Kefir fermented milk is produced from grains, a specific and complex mixture of bacteria and yeast. Kefir produces a sour taste from lactic and acetic acids1,2. It also contains CO2, ethyl alcohol, and aromatic compounds that make it is unique organoleptic3. Kefir can be made from nuts milk, and it does not contain cholesterol4.

Arachi or peanut (Arachis hypogaea L.) is one of the raw materials that can be processed into kefir milk5. Arachis hypogaea juice is proven to have higher nutrition than red bean and soybean extract and is a source of vitamin E and magnesium6. Arachis hypogaea were evaluated for total phenolic and flavonoid contents, antioxidants, and vitamins essential for optimum health7.

Arachis hypogaea contain Vitamin E, known as α-tocopherol. Vitamin E has been in use for more than 50 years in dermatology and is an essential ingredient in many cosmetic products8. They are associated with obstructing the formation of free radicals by preventing oxidation and are believed to prevent damage to collagen and elastin fibers, increase skin cell regeneration, treat acne, and reduce the risk of decreased skin firmness and wrinkles9.

The cosmetic preparation chosen in this study is in the form of a peel-off mask. This form was chosen because the peel-off mask is a popular facial treatment, does not cause dependence, and is easy to apply10. The peel-off masks can minimize pores and is valuable for recovering and treating facial skin11. Peel-off masks are usually made from polyvinyl alcohol (PVA) or hydroxypropyl methylcellulose (HPMC), developed in a hot aquadest of 80℃. This material has adhesive properties to form a film membrane that can be peeled off when dry12.

The research on peanut kefir has been carried out, in which A. hypogaea kefir with a concentration starter lactic acid bacteria of 2% (w/v) with the fermentation of 48 hours was the best composition reported13. However, antioxidant activity and the development of kefir in cosmetic product has not been carried out. This research will continue making formulation peel-off kefir mask with 2% yeast (w/v) and fermentation for 48 hours to have good physical stability and antioxidant activity.

Materials

Arachis hypogaea was obtained and determined in Badan Penelitian dan Pengembangan Daerah Kebun Raya Banua (050/492-LIT/KRB) and the yeast “Fermipan” used in the study were obtained from the Banjarbaru market, South Kalimantan. Meanwhile, D-glucose was purchased in Prida Lab (Central Jakarta) and De Man, Rogosa and Sharpe (MRS) agar Merck was purchased in Nitrakimia (Yogyakarta). The antioxidant activity test was performed with UV-Vis Spectrophotometry (Genesys 10 UV-Vis).

Methods

Material preparation

Arachis hypogaea were peeled and washed with clean water. Arachis hypogaea were grounded using a blender with a ratio of water : beans (8 L : 1 kg of beans). The resulting A. hypogaea slurry was filtered and given the A. hypogaea filtrate. A total of 300 mL of A. hypogaea milk in Erlenmeyer was added with 12 g of D-glucose (4% w/v) and pasteurized at 80°C for 15 minutes. Then, 300 mL of A. hypogaea milk was added with 6 g of yeast (2% w/v) and fermented for 48 hours.

Preparation of peel-off mask

Nipagin was dissolved into CO2-free water with a ratio of 1 : 30 at 80°C while stirring continuously with a magnetic stirrer. The nipagin solution was removed from the water bath and mixed with glycerin while continuing to stir. The HPMC powder was dissolved in CO2-free water with a ratio of 1 : 15 at a temperature of 80°C, then left to stand until the HPMC expanded utterly. The two mixtures were stirred until homogeneous and added with the A. hypogaea extract kefir, then added with CO2-free aquadest until it reached 100% of the total weight. The formulations used are presented in Table I.

TableI. Peel-off mask formulation with A. hypogaea kefir

Physical evaluation of peel-off mask

Physical evaluation includes organoleptic test, homogeneity, spreadability test, and pH with the procedure as reported by Priani et al14.

