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OPEN ACCESS Pakistan Journal of Nutrition
ISSN 1680-5194DOI: 10.3923/pjn.2018.214.218
Research ArticlePhytochemical Screening and in vitro Antimicrobial Effect ofOrange (Citrus sinensis) Ethyl Acetate Extract Silage
Ucop Haroen, Agus Budiansyah and Nelwida
Department of Nutrition and Animal Feed Science Technology, Faculty of Animal Science, Jambi University, Ma. Bulian KM 15,Mendalo Darat, Jambi, Indonesia
AbstractBackground and Objective: Secondary metabolites are complex compounds. Many citrus fruits contain such compounds in the skin,seeds and pulp that act as phytochemicals with bacterial growth-inhibiting, anti-fungal and anti-cancer activities. This study was designedto identify phytochemical compounds in ethyl acetate extracts of orange and assess their antibacterial activities. Methodology: An ethylacetate extract of orange silage (EAEOS) at 250, 500, 750 and 1000 ppm was fermented for 28 days. Treatments were replicated four times.The samples were placed in a jar serving as a silo under anaerobic conditions. At the end of fermentation, phytochemical screening wasperformed. Data were analysed using analysis of variance under a completely randomized design. Results: The EAEOS contains alkaloid,flavonoid, steroid, triterpenoid, phenolic, saponin and coumarin compounds. The antibacterial activity of EAEOS was assessed using disc and MIC (minimum inhibition concentration) methods with Escherichia coli (E. coli ), Staphylococcus aureus (S. aureus),Salmonella typhi (S. typhi ) and Bacillus subtilis (B. subtilis). The extract inhibited the growth of all test organisms, with zones of inhibitionranging from 9.75±0.00 to 16.75±0.14 mm (E. coli ), 8.00±0.23 to 12.50±0.24 mm (S. aureus), 8.50±0.24 to 11.75±0.00 mm(S. typhi ) and 7.75±0.11 to 11.75±0.12 mm (B. subtilis). The MICs were 38.72±0.23 to 59.54±0.23% (E. coli ), 15.08±0.54 to 23.25±0.59%(S. aureus), 10.46±0.12 to 19.65±0.02% (S. typhi ) and 9.64±0.45 to 11.28±0.44% (B. subtilis). Conclusion: The tested EAEOS compoundsexhibited inhibitory activities against both gram-positive (S. aureus, B. subtilis) and gram-negative (E. coli and S. typhi ) bacteria.
Key words: Ethyl acetate, inhibitory bacteria, orange, phytochemicals, silage, waste extract
Received: August 24, 2017 Accepted: January 30, 2018 Published: April 15, 2018
Citation: Ucop Haroen, Agus Budiansyah and Nelwida, 2018. Phytochemical screening and in vitro antimicrobial effect of orange (Citrus sinensis) ethylacetate extract silage. Pak. J. Nutr., 17: 214-218.
Corresponding Author: Ucop Haroen, Departement of Nutrition and Animal Feed Science Technology, Faculty of Animal Science, Jambi University,Ma. Bulian KM 15, Mendalo Darat, Jambi, Indonesia
Copyright: © 2018 Ucop Haroen et al. This is an open access article distributed under the terms of the creative commons attribution License, which permitsunrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Competing Interest: The authors have declared that no competing interest exists.
Data Availability: All relevant data are within the paper and its supporting information files.
Pak. J. Nutr., 17 (5): 214-218, 2018
INTRODUCTION
Secondary metabolites are complex compounds found inplants, occurring in the bark, leaves, roots and seeds, that canact as active phytochemicals. Moreover, phytochemicals incitrus plants show bacterial growth-inhibiting activities1. Thesecompounds can exert effects against various diseases throughanti-malarial, anti-cancer, anti-virus and anti-inflammatoryactivities2. Ethyl acetate extracts obtained from the wastegenerated from orange (Citrus sinensis) juice productioncontain complex compounds such as limonoid, alkaloids,flavonoids, steroids, triterpenoids, phenolic, saponins andcoumarin3. The major compounds in extracts of orange(Citrus sinensis) juice waste are limonoids, which are derivedfrom limonene and cause the bitter taste of citrus fruits. Thesecompounds can act as insecticides, antifeedants for insectsand inhibitors of the growth of bacteria and fungi2. Someresearchers evaluating the biological activity of Citrusphytochemical compounds have found that they can improvelivestock health and serve as a natural feed additive that canlower blood cholesterol4-6. In addition, phenolic compoundsexhibit considerable antimicrobial activity that can modulatethe gut ecosystem and feed efficiency7.
Orange waste has potential as an additive for poultryfeed8-10 and dairy cattle11. For example, orange waste can beused at up to 20% in broiler chicken rations and orange wasteextract used in drinking water up to 1000 ppm can increasebroiler chicken growth and feed efficiency12,13.Additionally, theuse of sweet orange peel flour as a feed additive at 1.5% inrations can improve the growth of broiler chickens14.
This study was conducted to investigate the possibilityto minimize waste in the fruit-processing industry. Wasteminimization during the production process and the recoveryof valuable by-products can substantially reduce the amountof waste. Here, it is evaluated that the antibacterial potentialand phytochemical compounds of orange extract silage as asource of natural feed additives for livestock.
MATERIALS AND METHODS
Plant material: The orange juice waste was obtained from ajuice merchant located in Jambi city. The citrus (C. sinensis)fruits used are local from Jambi. After collection, the orangewaste was cleaned, dried in an oven at temperature of 55ECfor 3-4 days until the water content reaches 10-15% andmilled into flour using a hammer mill. The material was furtherreduced to a powder before mixing with molasses and ricebran, wrapped in a plastic bag and stored at roomtemperature for 28 days. The samples were placed in a jar,which acted as an anaerobic silo.
Preparation of extracts: The powdered orange silage(up to 1000 g) was macerated in ethyl acetate (3×5 L) solventfor 3 days at room temperature and filtered through a funnelto separate the extract from the dregs. The extracts wereconcentrated using a rotary evaporator at 40EC and thenevaporated to dryness in a water bath. The ethyl acetateextract of orange silage (EAEOS) was added to petri dishescontaining the following bacteria: Escherichia coli,Staphylococcus aureus, Salmonella typhi or Bacillus subtilis.Coleridin (antibiotic) was used as a control. The Petri disheswere covered with glass to prevent evaporation andincubated at 28EC15. A completely randomized design with5 treatments was used and the EAEOS treatments werereplicated four times. The treatment groups were as follows:P0 = containing no antibiotic extract (control), P1 = 0 ppmEAEOS, P2 = 250 ppm EAEOS, P3 = 500 ppm EAEOS,P4 = 750 ppm EAEOS and P5 = 1000 ppm EAEOS. The meanzone of inhibition was calculated.
Aqueous extraction: About 20 g of orange waste silage flourwas added to a test tube and macerated with ethanol withheating (in a water bath) for 15 min. The extract was filteredthrough Whatman No. 1 filter paper under hot conditions.Each extract was transferred to a test tube and the ethanolwas evaporated17.
Preliminary phytochemical analysis (Qualitative test8): Thepowdered material and extract were subjected to preliminaryphytochemical screening following previously describedmethodology9-11.
