The Effect of Binahong Leaf Extract as a Disinfectant in Duck Egg Incubator
Eulis Tanti Marlina*1, Ellin Harlia1, Yuli Astuti Hidayati1
1 Microbiology and Waste Management Laboratory, Department of Animal products Technology, Universitas Padjadjaran Bandung
Submitted: 29 September 2022, Accepted: 1 March 2023
ABSTRACT: Binahong is a native South American plant that grows well in Indonesia. The active compounds contained in the binahong plant are widely used as traditional medicine.
Active compounds such as flavonoids, alkaloids, saponins, triterpenoids, comarins and phenolic acids are antimicrobial. This study aims to determine the antimicrobial effectiveness of binahong leaves in inhibiting bacteria and fungi in duck egg incubators. The concentration of binahong extract used was 30%, 40%, 50%, 100% binahong leaf solution, and 1% chemical disinfectant Cethylpiridium Chloride (CPC). Evaluation was carried out on decreasing the number of bacteria, fungi, inhibition zones on gram-positive and gram-negative bacteria and identification of fungi reduced by binahong leaf extract in duck egg incubators. Data were analyzed using ANOVA and Tukey's test. The results showed the concentration of 50%
binahong leaf extract had the option to decrease the number of bacteria and fungi 88.48% and 53.81%, respectively, 100% binahong solution could decrease the quantity of bacteria and fungi 56.75% and 34.95% respectively and 1% CPC chemical disinfectant reduced the number of bacteria and fungi 91.65% and 62.60%, respectively. Binahong leaf extract produced inhibition zones on gram-positive and gram-negative bacteria with a strong interpretation, namely 19.2 mm and 16.2 mm, respectively.
Keywords: Binahong leaf extract; Disinfectant; Egg incubator
*Corresponding Author: eulis.tanti@unpad.ac.id
INTRODUCTION
Binahong is a plant native to South America, such as Paraguay, Southern Brazil, Uruguay and Northern Argentina. This plant is a shrub with heart-shaped leaves that have slender stems climbing about 30 cm, and dark green leaves with small white flowers and many. The Binahong plant belongs to the Kingdom Plantae with the species name Anredera cordifolia (Ten.) steenis.
Binahong is known by several names, such as in South America it is known as Madeira vine, in China it is known as Dhen San Chi, in Australia it is known as potato vine, and in Indonesia it is known as Binahong (Queensland Government, 2022). This plant is very easy to grow in humid areas and can invade other plants (Webb and Harrington, 2005). In Indonesia, binahong leaves have been widely used to treat various diseases such as diabetes mellitus, burns, and infections (Larissa et al., 2017; Gusnimar et al., 2021). This is due to the antimicrobial compounds contained in the binahong plant such as flavonoids, saponins, alkaloids, and terpenoids (Salimi et al., 2014; Silvana, et al., 2015; Mulangsri et al., 2020).
Flavonoids have a system of activity in restraining bacterial development by inactivating proteins in cell membranes, hindering lipoxygenase and cyclooxygenase pathways in arachidonic corrosive digestion.
Saponins fill in as antibacterial by disturbing the security of the bacterial cell film, causing bacterial cell lysis, so the system of activity of saponins is remembered for the antibacterial gathering that disrupts the porousness of bacterial cell layers, which brings about cell layer harm and causes the arrival of different significant parts from the bacterial cell, in particular proteins, nucleic acids. also, nucleotides.
The flavonoid content in the binahong leaf extract from the fresh sample was 11.263 mg/kg while the dry sample was 7.81 mg/kg.
The type of flavonoid acquired from the isolation and recognizable proof of new powder and dry powder of ethanol concentrate of binahong leaves is flavonol (Widya, et al, 2013). Binahong leaf extract
concentration 25% can inhibit the growth of Staphylococcus aureus bacteria and at a concentration of 50% can inhibit the growth of Pseudomonas aeruginosa bacteria and inhibit the growth of Shigella Flexner (Anwar and Soleha, 2016). In addition to antibacterial, binahong plants also contain antifungal compounds. Binahong leaf extract is able to inhibit the growth of Candida albicans (Kumalasari and Sulistyani, 2011; De Leilus Homans and Nahusona, 2020).
