Antimicrobial Activity and Phytochemical Analysis of Solvent Extraction of Citrus Limon Peels

Lemons make up the bulk of citrus fruits, and they're frequently employed in the juice business. These enterprises generate a lot of garbage because these peels contain a wide range of bioactive components, they may be used to create a variety of value-added products that help to reduce pollution. This study aims to determine the antibacterial activity and phytochemical analyses of Citrus limon peels extracted with a solvent. The antibacterial activity of Citrus limon peels was tested against Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, as well as Gram-negative like Salmonella typhi, utilizing different solvents including acetone, ethanol, ethyl acetate, and petroleum ether. To determine whether specific bio-reactive chemicals are present or absent in solvent extraction, a phytochemical analysis was performed. Antimicrobial tests such as agar well diffusion assay, the minimum inhibitory concentration (MIC), and the minimum bacterial concentration were performed, and the results revealed that the ethanol extract of Citrus limon peel was more effective than peel extracts prepared with other solvents. Essentially digesting chemical and organic waste, enzyme bio cleaner is a type of natural cleaning product that includes certain numbers and types of bacteria, as well as enzymes and microbial nutrients. The primary objective of the research is to create a natural cleaning agent from citrus fruit utilizing lemon.


INTRODUCTION
Lemons make up the bulk of citrus fruits, and they're frequently employed in the juice business.It is one of the most significant commercial fruits cultivated on all continents with a global output of 102.64 million tons (Patsalou et al., 2017).These enterprises generate a lot of garbage, which mostly consists of lemon peels.Because these peels contain a wide range of bioactive components, they may be used to create value-added products that help to reduce pollution.Peels contribute the most to this waste with 50-60% (Jimenez et al., 2020).Citrus peels are high in flavonoids, flavones glycosides, phenols, essential oils, and organic acids which are uncommon in other plants, according to studies (Kanedi et al., 2021;Mitsagga et al., 2021).These chemicals are not only crucial for human health and the environment, but they also have a wide range of economic uses in the food and pharmaceutical sectors (Harfouch et al., 2019;Kryukova et al., 2021).Other active terpenes as well as aldehydes, esters, and alcohols contribute to the overall antimicrobial effects of the essential oils (Samarakoon et al., 2012).Citrus fruit has been used in traditional Asian medicine for thousands of years to treat gastrointestinal, bronchial, and asthmatic illnesses (Rafiq et al., 2018;Bottalico et al., 2020).The most important fruits in the Citrus genus are lemon (citrus limoniuym), lime (citrus aurantifulia ), grapefruit (citrus vitis), tangerine or mandarin (citrus reticulata) and sweet orange (citrus sinesis) (Samarakoon et al., 2012).Flavedo or epicarp (colored peripheral surface) and albedo or mesocarp (white soft inner layer) are the two types of citrus peels (Muliarta &Darmawan, 2021).This research aimed to concentrate on minimizing the waste in the fruit juice manufacturing business.The combined efforts of waste reduction throughout the manufacturing process and product recovery significantly lower the waste and improve the environmental profile of the juice processing business.By analyzing the 3 chemical contents and calculating the yield percentage of crude phytochemicals, the study analyses the antimicrobial activity and fundamental scientific foundation for the usage of citrus peels (Henderson et al., 2018).fruit solutions are an example of a natural cleaning product.Natural cleaning solutions are less expensive and damaging to the environment.Cleaning supplies are frequently utilized in daily life, whether it be for household chores, laundry, or other industrial uses.Since many of these products contain substances made of petrochemicals, most conventional cleaning solutions employ chemicals that are relatively harmful to human health and the environment.Garbage enzymes are an organic substance made by simply fermenting fresh vegetable and fruit wastes with water, brown sugar, and specialized yeast and bacteria (Satari & Karimi, 2018).