Test for alkaloids: Filtrate (2 mL) was mixed with 1% HCland approximately six drops of Mayor’s reagents. Acream-coloured or pale yellow precipitate indicated thepresence of alkaloids.
Test for steroids: Acetic anhydride (2 mL) was added to 0.5 gethanolic extract of each sample plus 2 mL of H2SO4. Colourchange from violet to blue or green indicated the presence ofsteroids.
Test for flavonoids: Filtrate (2 mL) was added toconcentrated HCl and magnesium ribbon. A pink-tomatored colour indicated the presence of flavonoids.
Test for saponins: A 4th test was used to detect saponins.About 2 g of fresh sample was added to a test tube andmacerated with ethanol with heating in a water bath for
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15 min. The mixture was filtered under hot conditions into atest tube and the ethanol was evaporated. Chloroform anddistilled water were added at a ratio of 1:1 for a total of 5 mL,chloroform and aqueous layers formed. The tube was shakento generate a foam, retention of the foam after the addition ofa few drops of concentrated HCl indicated the presence ofsaponins18.
Test for terpenoids: The chloroform layer was dripped ontoa plate consisting of three holes and left to dry. ConcentratedH2SO4 was added to one hole, either one drop of aceticanhydride or one drop of concentrated H2SO4 was added tothe other holes. The formation of green or blue-green colourindicated terpenoids19.
Test for phenolic compounds: Most of the water-methanolphase was removed with a pipette and placed in a small testtube, to which FeCl3 reagent was added. The formation ofblue/purple colour indicated the presence of phenoliccompounds18.
In vitro testing of extracts for antibacterial activityAntibacterial activity assay: The antibacterial activity ofEAEOS was tested via determination of inhibition zones usingthe disc diffusion method. Two paper discs were dipped intomedium containing each concentration of EAEOS, 250, 500,750 or 1000 ppm or coleridin (control). The discs were placedon the surface of a petri dish containing the test bacteria andincubated for 24 h at 37EC, after which the inhibition zone wasmeasured. If the zone of inhibition formed was larger than thatof the control, the sample was considered to possessantibacterial activity20.
Determination of the minimum inhibitory concentration(MIC): The MIC of EAEOS was determined by the serial dilutionmethod. EAEOS was serially diluted to 10, 20, 30, 40 and 50%.Bacterial suspensions (106 colony forming units [CFU]) wereadded to the tubes, which were incubated at 37EC for 24 h.The MIC was taken as the lowest concentration of EAEOS thatinhibited growth after a period of 24 h.
Statistical analysis: Data were statistically analysed usingone-way analysis of variance (ANOVA). If necessary Duncan’smultiple range test was applied to compare differencesbetween means16.
RESULTS AND DISCUSSION
Table 1 presents the phytochemicals detected in EAEOS.Tests for alkaloids, flavonoids, steroids, triterpenoids andphenolics were positive. These metabolites are known to haveantibacterial activity against E. coli, S. aureus, S. typhi andB. subtilis. Table 2 and 3 compare the antibacterial activity ofEAEOS and coleridin, as assayed by the disc diffusion method.EAEOS showed activity against E. coli, S. aureus, S. typhi and B. Subtilis and inhibited bacterial growth at all testedconcentrations. However, the antibacterial activity of EAEOSwas lower than that of coleridin.
The EAEOS showed significant antibacterial activityagainst all tested organisms, with better antibacterial activityat 1000 ppm than at 500 and 750 ppm, no antibacterialactivity was detected at 250 ppm. The greatest zone ofinhibition by EAEOS (1000 ppm) was against E. coli(16.75 mm), followed by S. aureus (12.50 mm), S. typhi(11.75 mm) and B. subtilis (11.75 mm). This antibacterialactivity might be due to the broad spectrum of phytochemical
Table 1: Phytochemical compounds in ethyl acetate extract of orange silageSecondary metabolites Reagent Observation ResultAlkaloids Meyer White precipitate formed (+)Flavonoids Sianidin test Orange solution (+)Steroids Liebermann-Burchard Blue solution (+)Triterpenoids Liebermann-Burchard Red-brown solution (+)Phenolic FeCl3 Solution blue/purple (+)
Table 2: Zone of inhibition (mm) of crude ethyl acetate extract of orange silage against test bacteria on Mueller-Hinton agar using the disc diffusion methodZone of inhibition (mm)----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Crude ethyl acetate extract of orange silage waste (ppm)--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Bacteria Control (coleridin) 250 500 750 1000E. coli 24.50d 9.75±0.00a 10.50±0.00a 12.50±0.27b 16.75±0.14c
S. aureus 23.65d 8.00±0.230a 10.75±0.12b 11.75±0.18b 12.50±0.24b
S. typhi 23.65d 8.50±0.24a 10.75±0.54b 11.00±0.00b 11.75±0.00b
B. subtlis 22.67d 7.75±0.11a 9.50±0.00b 11.50±0.13c 11.75±0.12c
Different superscripts in the same row refer to significantly different data (p<0.05)
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Table 3: Minimum inhibition concentration of crude ethyl acetate extract of orange silage for E. coli, S. aureus, S. typhi and B. subtilisPercentage of minimum inhibition concentration------------------------------------------------------------------------------------------------------------------------------------------------------------------------Crude ethyl acetate extract of orange silage waste (ppm)------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Bacteria 250 500 750 1000E. coli 38.72±0.23a 44.54±0.00b 57.15±0.98c 59.54±0.23c
S. aureus 15.08±0.54a 17.96±0.00b 18.15±0.14b 23.25±0.59c
S. typhi 10.46±0.12a 11.28±0.27a 18.15±0.54b 19.65±0.01b
B. subtlis 9.64±0.45a 10.05±0.76a 10.46±0.27a 11.28±0.44a
Different superscripts in the same row refer to significantly different data (p<0.05)
compounds present in EAEOS. The EAEOS at 250 and 500 ppmshowed very similar antibacterial activities against all testedorganisms.
Table 1 shows the presence of various constituents inorange juice waste. No effect of silage treatment on thecontents of citrus waste phytochemical compounds wasobserved, likely because a maceration method wasemployed, i.e., maceration does not alter chemical natures orstructures21. Indeed, maceration can lead to more extractedmaterial and avoid chemical changes to certain compoundsdue to heating. Factors that affect the phytochemical contentof a plant are the nature of the plant itself17 and the soil profile,harvest time, extraction method, temperature and solventproperties22.
The results of inhibitory zone measurements caused byEAEOS are shown in Table 2 and 3. Table 2 lists the EAEOSinhibitory zones for E. coli, S.aureus, S.typhi and B. subtlis,which ranged from 38.72±0.23 to 59.54±0.23% (E. coli ),15.08±0.54 to23.25±0.59% (S. aureus), 10.46±0.12 to19.65±0.02% (S. typhi) and 9.64±0.45 to 11.28±0.44%(B. subtilis). The ANOVA results showed that treatment withEAEOS (p<0.05) had an effect on bacterial growth, withhigher inhibition against E. coli and Salmonella bacteria(3 and 7 mm)23. For example, inhibition by etiacetic-extractedlemon peel and orange peel against E. coli was reportedly14 and 13 mm, respectively24,25. Variability in inhibitory powermay be due to differences in the phytochemical compositionof each extract and the composition of phytochemicals inplant extracts is affected by the soil profile, harvest time,extraction method, concentration, time, temperature andsolvent properties of the extract26.
