Inhibition of Carica Solid Soap to the Growth of Staphylococcus epidermidis Bacteria

. Carica ( Carica pubescens Lenne & K. Koch.) contains various nutrients such as vitamins, minerals, and active substances that can be used for skin care, and to bath soap products. The purpose of this research was to determine the inhibition of Carica soap to the growth of Staphylococcus epidermidis bacteria. The method used in this study was agar diffusion. Samples in this research were 12 Carica soaps with different formulation, and a control using papaya soap that has been circulating on the market. The results of the study were analyzed using ANOVA with a confidence interval of 5 %. The results showed that the significance value was 0.472 > 0.05 so there was no significant difference between the soap formulations on the inhibition zone of the S. epidermidis bacteria. The highest inhibitory zone is in soap with formula 1 (1.38 cm), the lowest inhibitory zone is in soap with formula 7 (1.1 cm), while the papaya soap inhibition zone (positive control) is 1.7 cm. Suggestion from this research is to reformulate Carica soap to improve its quality.


Introduction
Carica (Carica pubescens Lenne & K. Koch.) is commonly known as mountain papaya.This plant thrives in mountainous regions.In Indonesia, Carica can be found in the Dieng plateau of Central Java and Tengger plateau of East Java.Carica has a close kinship with papaya (Carica papaya L.).Carica contains various nutrients such as vitamins, minerals, and active substances that can be used for skin care.
Kusnadi et al. [1] states that Carica fruit contains vitamin C, vitamin A, Calcium (Ca), and Phosphorus (P).While Minarno [2] states that Carica fruit contains saponins, antioxidants, flavonoids, and antibacterial compounds because they contain polyphenol, tannin and triterpenoid compounds.Total flavanoid of Tengger Carica (816.65 mg L -1 ) is higher than that of Dieng Carica (633.35 mg L -1 ).With the various contents of the fruit Carica is rich in nutrients needed by the skin so it is suitable to be applied to skin care products.This is the basis for conducting research by utilizing Carica fruit as an additional ingredient in making bath soap.
Bath soap is a mixture of sodium compounds with fatty acids that are used as body cleansing agents, in the form of solid, foam, with or without other additives and does not cause irritation [3].Soap quality standards have been set based on SNI 3532:2021 (Standar Nasional Indonesia -Indonesian National Standard for solid bath soap.The quality of soap products can be determined and anticipated by looking at the quality of the Iodide Value, Peroxide Value, and Saponification Value of the crude oil used as raw material [4]. As a skin cleansing agent, ideally soap can not only cleanse the skin of impurities but also clean the skin of various pathogenic microorganisms that normally live on the surface of human skin.One of the bacteria found on the surface of human skin is Staphylococcus epidermidis (Winslow & Winslow 1908, Evans 1916).Therefore this study aims to determine the inhibition of Carica soap on the growth of S. epidermidis bacteria.

Soap making
The soap ingredients consisted of oil, alkaline solution (NaOH), cocamide-DEA, and Carica fruit extract.Carica as an additional ingredient of soap was taken from the Tengger plateau.Carica fruit extract was taken as a concentration of 100 %.The extract was then diluted by distilled water until the concentration of 80 % and 60 %.The palm oil was heated to a temperature of 50 °C to 60 °C then cocamide-DEA was added.Then it was mixed with an alkaline solution at a concentration according to the design of the experiment (the mixing temperature of the alkaline solution and oil were the same).Carica juice was added to the mixture at respective concentration.Furthermore, it was heated again with temperatures above 70 °C.Then poured into molds and allowed to stand for 2 wk so that the saponification process run perfectly.The experimental design of making Carica soap was presented in Table 1.

Soap quality test
The quality of the soap was determined based on chemical variables (water content, amount of fatty acids, free alkaline, free fatty acids, non-soapy fats, pH), physical variables (hardness, foam stability), Hedonic tests (color, aroma, thickness, lots of foam), and the antibacterial activity of soap against to the S. epidermidis bacteria.