Microorganism selection
Extractions of Staphylococcus aureus; Salmonella typhi and Enterococcus faecalis were used for antibacterial testing as Gram-positive and Gram-negative bacteria, respectively.The pure cultures of Staphylococcus aureus; Salmonella typhi and Enterococcus faecalis are taken from the master plate and then subcultures into nutrient broth by inoculation technique.Overnight cultures of bacteria were prepared on nutrient broth.
Microbiological media and agar plate preparation 28 grams of nutrient agar was liquefied in one liter of distilled water.A small amount of agar-agar was added to it.The mixture was stirred to dissolve all the components.The mixture was autoclaved for 15 mins at 15 lbs pressure.The nutrient agar was cooled after autoclaving.Once the media is cooled, it is poured onto the Petri plates and allowed to solidify.

Collection of plant extracts
The peel of the plant used in the is Citrus limon (common name: Lemon).The peels were obtained from local juice shops.The peels were washed with distilled water to remove dust.They were then shade dried at a temperature of 32-35⁰C for 5 days.The dried peels were ground and made to powder.The powder was stored in a closed container.
Preparation of extracts 25 grams of dried and powdered orange peel was extracted using 100 ml of each solvent (acetone, ethanol, ethyl acetate, and petroleum ether).In a conical flask, the peel powder and solvents were placed and thoroughly mixed using a shaking machine for 48 hrs. at 150 rpm (except Petroleum ether-24 hrs).The extracts were then filtered using the Whatman filter paper.The filtrate obtained was then dried using the open-dish evaporation method and the dried extract obtained was placed in small vials and stored in the refrigerator at 4 ⁰C (El-Desoukey et al., 2018).

Estimation of percentage yield
The given formula was used to calculate the percentage yield of the extract that was obtained using different solvents (Giwa et al., 2018).

Antimicrobial activity/assay Agar well diffusion assay
The extracts agar well diffusion assay was conducted to determine the antimicrobial assay.Two Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and one Gram-negative bacterium (Salmonella typhi) were used to test for antimicrobial activity.Petri plates used for this assay were autoclaved and all the steps were carried inside the laminar airflow chamber.Nutrient agar was used as a nutrient medium for assay.Bacteria was poured and spread on the solidified nutrient agar and wells of 6 mm diameter were punched on the agar.100 μL of 100 mg/ml of test samples were transferred to the labeled wells.The test samples were allowed to diffuse through the media and the plates were incubated in the upright position for 24 hrs at 37 ⁰C. 1 mg/ml of ciprofloxacin was used as positive control and 25% Dimethyl sulfoxide (DMSO) was used as a negative control.The diameter of the zone of inhibition around the wells was measured to evaluate the antimicrobial assay.

Determination of minimum inhibitory concentration (MIC)
The final extraction concentration with no apparent microbial growth is known as minimum inhibitory concentration (MIC) (Guo et al., 2018).A sterile 96-well microtiter plate was labeled.To all the wells, a nutrient broth of 100 μL was added.Serial dilutions of 6.25 mg/ml, 12.5 mg/ml, 25 mg/ml, and 50 mg/ml of test samples were prepared with a micropipette.100 μL of the test sample was added to each well in ascending order and tips were discarded after each use.Finally, 25 μL of each bacterial suspension was added to every well.The plate was wrapped loosely with parafilm to avoid contamination and dehydration of bacteria.Added positive control with vegetative broth and test organisms and negative control with the nutrient broth was added.The plate was then kept in the incubator for 24 hrs at 37 ⁰C.After 24 hrs the microtiter plate was read using a microtiter plate reader and the MIC of the extract is recorded.

Determination of minimum bactericidal concentration (MBC)
MBC was determined by taking a loopful of broth from the wells with no evident signs of bacterial growth.The broth was streaked onto the sterile nutrient agar plates by streaking.The plates were then incubated for 24 hrs at 37 ⁰C.The concentration in which no visible growth of bacteria was seen was taken as the minimum bactericidal concentration.