The MIC of EAEOS against E. coli, S. aureus, S. typhi andB. subtlis ranged from 38.72±0.23 to 59.54±0.23% (E. coli ),15.08±0.54 to 23.25±0.59 (S. typhi ) and 9.64±0.45 to11.28±0.44% (B. subtilis). Duncan’s test results for theminimum EAEOS inhibitory level at 1000 ppm was significantly(p<0.05) higher than that at 750, 500 and 250 ppm. This is inline with the results of the EAEOS inhibitory zone test,
where the 1000 ppm EAEOS inhibition zone was larger for alltested bacteria. This is because at 1000 ppm, EAEOS containsa high concentration of phytochemical compounds, withconcomitant high antibacterial activity.
CONCLUSION
Fruit waste can be recycled in various innovative ways.Here, we report the presence of multiple antibacterialcompounds in Citrus sinensis extract silage.
SIGNIFICANCE STATEMENT
This study reveals that Citrus sinensis extract silagecontains phytochemical compounds that might be beneficialfor the livestock industry as natural feed additives.
ACKNOWLEDGMENT
The authors would like to thank the Directorate GeneralStrengthening Research and Development, Ministry ofResearch, Technology and Higher Education, Republic ofIndonesia, for funding this research through the HibahFundamental Project 2017.
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Phytochemical Screening and in vitro Antimicrobial Effect of the
Ethyl acetate Extract of silages waste of orange (Citrus sinensis) 1Ucop Haroen,
1Agus Budiansyah and
1Nelwida
1Departement of Nutrition and Animal Feed Science Technology
Faculty of Animal Science, Jambi University, Jambi
Jl. Jambi - Ma. Bulian KM 15, Mendalo Darat, Jambi.
email: [email protected]
ABSTRACT
This study was done to determine phytochemical compounds and antibacterial
activity of ethyl acetate extract of silages waste of orange (Citrus sinensis). Extract was
applied at 250, 500, 750 and 1000 ppm, then fermented for 28 days. Each treatment
was conducted on four replications. Samples were put in a jar as a silo in anaerobic
condition. At the end of fermentation phytochemical screening. Data were analyzed
using variance analysis of completely randomized design.
The result showed the ethyl acetate extract of silages waste of orange, revealed the
presence alkaloid, flavonoid, steroid, triterpenoid, fenolic, saponin, cumarin. The
antibacterial activity of the ethyl acetae extract of silages waste of orange, was assayed
using Disc method and MIC (Minimum Inhibition Concentration).Test microorganism
were Escherichia coli, Staphylococcus aureus, Salmonella tyhi and Bacillus subtilis.
The extract inhibited the growth of all the tests organisms at different concentrations.
Zone of inhibition for E. coli ranging 9.75±0.00mm to 16.75±0.14 mm, S. aureus
ranging 8.00±0.23mm to12.50±0.24 mm, S. typhi ranging 8.50±0.24mm to
11.75±0.00mm and B. Subtilis ranging 7.75±0.11mm to 11.75±0.12mm. Minimum
Inhibitory Concentration (MIC) for E.coli ranging 38.72±0.23% to 59.54±0.23%,
S.aureus ranging 15.08±0.54% to 23.25±0.59%, S.typhy ranging 10.46±0.12% to
19.65±0.02% and B. Subtilis 9.64±0.45% to 11.28±0.44%. The extract showed varied
inhibitory activities against the entire organism studied. The study has justifed the ethyl
acetate extract of silages waste of orange use for the inhibition activities antibacterial,
especially microbial culture of the Gram-positive strains S. aureus, B. Subtilis and the
Gram negative strains E. coli and S.typhy.
Keywords : orange waste extract, silages, phytochemical, inhibitory bacteria, ethyl acetate
Introduction
The use plants for medicines is an ancient practice common to all societies
espenciallly the Indonesia society. Plants show enormous versatility in synthesizing
complex materials which have no immediate abvious growth or metabolic fuctions.
These complex materials are called secondary metabolites (Akinmoladum et al., 2007).
Plants secondary metabolites have recently been raferred to as phytochemicals.
Phytochemicals are naturally accurring and biological active plant compounds that have
potential bacterial growth inhibiting activities. It is bellived that phytochemical
compounds may be effective combating or preventing disease due to their antioxidants
effect and bacterial growth (Miller et al., 2004). Antioxidants protect other molecule
(in vivo) from oxidation when they are exposed to free radicals and oxygen species
which have been implicated in the etiology of many disease and in food deterioration
and spoilage species.
Oranges (Citrus sinensis) are one of the most important and oldest horticulture
products in many tropical and subtropical areas. Orange waste is a primary by product
produced by the fruit processing industry and attempts have been made to use orange
waste extracts as natural antibiotic and feed additive for animals (Callaway et al., 2008;
Comment [WU1]: Please note that the journal requires the Abstract to be divided into the following sections: Background/Objective, materials and methods, results and Conclusion.
Comment [WU2]: References must be cited in the text in superscript digits at the end of sentence or paragraph before punctuation or full stop1. In case of two or more references, separate the superscript digits by comma1,2,6. Moreover, If there are more references but in continuous numbers then use dash between superscript digits2-6.
5
Haroen et al., 2013). As bioactive compounds are a valuable source of flavonoids,
steroids, triterpenoids, phenolic, saponin, cumarin and vitamin C (Miller et al., 2008)
and especially limonoids compounds (Haroen et al., 2013). Recent phytochemical
studies on the silages waste of orange juice revealed the presence of flavonoids,
steroids, triterpenoids, phenolic and saponin. The orange waste extracts are rich in
nutrients and contain many phytochemicals, they can be efficiently used as antibiotics
or as feed supplements too. Sice there is an increase in the number of antibiotic
resistance pathogens, there is always a search of an alternative feed supplements that is
regarded as safe. The orange waste extracts if proved to have antibacterial activity, they
can be also used in same food industry which generate large pell waste as a food
preservative.
This study was aimed to focus on waste minimization in fruit processing industry.
The combined efforts of waste minimization during the production process and recovery
of valuable product substantially reduce the amount of waste, as well as boost the
environmental scientific basis for the use of pells of citrus fruits by determining the
chemical constituents as well as qualitative analysis of crude phytochemicals. The
present work has been designed to evaluated the antibacterial potential and
phytochemical compounds of the extract from the orange (Citrus sinensis) waste
silages. Culture of the Gram-positive strains S. aureus, B. Subtilis and the Gram
negative strains E. coli and S. typhy were prepared on nutrient agar plates.
Materials and Methods
Plant material:
The plants used in this study were Citrus sinensis (common name: sweet orange).
The waste were collected from the local fruit juice shops. Affter colection the orange
waste were shade dried at oven temperature (45-50 oC) to constant weight over a perior
of 5 days. Each of the plant parts were coarsely powdered using a mortar and pestle and
were futher re duced to powder and were used to prepare the products, 2 kg orange
waste then mixture was stirred with the addition molases and rice bran and then
wrapped with plastic bag and stored at 28 days, samples were put in a jar as a silo in
anaerobic condition. The dried silage waste of orange was pulverized orange waste
were kept separately in an air-tight cellophane bag until used.