Bacterial inhibition test
Bacterial inhibition test was carried out on 12 sample to the S. epidermidis with agar diffusion method (diffusion of wells).Bacteria were grown on Nutrient Broth (NB) medium for 24 h at 37 °C.Furthermore, for testing the inhibition of Carica soap against the S.epidermidis using the medium Nutrient Agar (NA).Bacteria were inoculated on NA media in Petri dishes by using the spread plate method.The soap sample was diluted using distilled water with a ratio of 1:1, then put into the wellbore.The positive control used papaya soap produced by RDL Pharmaceutical Laboratory, while negative control used distilled water.Petri dishes were incubated for 24 h at 37 °C.Measurement of clear zone diameter at 6 th , 12 nd , and 24 th h.The data of antimicrobial test was carried out using the ANOVA with a confidence interval of 5 % [5,6].

Chemistry and physics of Carica soap
Soap water content was tested by the gravimetry method.Table 2 showed that almost all the water content of the treatment soap is not fullied SNI 3532:2021 provisions (maximum 15 %).Analysis with ANOVA showed a significance value of 0.000 < 0.05, meaning that there were significant differences between treatments.The treatment without the addition of Carica juice had the lowest water content.Kenna [7] stated that the proportion of water in the soap base material influenced the saponification process.The higher the water content, the slower the saponification reaction rate.Moisture content that was too high also causes the soap structure to form a gel.The higher reaction temperature used, the water content in soap would be lesser, it is caused by some evaporated water during reaction and mixing process [8].The measurement of the pH of Carica soap presented in Table 2 are high (pH 8.60 to 23.76).Generally, the pH of bath soap is safe to use at a pH of 7 to 10. Dlova et al. [9] stated that 69.4 % of the soap and cleaning product samples studied had an alkaline pH in the range of 9.3 to 10.7.The high pH of the soap can be caused by the concentration of NaOH used is too high or the saponification reaction does not run optimally.This can also be seen from the high level of soap-free alkali (Table 3) which exceeds the SNI standard (< 2.5 %).
If it is associated with a fairly high level of unsaponifiable fat (Table 3), it can be assumed that the saponification process is not running optimally, because if it runs optimally, the high enough free alkali level can saponify all the unsaponifiable fat.The inhibition of this saponification reaction can be due to the total amount of water from the ingredients used is too high.High pH soap can disrupt the pH balance of the skin (5.4 to 5.9).Soap alkaline substances will damage the acid coat on the skin which plays a role in protecting the skin from bacteria and viruses, and makes the skin dry so that it is susceptible to irritation and allergies [10].In addition, the amount of water contained in soap can affect the characteristics of soap during storage [11].Soap with a high water content will have a softer texture than soap with a low water content [12].The results of the analysis using ANOVA showed a significance value of 0.239 > 0.05, which means that there was no significant difference between the formulations of Carica soap.Both from pH, free alkali, and unsaponifiable fat, the three parameters showed a much higher number than that set in SNI 3532: 2021.This means that Carica soap has not met the established standard.The high pH and free alkali can harm the skin, while the unsaponifiable fat indicates the saponification process is not going well.
The high pH of the soap sample can be caused by several things, firstly, the amount of alkali added is too much so that when the saponification process is complete there is still residual alkali, this is also reflected in the results of the free alkali content test which tend to be high.Second, the saponification process does not run perfectly due to the presence of inhibitors.The saponification process is mainly influenced by the type of oil used, one of which is determined by the iodine value of the oil.Oils with high saponification values have low iodine values and vice versa [13].Palm kernel oil has free fatty acids below 1 % which indicates that the palm kernel oil used is of good quality, the higher the free fatty acid content of an oil, the quality is not good because the free fatty acids in the oil come from oxidation reactions, hydrolysis, heating, during processing and [14].Foam is a gas trapped by a thin layer of liquid containing a number of soap molecules adsorbed on the thin layer.In bubbles, the hydrophobic group of the surfactant will point to the gas, while the hydrophilic part will point to the solution and then the bubbles will come out of the liquid body [15], Table 4 shows the foam stability ranging from 94.2 % to 99.9 %.The stability of the foam is very good, this can be due to the addition of cocomid , 00029 (2023) https://doi.org/10.1051/e3sconf/202337400029E3S Web of Conferences 374 3 r d NRLS DEA which functions as a surfactant and foam stabilizer.Test the hardness of soap using a penetrometer.Table 4 shows that the hardness of soap ranges from 1.13 N m -2 to 4.5 N m -2  The lower the liquid content in the soap-making material, the higher the hardness of the soap.The use of olive oil as an ingredient will produce soap with a soft texture on the skin but has low foaming ability, coconut oil makes the resulting soap has a slightly hard texture but has high foaming ability, and palm oil will produce soap with very dense properties and foaming ability low [16].In this study, palm oil was used, so to increase the foam, cocomid DEA was added.Good quality soap has a high total fat content and low alkali content.The lower the total fat content, the higher the hardness of the soap [17].

