of resistance and belongs to the strong category.

Figure 1 .
Figure 1.Diameter of inhibitory power of Staphylococcus aureus bacteria.

Figure 2 .
Figure 2. Diameter of inhibitory power of Bacillus cereus bacteria.

Figure 3 .
Figure 3. Diameter of inhibitory power of Salmonella typhimurium bacteria.

Figure 4 .
Figure 4. Inhibitory power of food pathogenic bacteria.

Table 2 .
Qualitative test results of tannin compounds.

Table 3 .
Qualitative test results of phenol compounds.
If the letters are the same, it has no effect **.If the letters are the same, it has no effect