Preparation of extracts:
Waste silages mashed with orange using a mortal and pestle as much as 2 kg then
at room temperature. Then filtered to obtain the extract, the extract was concentrated
using a rotary evaporator at a temperature of 40 oC. Exactly 700 g each of the
pulverized plant was macerated successively in ethyl acetate 95% for 36 h each. The
mixture were then filtered under vacum and the filrates concentrated using a rotary
evaporator. The ethyl acetate concentrate was evaporated to dryness in a water bath.
The ethyl acetate extract of silages waste orange at 250, 500, 750 and 1000 ppm
concentrations 10, 20, 30, 40 and 50% aqueous ethyl acetate extract of silage waste of
orange were introduced into each of the petridishes. Coleridin (antibiotic) only served as
control . The contents of the petridishes were incubated at a temperature of 28 oC. The
petrdishes were covered with glass to prevent evaportion. Each treatment was replicated
four times in Completely Randomized Design. If necessary, a Duncan´s multiple range
test was applied to compare differences between means Steel and Torrie (1997).
Aqueous Extraction
The method of Dupont et al. (2005) was adopted for extraction with little
modification. Briefly, 15g of the powdered plant were soaked separately in 200 ml of
Comment [WU3]: Describe the Statistical Analysis at the end of the Materials and Methods section.
Comment [WU4]: What probability was used to decide the level of significance?.
6
distilled water at ambient temperature for 24 hour under shaking condition at 130 rpm.
The extract was then filtered using Whatman filter paper No 1. Each extracts transferred
to glass vials and kept at 4 ºC before use.
Preliminary phytochemical analysis (qualitative test)
The powered plant parts as well as the extracts were subjected to preliminary
phytochemical screening following the methodology of Sofowora (1994), Harborne
(1998) and Kokate (2001).
Test for alkaloids: Two milliliter filtrate was mixed with 1% HCl and about 6 drops of
Mayor’s reagents. A Creamish or pale yellow precipitate indicated the presence of
respective alkaloids.
Test for steroids: Two milliter of acetic anhydride was added to 0.5 g ethanolic extract
of each sample with 2 ml H2SO4. The color changed from violet to blue or green in
some sample indicating the presence of steroids.
Test for flavonoids: Two milliliter filtrate was added to concentration HCl and
magnesium ribbon. Pink-tomato red colour indicated the presence of flavonoids.
Test for saponins: Froth test for saponins was used. 1 g of the sample was weighed into
a connical flask in which 10 ml of sterile distiled water was added and boiled for 5 min.
The mixture was filtered and 2.5 ml of the filtrate was added to 10 ml, of sterile distilled
water in a test tube. The test tube was stopped and shaken vigorously for about 30 sec. It
was then allowed to stand for half an hour. Honeycomb froth indicated the presence of
saponins.
Test for terpenoids (Salkowski test): Five milliliter of each extract was mixed in 2 ml,
of chloroform and concentrated H2SO4 (3 ml) was carefully added to form a layer.
Areddish brown coloration of the inter face was formed to show positive results for the
presence of terpenoids.
Test for phenolic compounds: Most of the water-methanol phase were removed with a
pipette into a small test tube, then added FeCl3 reagent the formation of blue/purple
indicate the content of phenolic compounds.
In vitro testing of extracts for antibacterial activity
Antibacterial Activity Assay:
Antibacterial activity of ethyl accetate extract of silages waste of orange juice was
determined by Disc diffusion and broth dilution method (Long et al., 2003). Nutient
Agar (NA) and Muller Hinton broth (MHB) were used for the tests. Overnight culture
were grown at 37 oC in MHB. About 2 ml of test organisms was aseptically injected
into sterilized plates shaken to spread round the plates. Sterilized nutrient agar was
poured on top of the test organisms aseptically after it was cooled to about 45 oC. It was
shaken immediately for even distribution of the test organisms. The nutrients agar was
allowed to solidify sterilized cup borer of 8 mm diameter was used to make wells on the
solidified agar into which 0.5 ml diluted ethyl accetate extract of silage waste of orange
of the sample were aseptically introduced using pipette under strerile conditions. The
plates were incubated at 37 oC for 48 hours under room temperature.
Zone of inhibitions were measured around each well. Discs with 250 ppm coleridin
served as controls. The antibacterial activity against each test organism was quantified
by determining by average diameter of the zone of inhibition around the paper discs in
millimeters. The tests were performed twice and average diameters of zones were
measured.
Determination of Minimum Inhibitory Concentration (MIC)
MIC of ethyl accetate extract of silage waste of orange, was determined by serial
dilution method. Stock serial dilution of ethyl accetate extract of silages waste of
7
orange, was subjected to fold dilutions such that concentration ranged 10, 20, 30, 40,
and 50%. Again 106 CFU bacterial suspensions were added to the tubes. The tubes
were incubated at 37 oC for 24 h. MIC was take as the highest dilution of the extract
that inhibited the growth of the bacterial lowest concentrations of the ethyl accetate
extract of silages waste of orange which inhibited the growth after a period of 24 h of
incubation at 37 oC, were recorded as MIC.
Results and Discussion
Tabel 1. Phytochemical compounds in ethyl acetate extracts of silages waste of orange
Secondary
metabolites Reagent Observation Result
Alkaloids Meyer White mist formed (+)
Flavonoids Sianidin test Orange solution (+)
Steroids Liebermann-burchard Blue solution (+)
Triterpenoids Liebermann-burchard Red-brown solution (+)
Phenolic FeCl3 Solution blue/purple (+)
Table 1. Shows the phytocemicals detected in ethyl acetate extracts of silages waste
of orange. Test for alkaloids, flavonoids, steroids, triterpenoids and phenolics were
positive. These metabolites compounds (alkaloids, flavonoids, steroids, triterpenoids
and phenolic) are know to have curatives activity against E. coli, S. aureus, S. typhi and
B. Subtilis. The antibacterial activity of ethyl acetate extract of silages waste of orange,
assayed by Disc diffution method is compared with the antibiotic coleridin (controls)
Table 2 and Table 3. The results of the study showed that the ethyl accetae extracts of
silage waste of orange demonstrated activity on bacteria E. coli, S. aureus, S. typhy and
B. Subtilis. This showed the susceptibility tests against both organisms (E. coli, S.
aureus, S. typhi and B. Subtilis). The ethyl accetate extract of silages waste of orange
exhibited considerable levels of inhibition against all the levels. This was found to be
significantly lowers compared to coleridin (antibiotic) against E. coli, S. aureus, S. typhi
and B. subtilis. This suggestive of the presence of some phytochemicals compounds in
the extracts of orange waste juice silages.
Table 2. Zone of inhibition (mm) of crude ethyl acetate extract of silages waste of
orange against test bacteria on Mueller-Hinton agar medium using Disc
diffusion method.