The disinfection process in the incubator is an important thing to do because it will affect the hatchability of eggs. Poor hatchery sanitation can be the main cause of low hatchability and poor chick quality (Boleli et al., 2016). Every effort should be made to make hatching eggs free from the risk of contamination from the time the eggs are released until the chicks are obtained (Mulyantini, 2010). Disinfection using 10%
betel leaf solution resulted in hatchability reaching 97.22% (Septiyani et al., 2016).
The incubator that will be used to incubate poultry eggs needs to be disinfected using a disinfectant as a way to prevent the spread of disease from the incubator room into the eggs (Yunaldi, 2021). If disinfection is carried out properly, it can break the path of the spread of disease that can be harmful. It has been demonstrated that hatcheries without being sterilized prior to incubation will result in excessive bacterial contamination and subsequent growth will result in reduced hatchability and poor chick quality (Mousa-Balabel et al., 2016). In some cases, as much as 20% of livestock die due to poor sanitation (Peric et al., 2022).
Microorganisms that are often encountered in incubators include Escherichia coli, Salmonella sp., Mycobacterium avium, and Aspergillus sp.
This is because incubators and hatching eggs that have been contaminated with pathogenic bacteria can be a source of infection in embryos (Kim and Kim, 2022).
One of the sources of bacteria can come from feces that stick to the hatching eggs and dry into dust so that it spreads in the
hatching machine room. Until now, many breeders still use KMnO4 and formalin as a disinfectant in hatching machines. However, due to the high price and limited sales, many farmers use liquid disinfectants with the active ingredients Chetypyridinium Chloride (CPC), Cetyl Trimethyl Ammonium Bromide (CTAB), and Benzalkonium Chloride (BC). The use of CPC to prevent contamination of pathogenic bacteria in the poultry processing industry was approved by the FDA in 2004 (Donabeth et al., 2013) and the European Union Commission in 2012 with a concentration of 0.8 to 1.0% (European Food Safety Authority, 2012). However, the use of CPC in excess of the dose will result in irritation and necrosis of open wounds (Mao X et al., 2020). The study of the effectiveness of binahong leaves in reducing microbes in duck egg incubators is an effort to reduce the negative impact of the more massive use of chemical disinfectants.
MATERIALS AND METHODS Hatching Egg Incubator specifications
Machine capacity for duck eggs is 60 egg tray. Machine dimension length x width x height = 60 cm x 40 cm x 32cm. Selected metal materials with advanced anti-erosion ability for incubator trolleys. Water proof insulation material for wall panels.
Binahong Leaf Extract Preparation Binahong leaf extract was obtained using the maceration method using 96%
ethanol as solvent 1000 ml of binahong gram leaves were soaked in 4000 ml of ethanol then stirred using a magnetic stirrer for 3 hours at a speed of 400 rpm, allowed to stand for 3 x 24 hours (Cacique et al., 2020;
Souhoka et al., 2021).
The binahong leaf filtrate was evaporated using a vacuum evaporator at a temperature of 40˚C and a pressure of 76 mmHg for 3 hours to free ethanol from the filtrate, in order to obtain a green binahong extract. Binahong leaf extract is ready to use. A chemical disinfectant solution as a comparison test was used Rodalon which contains the active ingredients
Chetypyridinium Chloride (CPC), Cetyl Trimethyl Ammonium Bromide (CTAB), and Benzalkonium Chloride.
Preparation of Agar Media and Gram's Stain
The agar media used were Nutrient Agar (NA) to grow bacteria and Potatos Dextroes Agar (PDA) to grow fungi. A total of 28 grams of NA and 39 grams of PDA were each dissolved in 1000 ml of distilled water, then sterilized by autoclave at 121oC for 15 minutes. Bacterial identification was carried out by gram staining using carbol gentiana violet, fuchsin water, lugol, and 96% alcohol.
Disinfection Treatment
The process of disinfection of the duck egg incubator is carried out by spraying chemical disinfectant and binahong leaf extract on different incubators. Testing the inhibition of bacteria and fungi using the open cup method. Each cup was placed separately in the incubator room and left for 30 minutes. After 30 minutes, the cup was closed and put in an incubator to be incubated for 24 hours at 37˚C.