Hedonic of Carica soap
The hedonic test was carried out on 30 untrained panelists [18] with the aim of obtaining information on the level of panelists' preference for Carica soap.The test results show that on average the panelists do not like or are neutral towards Carica soap products (Figure 1).In the process of making soap, no dye or perfume is added, assuming the Carica fruit has a bright orange color and a fragrant aroma.However, once applied to the soap, the color and aroma of the carica fruit disappears.This is in line with Minarno [2] explanation that Carica fruit does not contain essential oils.The original aroma of Carica fruit evaporates during the soap-making process.

Antibacterial activity of Carica soap
The analysis showed that the significance value was 0.472 > 0.05 so there was no significant difference among treatments for bacterial inhibition zones.The highest inhibitory zone was in soap with formula 1 (1.38 cm), the lowest inhibitory zone was in , 00029 (2023) https://doi.org/10.1051/e3sconf/202337400029E3S Web of Conferences 374 3 r d NRLS soap with formula 7 (1.1 cm) (Figure 2), while papaya soap inhibition zone (positive control) was 1.7 cm and distilled water (control negative) 0.6 cm.

Fig. 2. Bar chart of Carica soap inhibition zone for S. epidermidis bacteria
The results of the analysis showed that the significance value was 0.472 > 0.05 so that there was no significant difference between treatments on the bacterial inhibition zone.The highest inhibition zone was in soap with formula 1 (1.38 cm), the lowest inhibition zone was in soap with formula 7 (1.1 cm), while the inhibition zone for papaya soap (positive control) was 1.7 cm and distilled water (control) was negative) 0.6 cm.Some compounds that include antimicrobial compounds are saponins, flavonoids, tannins, terpenoids, xanthones, alkaloids, and essential oils [19,20,10] which can be used to replace synthetic antimicrobial compounds.Many synthetic antibacterial agents such as triclosan and chloroxylenol are used to produce antibacterial soaps [21].Minarno [2] states that Carica that grow in the Cangar, Bromo and Dieng regions have a total flavanoid content ranging from 600 mg L -1 to 800 mg L -1 .Kusnadi et al. [1] stated that the fresh fruit of Carica Dieng has vitamin C content of 65.12 mg 100 g -1 and vitamin A 1 771.1 μg 100 g -1 .Laili [10] states that the Carica fruit in the Dieng plateau contains antioxidant flavonoids, the higher the vitamin C content, the higher the antioxidant activity.The nhexane fraction of C. pubescens fruit peel contains alkaloids and tannins [22].From some of the results of these studies indicate that Carica fruit basically contains antimicrobial compounds.
Insignificant test results can be caused by several things, firstly there is another reaction (outside the discussion) of how the basic ingredients of soap to the juice of Carica is synergistic or antagonistic, both samples of Carica soap when diluted have a thick texture and the longer it forms a gel which prevents the diffusion process of the solution and active ingredient of Carica soap into the agar medium, and thirdly it requires a higher concentration of Carica fruit for example by adding fruit extracts.

Conclusion
The results of the inhibition test of Carica soap on the growth of Staphylococcus epidermidis bacteria showed no significant differences between treatments for bacterial , 00029 (2023) https://doi.org/10.1051/e3sconf/202337400029E3S Web of Conferences 374 3 r d NRLS inhibition zones.Bacterial inhibition zones range from 1.19 cm to 1.38 cm.Some suggestions from this study are that it is necessary to test the antibacterial power of Carica fruit extracts against Staphylococcus epidermidis bacteria, to reformulate the making of Carica soap, and test bacteria can use other bacteria that are usually present on human skin.

Tabel 2 .
The result of carica soap water content analysis Tabel 3. The result of carica soap pH, alkali free, and non-soapy soap test *AF =Alkali Free, NSS = Non-Soapy Soap