Bacteria
Zone of inhibition (mm)
Crude ethyl acetate extract of silages waste of orange (ppm)
Control
(coleridin) 250 500 750 1000
E. coli 24.50d 9.75±0.00
a 10.50±0.00
a 12.50±0.27
b 16.75±0.14
c
S. aureus 23.65d 8.00±0.230
a 10.75±0.12
b 11.75±0.18
b 12.50±0.24
b
S. typhi 23.65d 8.50±0.24
a 10.75±0.54
b 11.00±0.00
b 11.75±0.00
b
B. subtlis 22.67d 7.75±0.11
a 9.50±0.00
b 11.50±0.13
c 11.75±0.12
c
Different superscripts in the same row refer to significantly different data (P<0.05)
Comment [WU5]: It is observed that results are neither compared nor contrasted properly with relevant findings from other published work. Discuss the Results in more detail.
8
Table 3. Minimum Inhibition Concentration crude ethyl acetate extract of silages
waste of orange for different bacterial strains.
Bacteria
Percentage of Minimum Inhibition Concentration
Crude ethyl acetate extract of silages waste of orange (ppm)
250 500 750 1000
E. coli 38.72±0.23a 44.54±0.00
b 57.15±0.98
c 59.54±0.23
c
S. aureus 15.08±0.54a 17.96±0.00
b 18.15±0.14
b 23.25±0.59
c
S. typhi 10.46±0.12a 11.28±0.27
a 18.15±0.54
b 19.65±0.01
b
B. subtlis 9.64±0.45a 10.05±0.76
a 10.46±0.27
a 11.28±0.44
a
Different superscripts in the same row refer to significantly different data (P<0.05)
Ethyl acetate extract of silages waste of orange orange showed a significant
antibacterial activity against all the test organisms. Ethyl acetate extract of silages waste
of orange levels 1000 ppm showed a very good antibacterial activity when compared to
ethyl acetate extract of silages waste of orange 250, 500 and 750 ppm. Ethyl acetate
extract of silage waste of orange level 1000 ppm showed a maximum zone of inhibition
against E. coli (16.75 mm) followed by S. aureus (12.50 mm), S. typhi (11.75 mm) and
B. subtilis (11.75 mm) whereas the Ethyl acetate extract of silage waste of orange level
250 ppm did not show such high antibacterial activity. This antibacterial activity may be
indicative of broad spectrum phytochemical compounds. In case of ethyl acetate extract
of silage waste of orange levels 250 ppm and 500 ppm showed more or less the same
antibacterial activity all the test organisms. Ethyl acetate extract of silage waste of
orange levels 250 ppm all the test organisms showed very less antibacterial activity,
when compared to other treatments. Ethyl acetate extract of silage waste of orange
level 250 ppm did not show any significant effect against all the tested strains except all
the test organisms. This show that ethyl acetate extract of silage waste of orange level
250 ppm has the capability of zone inhibition very less the same antibacterial activity
all the test organisms. This it indicate that different ethyl acetate extract of silage waste
of orange level may have diverse antibacterial agent that has different modes of action
or the bacteria may have a special metabolism to overcome or adapt its activity. Ethyl
acetate extract of silage waste of orange level 1000 ppm proves to be a good for the
inhibition of anti-bacterial agents from to other teatments as it has shown better as
antibacterial activity relating that higher zone inhibition means high concentration of
single or variety of phytochemical and there fore high antibacterial activity. This
statement can be validated as ethyl acetate extract of silage waste of orange level 1000
ppm has show highest zone inhibition a well antibacterial activity. Haroen et al. (2013)
reported antibacterial activity screening of crude (limonoid-ethyl acetate), (limonoid-
methanol) and (limonoid-n-hexane) of orange juice waste with concentration 250 ppm
were test showed the zone inhibition in millimeter (11.75 for the bacterial E. coli and
10.25 for the S. enteridis) than limonoid-methanol (8.00 and 9.00) and limonoid n-
hexane (8.00 and 9.25). However, this difference may be becauce of the difference in
the phytochemical composition in various part of the plant or may be also due to the
concentration and the extraction method used or environmental factors or difference in
the genotypes of the citrus plant used. The ethyl acetate extract of silage waste of
orange level 1000 ppm higher anti-bacterial activity as that standar antibiotic used in
the study. However, coleridin showed higher activity relatively.
MIC of ethyl acetate extract of silage waste of orange are shown in Table 3
respectively. The ethyl acetate extract of silage waste of orange showed significant
activity. The hydrocarbon components either remain on the surface of the medium or
evaporate (Griffin et al., 2000). That could be the reason for the better results obtained
9
by the microdilution method. Use of small volumes of the test substance and growth
medium (Sokovic et al., 2007). The difference in the antibacterial activity with the
source different concentration. Solvent has proven that not all phytochemical that are
responsible for antibacterial activity are soluble in a singel solvent. The preliminary
phytochemical investigation revealed the presence of various constituents of orange
waste. The results are shown in the Table 1.
Conclusions
Recycling of fruit waste is one of the most important means of utilizing it in a
number of innovative ways, yielding new products and meeting the requirements of
essential products required in human, animal and plant. Conclusion of the bioactive
compounds contributing to the antibacterial activity.
Acknowledgement The authors are very grateful to Directorate General Strengthening Research and
Development Ministry of Research, Technology and Higher Education, Republic
Indonesia that was Funded this experiment by Hibah Fundamental Project 2017.
References Akinmoladun. A. C., Ibukun. E. O., Afor. E., Obuotor. E. M., Farombi. E. O. 2007.
Phytochemical constituent and antioxidant activity of extract from the leaves of ocimum
gratissimum. Sci. Res. Essay. (2) pp (163-166).
Callaway. T. R., J. A. Carrol. J. D. Arthington., C. Pratt and T. S. Edrington. 2008. Citrus
Products Decrease Growth of E. Coli O157: H7 and Salmonella Thyphimurium in Pure
Culture and in Fermentation with Mixed Ruminal Microorganisms in vitro. Foodborne
Pathog. Dis (5): 621-627.
Dupont. J., C. S. Consorti., P. A. Z. Suarez. (2002): Organic Syntheses. John Wiley, Chichester
. (79) pp: 236.
Griffin. G. S., L. J. Markham., N. D. Leach (2000). An agar dilution method for the
determination of the minimum inhibitory concentration of essential oils. Journal of
Essential oil Research (12): 149-255.
Harborne. J. B and A. J. Harborne. (1998). Phytochemical Methods A Guide to Modern
Techniques of Plant Analysis. Kluwer Academic Publishers London. UK.
Haroen. U., Y. Marlida., Mirzah., A. Budiansyah. 2013. Extraction and isolation
phytochemical and antimicrobial activity of limonoid compounds from orange waste
juice. Pak. J. Nutr (12): 730-735.
Kokate. C. K. (2001): Practical Pharmacognosy. Vallabh Prakashan. pp. 218.
Long. H. H., N. Furuya., D. Kurose and Y. Takanami. 2003. Isolation of endophytic bacteria
from salanum sp. And their antibacterial activity against plant phatogenic bacteria. J. Fac.
Agr. Kejushu. Univ. 48(12): 21-28.
Miller. E. G., J. J. Peacock. T. C. Bourland. S. E. Taylor and J. M. Wright. 2008. Inhibition of
oral carcinogenesis by citrus flavonoids. Nutr. And Cancer. (60) 69-74.
Miller. E.G., J.L. Porter, W.H. Binnie. I. Y. Guo and S. Hasegawa. 2004. Futher studies on the
anticancer activity of citrus limonoids. J. Agric. Food Chem. (52) 4908-4912.