Counting and Identification of Bacteria and Fungi
Bacteria and fungi that grow in the duck egg incubator before and after the disinfection process are calculated using the following formula (Ben-David and Davidson, 2014): The number of colonies obtained is expressed in cells/cm²
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 x60 minutes
30 m𝑖𝑛𝑢𝑡𝑒𝑠 x Hatching machine area Petri dish area
The calculation of the percentage decrease in the number of bacteria and fungi in the duck egg incubator is carried out with the following (Kennan, 2018):
Σinitial bacterial −Σfinal bacterial
Σinitial bacterial x 100%
Identification of bacteria using gram staining and identification of fungi using the slide culture method by growing the fungi on a glass object for 72 hours and observing macroscopically and microscopically.
Zone of Inhibition Test
The test bacteria used in the inhibition test were in situ bacteria, namely bacteria that had been isolated from the duck egg incubator. Before use the bacteria are enriched in Lactose Broth for 24 hours.
Inhibition test using agar diffusion method.
The clear zone on agar media was measured using a caliper with units of measurement in millimeters (mm).
Statistical Analysis
The data obtained was calculated using ANOVA with further testing using the Tukey Test using the SPSS version 25 program (IBM, 2019).
RESULTS AND DISCUSSION Bacterial Inhibitory Activity
The disinfection process was carried out by spraying the inside of the incubator with binahong leaf extract according to the treatment and chemical disinfectant as a control. Chemical disinfectants commonly used by farmers. There was an increased of reduction of bacteria and fungi in the duck egg hatching machine room, after the disinfection process was carried out (Table 1) The reduction of bacteria and fungi gradually increased in line with the increase in the concentration of the binahong leaf extract (Figure 1).
Table 1. The increased reduction of bacteria and fungi in the duck hatchery after the disinfection process
Disinfectant Binahong Leaf Extract 5%
Binahong Leaf Extract 10%
Binahong Leaf Extract 15%
Chemical disinfectant**
Increased reduction
of Bacteria (%) 52.69±5.40a* 71.65±8.32b 87.50±11.31c 89.91±14.10c Increased reduction
of Fungi (%) 30.10±11.05a 51.61±16.46b 53.81±11.17b 62.60 ±5.36c
*Different letters show significant differences (P<0.05)
** Rodalon
The highest reduction in bacteria and fungi was achieved by treatment with 15%
concentration of binahong leaf extract.
Binahong leaf extract up to 15% has the same effectiveness as chemical disinfectants commonly used by breeders in reducing the total number of bacteria in hatching machines. The active compounds in binahong leaves work actively in inhibiting bacterial growth.
Alkaloid compounds, terpenoids, and saponins in binahong leaves are efficacious as antibacterial substances. The mechanism of inhibition of alkaloid compounds is by slowing down the peptidoglycan constituent parts in bacterial cells, so the cell wall layer isn’t full grown and causes cell passing (Anggraini et al., 2019; Nurhasanah et al., 2020; Egra et al., 2019). The mechanism of action of terpenoid or steroid compounds as antibacterial is through damage to cell membranes by lipophilic compounds.
Terpenoids respond with porins on the outer
membrane of bacterial cells to frame solid polymeric bonds that harm the porin and diminish the penetrability of the bacterial cell wall so microbes will need supplements and their development will be hindered (De Oliveira Junior et al., 2018; Guimaraes et al., 2019; Amirzakariya and Shakeri, 2020).
Saponins are active compounds that are widely contained in various kinds of plants that are often used for traditional medicine, including the binahong plant that thrives in Indonesia (Yao et al 2014; Ozogul et al., 2015; Trisunuwati and Setyawati, 2017).
The activity of saponins as antibacterial is based on their chemical structure (Dong et al., 2020).
Saponins in binahong leaves are able to work in destroying the permeability of bacterial cell walls, causing bacterial death (Indarto et al., 2019). The bacteria in the duck incubator can come from dust or feces attached to the egg shell. One of the most influential factors in the success of hatching
eggs is the cleanliness of the egg shell.
Microbes attached to the egg shell can attack the embryo which is the cause of low hatchability (Septiyani et al., 2016).
Currently, many breeders still use chemicals as a disinfectant in hatching machines.
However, there are several shortcomings in the use of chemical disinfectants, including
causing disruption of embryo development resulting in embryo abnormalities (Nandhra et al., 2015). Referring to the aftereffects of the review, the utilization of binahong leaf extract up to 15% can decrease the quantity of microbes and fungi in duck hatching machines the same as the use of chemical disinfectants (Table 1).