Phytochemical analysis (Qualitative analysis)
• Test for alkaloids 2 ml of filtrate was mixed with 2 ml of distilled water and a continuous shaker.The presence of alkaloids was indicated by the formation of a 1 cm layer of foam.

• Test for amino acids
A few drops of Ninhydrin reagent were added to 2 ml of filtrate.The presence of amino acids was indicated by the appearance of purple color.
• Test for flavonoids 2 ml of 2N sodium hydroxide was mixed with 2 ml of filtrate.The presence of flavonoids was indicated by the appearance of yellow color.
• Test for steroids 2 ml of filtrate was mixed with an equal volume of chloroform and a few drops of conc.Sulfuric acid.The presence of steroids is indicated by the formation of a brown ring.
• Test for tannins 2 ml of filtrate was mixed with a few drops of 0.1% ferric chloride.The presence of tannins was indicated by the formation of blue-black or brownish-green coloration.

RESULTS AND DISCUSSION
The solvent extract of the lemon peel using various solvents produced different outcomes in each of the tests conducted in this study.The percentage yield of the recovered extract was significantly varied when different solvents were used.The percentage yield of extracts obtained from various solvents is shown in Figure 1.The greatest percentage yield was ethanol (5.4 %), followed by acetone extract (4.3 %) and ethyl acetate extract (2.3 %) petroleum ether (1.4 %).The petroleum ether extract had the lowest percentage yield (1.4%).The variation in yield between various solvents explains the solubility of distinct plant components in various solvents.Studies conducted by Eldesukoy et al., (2018) showed that the percentage yield of ethanol extract from Citrus limon peels was greater than that of other citrus fruits such as Citrus limetta, Citrus aurantifolia, Citrus reticulate, and Citrus maxima.The color of the extract was brownish orange.Citrus peel extracts show considerable antibacterial activity against all of the species tested (Table 1).Because of its high yield and antibacterial action, ethyl acetate is a good solvent for extracting antibacterial compounds.Because of the enhanced production, a higher antibacterial agent is linked to a higher concentration of certain phytochemicals.Citrus limon ethyl acetate extract had the maximum zone of inhibition (16mm) against S. aureus and S. typhi (10mm), followed by S. aureus (13mm).Petroleum ether and ethanol both have antimicrobial properties.Petroleum ether has reduced antibacterial activity when compared to other solvents.This reveals that the solvents can extract antibiotic chemicals that are potentially dangerous to bacterial strains.As a result, different extracts may contain different antibacterial agents with different modes of action, or bacteria may develop a specialized metabolism to resist or adapt to their activity (Giwa et al., 2018).When compared to extracts from other solvents, ethanol extract contains more phytochemicals (alkaloids, tannins, flavonoids, steroids), which may have boosted bacterial activity.The antibacterial effects seen in this investigation might be attributable to the phytochemical group's synergistic action (Guo et al., 2018).When compared to Gram-positive bacteria, Gram-negative bacteria were less susceptible to the solvent extraction of the peels.The insensitivity of diverse test organisms to the investigated solvent extracts was attributed to variations in the content and structure of Gram-positive and Gram-negative bacteria's cell walls (Shakya et al., 2019).Gram-negative bacteria are less susceptible to antibacterial agents because they have an outer membrane that inhibits hydrophobic chemicals from diffusing through the lipopolysaccharide coating (Kumar et al., 2011).The antibacterial activity of extracts obtained using several solvents indicated that not all phytochemicals with antimicrobial activity are miscible in one solvent (Oikeh et al., 2020).
The MIC and MBC of several solvent extracts of Citrus limon are shown in (Tables 2 and 3).The extracts were shown to have significant antibacterial activity.The broth dilution technique was used to perform MIC.The broth approach, which is carried out in microtiter trays, has the advantage of requiring less effort for a larger number of duplicates and allowing for the use of very small amounts of test material and growth media.
The MIC and MBC data also reveal that ethyl acetate extracts are more effective than extracts from other solvents.The following are the MIC values for different solvent extracts against various bacteria: S. aureus (acetone extract-13 mg/ml, ethanol extract-15 mg/ml, ethyl acetate extract-16 mg/ml, petroleum ether extract-12mg/ml); S. typhi (acetone extract-9 mg/ml, ethanol extract-9 mg/ml, ethyl acetate extract-10 mg/ml, petroleum ether extract-0mg/ml); E. faecalis (acetone extract-0 mg/ml, ethanol extract-14mg/ml, ethyl acetate extract-13 mg/ml, petroleum ether extract-13mg/ml).MBC values if various solvent extract against different bacteria is as follows: E. faecalis (acetone extract-50mg/ml, ethanol extract-50mg/ml, ethyl acetate extract100mg/ml, petroleum ether extract-0mg/ml); S. aureus (acetone extract-50mg/ml, ethanol extract-75mg/ml, ethyl acetate-1mg/ml, petroleum ether extract-0mg/ml); S. typhi ((acetone extract-1mg/ml, ethanol extract-75mg/ml, ethyl acetate extract-50mg/ml, petroleum ether extract-0mg/ml).Less activity at low extract concentrations might imply that the active ingredient concentrations in the extracts are insufficient to have any antibacterial action (Abdella et al., 2019).To battle infections caused by E. faecalis, S. typhi, and S. aureus, ethyl acetate extract of the peel may be chosen over-extract from other solvents (Teneva et al., 2019).A phytochemical study revealed the presence of numerous components in lemon peels.The presence of different phytochemicals such as tannins, saponins, essential oils, flavonoids, and 22 phenolic compounds in lemon peel is connected to its antibacterial effect (Hsouna et al., 2017;Gorski et al., 2022).The phytochemical analysis findings are shown in (Table 4).Extracts from various solvents included phytochemicals of diverse types.All extracts included significant amounts of alkaloids, steroids, flavonoids, amino acids, and tannins (Musa & Hafiz, 2019).All solvents were able to dissolve alkaloids, steroids, flavonoids, amino acids, and tannins in this study.In the extracts of several solvents, amino acids were absent (Egbuonu & Osuji, 2016).The polarity of the chemical and the solvent might explain the presence and absence of distinct components in the extracts.Tannins, steroids, flavonoids, and alkaloids are secondary active metabolites found in plants that contribute to lemon peel's antibacterial activity (Rehab et al., 2018;Nawaz et al., 2020).Citrus limon peel contains flavonoids and tannins, indicating that the peel has antioxidant effects.Tannins are also said to have antibacterial properties (Mostafa & Essaway, 2021).