Comment [WU6]: What are the implications, applications, recommendations and limitations of the study? State clearly at the end of the Discussion section
Comment [WU7]: References are each must be numbered, ordered sequentially as they appear in the text
10
Sofowora. E. A., (1994). Medicinal plants and Traditional medicine in Africa. John Wiley and
Sons Ltd.
Sokovic. M., D. Ptar., Marim., D. Brkic., J. L. Leo., D. V. Griensven (2007). Chemical
composition and antibacterial activity of essential oil of ten aromatic plants against
human phathogenic bacteria. Global Science Books Food. 1(1).
Steel. R. G. D., H. J. Torrie 1997. Principles and Procedures of Statistics. A Biometrical
Approach 3rd Edition McGraw-Hill Series in Probability and Statistics
1
2
Phytochemical Screening and Iin Vvitro Antimicrobial Effect of Orange (Citrus
sinensis)Ethyl Acetate Extract Silage
Ethyl Aacetate Orange (Citrus sinensis) Silage Extracts. 1Ucop Haroen,
1Agus Budiansyah and
1Nelwida
1Departement of Nutrition and Animal Feed Science Technology
Faculty of Animal Science, Jambi University, Jambi
Jl. Jambi - Ma. Bulian KM 15, Mendalo Darat, Jambi.
email: [email protected]
Abstract
Background and Objective: Secondary metabolites are complex compounds.Many
citrus fruits containedin citrus fruitsthat are naturallyoaccurringsuch compoundsand
derived fromin the skin, seeds and pulp that;these compounds that act as
phytochemicals,that havewithpotential bacterial growth-inhibiting, activities, anti-
fungal and anti-cancer activities.This study was designed to identify the phytochemical
compounds in ethyl acetate extracts of orange and assess their antibacterial activitiesy
of ethyl accetateacetate extracts of orange silage. Methodology:An eEthyl acetate
extract of orange silage (EAEOS)was applied at 250, 500, 750 and 1000 ppm,and
thenwas fermented for 28 days. Treatments were replicated four times. The sSamples
were put placed in a jar serving as a silo under anaerobic conditions. At the end of
fermentation, phytochemical screening was performed. Data were analyzedanalysed
using analysis of variance under the a completely randomized design. Results: It is
observed that : EAEOSTheethyl acetate extract of orange silage contains alkaloid,
flavonoid, steroid, triterpenoid, phenolic, saponin and cumarincoumarin
compounds.The antibacterial activity of EAEOSthe ethyl acetate extract of orange
silage, was assessayed using the disc and MIC (minimum inhibition concentration)
methods with.Test microorganisms were Escherichia coli, Staphylococcus aureus,
Salmonella typhi and Bacillus subtilis. The extract inhibited the growth of all tests
organisms, with.The zones of inhibition ranginged from 9.75±0.00 to 16.75±0.14 mm
(E. coli), 8.00±0.23 to 12.50±0.24 mm (S. aureus), 8.50±0.24 to 11.75±0.00 mm (S.
typhi) and 7.75±0.11 to 11.75±0.12 mm (B. sSubtilis). The MICs ranged fromwere
38.72±0.23 to 59.54±0.23% (E. coli), 15.08±0.54 to 23.25±0.59% (S. aureus),
10.46±0.12 to 19.65±0.02% (S. typhi), and 9.64±0.45 to 11.28±0.44% (B. sSubtilis).
Conclusion: Thetested EAEOSethyl acetate extract of orange silage compounds had
exhibitedinhibitory activities against both gGram-positive (S. aureus, B. subtilis) and
gGram-negative (E. coli and S. typhi)bacteria.
Keywords:Ethyl acetate, inhibitory bacteria, orange, phytochemicals, silage, waste extract
Introduction
Secondary metabolites are complex compounds found in plants,;they and
naturallyoccurringring in the bark, leaves, roots and seeds,of plants andthat can act as
active phytochemicals. Moreover, pPhytochemicals in citrus plants have potentialshow
bacterial growth-inhibitingon activities1. Biologically,Tthese compounds can exert
eaffects against various diseases throughsuch as anti-malarial, anti-cancer, anti-virus
and anti-inflammatory activitiesy2.Ethyl acetate extracts obtained from the waste
generated from orange (Citrus sinensis) juiceproduction contain complex compounds
such as limonoid, alkaloids, flavonoids, steroids, triterpenoids, phenolic, saponins
andOrange (Citrus sinensis)wastejuice extractsusing ethyl acetate solvent still contains
complex compounds such as limonoid, alkaloids, flavonoids, steroids, triterpenoids,
phenolic, saponins and coumarin3. In addition thatTthe mostdominant majorcompounds
Comment [SE1]: Please ensure that the intended meaning has been maintained.
Comment [SE2]: Please ensure that the intended meaning has been maintained.
3
in extracts oforange (Citrus sinensis)wastejuicewaste extract areis limonoidss
compound, which .Limonoids are coppounds derived from limoneineand cause the
bitter taste ofn citrus fruits.; andTthese compounds can biologically act as insecticides,
antifeedants for insects and inhibitors oftion the growth of bacteria and fungi2. Some
researchers evaluating the biological activity of Ccitrus phytochemical compounds can
have found that itthey can improve livestock health and serve as a natural feed additive
and that can lower blood cholesterol4-6
. In addition, phenolic compounds exhibit
considerable antimicrobial activity.Their antimicrobial abilitythatmay canmodulate the
gut ecosystem and feed efficiency7.
In addition to containing phytochemical compounds such as alkaloids, flavonoids,
triterpenoids and limonoids,Oorange waste is alsohas potential as a mixture of an
additive toforpoultry feed Oluremi 8-10
and.Orange (Citrus sinensis) wastecan also be
used as aan mixture ofadditive to thefeed of dairy cattle11
.For example, oOrange(Citrus
sinensis) waste can be used at up to 20% in broiler chicken rations, and orange (Citrus
sinensisi) wastejuiceextract used in drinking water up to 1000 ppm level can increase
growthbroiler chickengrowth and feed efficiency12-13
.Furthermore Additionally, tThe
use of sweet orange peel flour as a feed additive atof 1.5% in rations can improve the
growth of broiler chickens14
.
This study was conducted to investigate the possibility for towaste
minimizewasteation in the fruit-processing industry. Waste minimizationduring the
production process and as well as the recovery of valuable by-products can,
substantially reduce the amount of waste. Here, we evaluate the antibacterial potential
and phytochemical compounds of orange(C. sinensis) silage extract silages.Which will
serve as a source of natural feed additives for livestock.
Materials and Methods
Plant material:
The orange juice waste is wastaken obtainedfrom a juice merchant located in Jambi
city. The cCitrus fruits used are local citrus fruits from Jambi (C.itrus sinensis)fruits
used are local from Jambi. After collected thecollection, the orange (Citrus
sinensis)waste is wascleaned, then dried withdried at an oven temperature of 55 oC5 °C
for 3-4 days until the water content reaches 10-15%, then and milled used asinto a flour
using a hammer mill. ItThe material wasand were further reduced to a powder before
mixing with molasses and rice bran, wrapped in a plastic bag and stored at room
temperature for 28 days.,The sSasamples were put placed in a jar, which acted as an
anaerobic silo.