Table 2. The inhibition of bacteria in duck egg incubator
No. Bacteria Type Zone of inhibition (mm)
Binahong leaf extract 15 % Chemical Disinfectant
1. Spored Bacilli 12.5±5.12 19.7±2.20
3. Coccus 19.2±2.30 23.0±2.90
5. Non-spored Bacilli 15.2±4.11 38.6±4.20
Interpretation Strong Very Strong
Spored Bacilli : purple bacteria color, Gram positive bacteria Coccus : purple bacteria color, Gram positive bacteria
Coccus : rod shape, purple bacteria color, Gram positive bacteria Coccus : purple bacteria color, Gram positive bacteria
Non-spored Bacilli : purple bacteria color, Gram positive bacteria
Figure 2. The zone of inhibition
0 20 40 60 80 100
0 0,5 1 1,5 2 2,5 3 3,5 4 4,5
Reduction Percentage
Disinfectant Consentrations
1. Binahong Leaf Extract 5%; 2. Binahong Leaf Extract 10%; 3. Binahong Leaf Extract 15%; 4.
Rodalon
The reduction of Bacteria (%) The reduction of Fungi (%)
Zone of Inhibition Testing is a fast, qualitative means to measure the ability of an antimicrobial agent to inhibit the growth of microorganisms. The inhibition of bacterial growth can be used to measure the susceptibility of the bacteria towards the disinfectant. The zone of inhibition is a circular area around the spot of the disinfectant in which the bacteria colonies do not grow (Figure 2). The bacteria that were incubated for the inhibition test were the bacteria that grew dominantly in the incubator. The inhibition zone at a dose of 15% binahong leaf extract on bacteria that grew dominantly in the incubator identified as sporing bacilli, coccus, and non-sporing bacilli resulted in a strong category of inhibition zone, with a clear zone diameter ranging from 12.5±5.12 mm to 19.2±2.30 mm, while chemical disinfectants were categorized as very strong (Table 2). The measurement of the hindrance zone describes the inhibitory power of the active compounds contained in the binahong leaf extract.
Fungi Inhibitory Activity
One of the fungi species that is often found contaminating the hatchery is Aspergillus fumigatus. This species can cause Aspergillosis in DOD (Day Old Duck) contaminated in incubators. Aspergillosis is an opportunistic disease that can increase mortality in DOD. Several researchers have studied the antifungal activity of flavonoid
active compounds from plants (Kanwal et al., 2010; Al Aboody and Mickymaray, 2020). The flavonoid compounds in binahong leaves were able to reduce the fungal population in duck egg incubators such as chemical disinfectants (Table 1).
Several antibacterial compounds are also known to have antifungal activity (Kumalasari and Sulistyani. 2011; De Leilus Homansand and Nahusona. 2020).
Flavonoids have been known to be effective as antifungal compounds that act widely on various fungal species, such as Candida albicans, Trichoderma rubrum, Candida glabrata, Candida krusei, Candida parapsilosis, Candida neoformans, Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius (Al Aboody and Mickymaray, 2020). Other active compounds such as terpenoids are able to inhibit the growth of Aspergillus flavus and Aspergillus niger (Bisht et al., 2011). The results showed that several species of fungi, such as Aspergillus sp, Penicillium sp, Rhizopus sp, Cladosporium sp, Trichoderma sp, and Mucor sp, could be eliminated from the incubator after disinfection by binahong leaf extract (Table 3).
However, there are species that are still found in the incubator after disinfection, namely Aspergillus sp. It is suspected that a higher concentration of binahong leaf extract is required to reduce Aspergillus sp.
Table 3. Antifungal resistance of binahong leaf extract against various species of fungi in duck egg incubator
Fungi Type Before Disinfection After Disinfection*
Aspergillus sp. √ √
Penicilium sp. √ −
Rhizopus sp. √ −
Cladosporium cladosporiodes √ −
Trichoderma sp. √ −
Mucor sp. √ −
Binahong Leaf Extract 15%
CONCLUSION
Binahong leaf extract is effective as antibacterial and antifungal in the process of disinfection of duck egg incubators.
Binahong leaf extract with a concentration
of 15% was able to match chemical disinfectants in reducing bacteria and fungi in duck egg incubators. However, the inhibitory power of binahong leaf extract against bacteria that grew dominantly in
hatching machines was in the strong category, while chemical disinfectants were very strong. The group of fungi that were resistant to disinfectants with 15%
concentration of binahong leaf extract was Aspergillus sp.
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