CONCLUSION
Fruit waste recycling is one of the most important strategies for repurposing it in several innovative ways, resulting in new products and meeting crucial demands in human, animal, and plant nutrition, as well as the pharmaceutical industry.Citrus limon peel extracts made with various solvents were tested for antibacterial activity and phytochemical content against test organisms.Although the kind and number of active components in each extract are not immediately apparent, they are encouraging; nonetheless, further research with pure compounds is required before a firm judgment can be made about the bioactive molecules that contribute to antimicrobial activity.However, further research is needed to understand how the bioactive component confers antimicrobial properties to the orange peel extract and how these extracts might be used to treat pathogenic bacteria like S. typhi.Four different fermentation samples namely T1, T2, T3, and T4 were prepared.Results obtained from PH and sugar analysis showed that T2 and T4 were more effective bio cleaners.

Figure 1 .
Figure 1.The percentage yield of Citrus limon peel extract

Table 1 .
Zone of inhibition of Citrus limon peel extract for various bacterial strains (Well concentration = 100mg/ml).

Table 2 .
MIC value of Citrus limon peel extract for various bacterial strains

Table 3 .
MBC values of Citrus limon peel extract for various bacterial strains

Table 4 .
Phytochemical analysis (qualitative analysis) of Citrus limon peel extracts