Preparation of extracts:
The pThe pPowdered orange (Citrus sinensis)silage(up to as much as one
thousand1000gramsg)is wasmacerated inby using ethyl acetate (3 x 5 L) solvent for 3
days at room temperature and.After that filtered byusing throughathe funnel and
obtainedto separate the extractsof ethyl acetate andfrom the dregs. The extracts were
then concentrated by using a rotary evaporator at 40 °C to obtain the citrus ethyl acetate
extract from orange silage.The ethyl acetate concentrateand thenwas evaporated to
dryness in a water bath. The ethyl acetate extract of orange silage (EAEOS) was
introduced addedinto each of the Petri dishes containing the following
bacteria:Escherichia coli, Staphylococcus aureus, Salmonella typhi orBacillus
subtilises. Coleridin (antibiotic) was used as a control. The Petri dishes were covered
with glass to prevent evaporation and incubated at 28 o
C8 °C15
.A cCompletely
randomized design with five treatments was used, and the. EAEOS treatments were
replicated four times. The treatment groups were as follows:; P0= containing only anno
Comment [SE3]: Please note that as written, this suggests that the cholesterol-lowering effect occurs in the livestock.
Comment [Ed.4]: Please ensure that the intended meaning has been maintained with this deletion.
Comment [QCE5]: Please ensure that the intended meaning has been maintained in this edit.
Comment [SE6]: Please ensure that the full intended meaning has been maintained; some redundancy was removed.
Comment [SE7]: Please note that this antibiotic is not found in the literature.
Comment [Ed.8]: Please ensure that the intended meaning has been maintained with this edit.
4
antibiotic extract (control); P1= 0 ppm EAEOS; P2= 250 ppm EAEOS; P3= 500 ppm
EAEOS; P4= 750 ppm EAEOS; and P5= 1000 ppm EAEOS. The mean zone of
inhibition was calculated for E. coli, S. aureus, S.typhi and B. subtilis.
Statistical analysis
Data were statistically analyzedanalysed using one-way analysis of variance (ANOVA).
If necessary, Duncan´s multiple range test was applied to compare the differences
between means16
.
Aqueous eExtraction
Twenty grams of orange waste silage flour then feed into thewas added to theatest
tube,then and macerated with ethanol that has beenwaswith heatinged (over inthe
awater bath) for 15 minutes. Then the extract was filtered through Whatman No.1 filter
paperunder hot conditions using Whatman No.1 filter paper. Each extract was is
transferred to the atest tube, and let thethe ethanol was evaporated17
.
Preliminary phytochemical analysis (Qualitative test8)
The powdered plant parts, as well as thematerial and extract, were subjected to
preliminary phytochemical screening following the previously described methodology9-
11.
Test for alkaloids:Filtrate (22m ml) was mixed with 1% HCl and
approximatelybout six drops of Mayor’s reagents. A cream-coloredcoloured or pale
yellow precipitate indicated the presence of respective alkaloids.
Test for steroids: Acetic anhydride (22m ml) was added to 0.5 g ethanolic extract of
each sample plus 2 ml of H2SO4.The color Ccolour changed from violet to blue or
green in some samples,indicateding the presence of steroids.
Test for flavonoids:Filtrate (2 ml) was added to concentrated HCl and magnesium
ribbon. A pPink-tomato red color colour indicated the presence of flavonoids.
Test for saponins: AA froth test was used to detectsaponins. Ttwo grams of fresh
samplesare fed intowerewas added tothe atest tube, then and macerated with ethanol
that has beenwaswith heatingedin a during the water bath for 15 minutes. Then filtered
inThe mixture was filtered under hot conditions into the atest tube, and let the wholethe
ethanol wasevaporated.Then add Cchloroform and distilled water were added at a ratio
of 1: 1 for a total ofevery 5 ml then input into the test tube, let briefly form;,and the two
layers of chloroform and wateraqueous layersformed. Then shake,The tube was shaken
toforminggenerate a foam;that is not lostretention of the foam afterby the addition of a
few drops of concentrated HCl,L indicateding the presence of saponins18
.
Test for terpenoids::Taken a littleThelayer of chloroformlayerand thenwas dripped
ointo a plate consisting of three holes and, left tot dry. In one holeTo one hole was
added Cconcentrated H2SO4H2SO4was added to one hole;either , to another hole plus
one drop of acetic anhydride and orone drop of concentrated H2SO4H2SO4 was added
to the other holes. . The formation of green or blue-green color colour indicateds
terpenoids19
.
Test for phenolic compounds:Most of the water-methanol phase was removed with
a pipette into and placed ina small test tube, to which FeCl3 reagent was added. The
formation of blue/purple color colour indicateds the presence of phenolic compounds18
.
In vitro testing of extracts for antibacterial activity
Antibacterial Activity Assay:
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Comment [SE10]: Please check for accuracy.
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5
Test The antibacterial activity of EAEOS iswas tested by viadetermination of the
inhibition zonesusing theby disc diffusion method. Into theTwo paper discs arewere
dipped into the hatching medium was dipped each of two paper discs atforcontaining
each concentration ofEAEOS,: is250 ppm, 500 ppm, 750 ppm, or 1000 ppm,and
orcoleridin (control). Place theThe discspaper waswere placed on the surface of the
adisc Petri dishcontaining the test bacteria. Incubate for and incubated for 24 hours at 37
°Cand, after which the inhibition zone was measured its inhibition zone. If the drag
zonezone of inhibition formed is waslarger than that of the control zone, the sample was
considered to possessit is presence to bethe treatment is antibacterially activitye.
Test the disc to see theTheCclear zone around the disc isdeterminationed:method20
.
1 ml of bacterial solution is wasfed addedto Petri dishes containing MHB medium and
left allowed todryfor 10 minutes to dry;, the remaining residue liquidis wastaken
removed with a pipette. Then the disc is wasimmersed in solution, and incubated for 48
hours at 37 °C,to andafter which aobserve the clear zonewas observed.
Determination of the Minimum Inhibitory Concentration (MIC)
The MIC of EAEOS, was determined by the serial dilution method. The EAEOS
was serially diluted to concentrations of 10, 20, 30, 40 and 50%. Bacterial suspensions
(106 colony forming units [CFU]) were added to the tubes, which were then incubated at
37 oC7 °C for 244h h. The MIC was taken as the lowest concentration of EAEOS which
that inhibitedthe growth after a period of 244h h.
Results and Discussion
TabelTable 1. Phytochemical compounds in ethyl acetate extract of orange silage.
Secondary
metabolites Reagent Observation Result
Alkaloids Meyer White mist
precipitateformed
(+)
Flavonoids Sianidin test Orangesolution (+)
Steroids Liebermann-Burchard Blue solution (+)
Triterpenoids Liebermann-Burchard Red-brown solution (+)
Phenolic FeCl3 Solution blue/purple (+)
Table 1.shows presentsthe phytochemicals detected in EAEOS. Tests for alkaloids,
flavonoids, steroids, triterpenoids and phenolics were positive. These metabolites are
knownto have antibacterial activity against E. coli, S. aureus,S. typhi and B. sSubtilis.
Tables 2 and 3 compare the antibacterial activity of EAEOS and coleridin, as, assayed
by thedisc diffusion method. It is observed that EAEOS had showedactivity against E.
coli, S. aureus, S. typhi and B. subtilis showing .This showed the susceptibility against
bothof all these organisms (E. coli, S. aureus, S. typhi and B. subtilis).and The EAEOS
inhibited bacterial growth at all tested concentrations.,but However,the antibacterial
activity of EAEOS was lower than that of that of coleridin against E. coli, S. aureus, S.
typhi and B. subtilis.
Table 2.Zone of inhibition (mm) ofcrudeethyl acetate extract of orange silageagainst
test bacteria on Mueller-Hinton agar medium using the disc diffusion method.
Comment [QCE11]: Please ensure this word choice is correct.
Comment [SE12]: Please check for accuracy.
Comment [SE13]: Please consider removing this content. The disc diffusion method is a common technique, and these details (except for the medium used) are not necessary.
Formatted: Font: Bold
Formatted: Font: Bold
Comment [Ed.14]: Please use a consistent tense (present or past) for all experimental methods.
6
Bacteria
Zone of inhibition (mm)
Crudeethyl acetate extract of orange silages waste of orange (ppm)
Control
(coleridin) 250 500 750 1000
E. coli 24.50d 9.75±0.00
a 10.50±0.00
a 12.50±0.27
b 16.75±0.14
c
S. aureus 23.65d 8.00±0.230
a 10.75±0.12
b 11.75±0.18
b 12.50±0.24
b
S. typhi 23.65d 8.50±0.24
a 10.75±0.54
b 11.00±0.00
b 11.75±0.00
b
B. subtlis 22.67d 7.75±0.11
a 9.50±0.00
b 11.50±0.13
c 11.75±0.12
c
Different superscripts in the same row refer to significantly different data (P<0.05)
Table 3.Minimum inhibition concentration of crudeethyl acetate extract of orange silage
for E. coli, S. aureus, S. typhi and B. subtilis.
Bacteria
Percentage of Minimum Inhibition Concentration
Crudeethyl acetate extract of orange silages waste of orange
(ppm)
250 500 750 1000
E. coli 38.72±0.23a 44.54±0.00
b 57.15±0.98
c 59.54±0.23
c
S. aureus 15.08±0.54a 17.96±0.00
b 18.15±0.14
b 23.25±0.59
c
S. typhi 10.46±0.12a 11.28±0.27
a 18.15±0.54
b 19.65±0.01
b
B. subtlis 9.64±0.45a 10.05±0.76
a 10.46±0.27
a 11.28±0.44
a
Different superscripts in the same row refer to significantly different data (P<0.05)
EAEOS showed a significant antibacterial activity against all of the
testedorganisms, with.At 1000 ppm, EAEOS had better antibacterial activity at 1000
ppm than at 500 and 750 ppm;,and no antibacterial activity was detected at 250 ppm.
The greatest zone of inhibition by EAEOS (1000 ppm)was against E. coli (16.75 mm),
followed by S. aureus (12.50 mm), S. typhi (11.75 mm) and B. subtilis (11.75 mm). This
antibacterial activity might be due to the broad spectrum of phytochemical compounds,
present in EAEOS. In case ofEAEOSThe ethyl acetate orange waste of silage
extractlevels at 250 ppm and 500 ppm showed more or less the samevery similar
antibacterial activitiesyof all theagainst all tested organisms.
Table 1 shows the phytochemical investigation and the presence of various
constituents of inorange juice waste.This situationIt shows Nno effect of silage
treatment on thecontents of citrus waste phytochemical compounds was observed
content, likely because a maceration.This is due to the method was usedemployed, i.e.,;
maceration does not alter chemical natures or structures is the method of
maceration.With the maceration method of the desired chemical component does not
change the nature and structure21
.The advantages ofIndeed, mMaceration can leads to
more extracted material andthe maceration method are more extraction and can avoid
chemical changes to certain compounds due to heating. Factors that affect the
phytochemical content of a the plant are the properties of the solvent used,the nature of
the plant itself17
a andthe soil profile soil, harvest time, extraction method, temperature
and solvent properties22
.
The results of the inhibitory zone measurements from caused byEAEOS are shown
in Tables2 and 3. Table 2 shows liststhe inhibitory zone of EAEOS inhibitory
zonesforfortoE. coli, S.aureus, S.typhi andandB. subtlis, which bacteria ranged from
38.72 ± 0.23 to 59.54 ± 0.23% E. coli), 15.08 ± 0.54 to23.25 ± 0.59% (S.aureus), 10.46
± 0.12 to 19.65 ± 0.02% (S. typhi), and 9.64 ± 0.45 to11.28 ± 0.44% (B. subtilis). The
ANOVAresultsof variance analysis showed that treatment withofrealEAEOS (P <0.,05)
had an effect on bacterial growth, with.This result is higher thaninhibitionthe use ofdue
Comment [QCE15]: Please ensure that the intended meaning has been maintained in this edit.
7
toan ethanolic orange peel extract using ethanol against E. coli and Salmonella bacteria
of(3 mm and 7 mm)23
.For example, The iInhibitionthe use ofofbylemon peel extract by
using etiacetic-extractedlemon peel solvent and orange peel extract from C.
sSinensisagainst the development of E. colibacteria wasofreportedly 14 mm and 13 mm,
respectively24-25
. The Vvariabilitytionof in inhibitory powerobtained wasmay bedue to
differences in the phytochemical composition of each extract produced, and.tThe
composition of phytochemicalscompounds fromin plant extracts wasisaffected by the
soil profile, harvest time, extraction method, concentration, time, temperature and
solvent propertiesof the extract.used26
The results of the minimum inhibitory concentrationMIC of EAEOSon againstE.
coli, S. aureus, S. typhi and B. subtlis ranged from 38.72 ± 0.23 to 59.54 ± 0.23% (E.
coli), 15.08 ± 0.54 to 23.25 ± 0.59 (S. typhi), and 9.64 ± 0.45 to 11.28 ± 0.44% (B.
subtilis). The Duncan test resultssfor the minimum EAEOS inhibitory level of at1000
pppm is wassignificantly different (P <0.,05) higher than thattreatment at 750 ppm, 500
ppm and 250 ppm. This is in line with the results of the EAEOS inhibitory zone test,
where the 1000 ppm EAEOS inhibition zone is wasbetter largeragainst forall tested
bacteria. This is because the atEAEOS level of 1000 ppm,EAEOS contains has a high
concentration of phytochemical compounds,so with concomitanthightheat antibacterial
activity is also high.
Conclusions
Fruit waste can be recycled in various innovativeways. Here, we
demonstratedreport the presence of multiple antibacterial compounds in Citrus
sinensissilage extractsilages.
Significance Statements
This study discovered revealsthat Citrus sinensissilage extractsilages contains
phytochemical compounds, that might be beneficial for the livestock industry as natural
feed additives.
Acknowledgements The authors would like to thank the Directorate General Strengthening Research
and Development, Ministry of Research, Technology and Higher Education, Republic
of Indonesia, for funding this research through the Hibah Fundamental Project 2017.
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PJN 214-218by Mirzah Mirzah
Submission date: 17-Aug-2018 11:38PM (UTC+0800)Submission ID: 990745489File name: PJN_214-218 (150.31K)Word count: 3106Character count: 18210
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