Antioxidant activity of peel-off mask

Antioxidant activity was tested using the DPPH method. Arachis hypogaea kefir was diluted and made at a concentration of 1000 ppm. Peel-off mask preparation with concentrations 0.5% and 2% each dissolved in 25 mL of ethanol. A series of solutions invariant peel–off mask concentrations were prepared (10, 15, 20, 25, and 30 ppm) until 10 mL volumetric flask. Furthermore, 1 mL of DPPH 0.4 mM was added to the solutions and incubated at room temperature and avoid light. The absorbance was measured at a maximum wavelength to calculate the inhibition percentage using the equation [1]. A0 was the absorbance of the blank solution, and A1 was the absorbance of the sample solution. The inhibition concentration of 50% (IC50) was determined using the linear equation y= bx+ a.

… [1]

Physical and chemical evaluation of A. hypogaea kefir and peel-off mask

Arachis hypogaea kefir was optimized with the addition of 2% yeast within 48 hours of fermentation and got pH of the kefir was 3.646. After the kefir was formed, the peel-off mask was formulated into two variance concentrations of A. hypogaea kefir as an active ingredient. Varian of kefir concentrations in the formula were 0.5% (F1), and 2% (F2) w/w. The various kefir concentrations were aimed to get the best formulation of mask and activity antioxidant. The physical evaluation of the peel-off mask was organoleptic, pH, drying time, and gel spread15. The peel-off masks in the two formulas were transparent yellow tended to be precise. For the two formulas, the distinctive aroma of A. hypogaea was produced. When applied to the skin layer, the gel was easily distributed and did not feel hot. Both F1 and F2 formulas had a good consistency. The physical appearance of the peel-off mask can be seen in Figure 1.

a b

Figure1. Peel-off mask with (a) 0.5% and (b) 2% A. hypogaea kefir concentration

The organoleptic test was aimed to see the physical appearance by observing the color, smell, consistency, and homogeneity. The results of organoleptic observations can be seen in Table II. The homogeneity test was conducted to determine that the resulting peel-off mask preparation did not experience clumping when the active substance mixed with the base. Peel-off kefir mask, when applied to the skin layer, was homogeneous. There were no fibers and lumps or color differences.

TableII. Organoleptic observations of peel-off mask formulation with A. hypogaea kefir

The spreadability test determines the ability to spread the gel on the skin layer. The peel-off mask is good if, when applied to the skin, the dispersibility of the gel has a standard diameter of 5-7 cm, and it can spread evenly that the effect can work optimally16,17. The peel-off mask spreadability test obtained for both formulas was 5.33 and 5.45 cm.

The dry time test in a peel-off mask preparation aims to determine the speed at which the mask forms a film when applied to the skin. The dry time of both peel-off mask formulas was 25 minutes. The drying time requirement for peel-off mask preparation is 15-30 minutes18. If the film forms faster, so active substances will be released so that consumers can immediately benefit from using these masks. The peel-off masks' pH was around 4.5-7.0, and it was appropriate with Standar Nasional Indonesia (SNI) and the pH balance of normal human skin19. The physical and chemical properties of the peel-off mask can be seen in Table III.

TableIII. Physical and chemical properties of peel-off mask formulation with A. hypogaea kefir

Antioxidant activity test of peel-off mask

The antioxidant activity test for the peel-off mask of A. hypogaea kefir was performed using the DPPH radical scavenging method at λ 519 nm. The proton radical scavenging activity with DPPH is quite reproducible and relatively simple20. The absorbance of the peel-off mask was linear with concentration according to the Lambert-Beer law (Table IV). The absorbance value was then used to calculate the % inhibition based on equation [1], and the results were presented in Table V. The IC50 value was then calculated based on a linear regression between concentration vs. %inhibition. The result shows that the peel-off mask F2 had better antioxidant activity than F1 with an IC50 value of 1.865 ppm and is included as a very strong antioxidant21. Meanwhile, F1 had an IC50 value of 6.950 ppm. This result shows that the concentration of A. hypogaea kefir added to the peel-off mask affects the antioxidant activity of the mask, with a higher concentration increasing its antioxidant activity. This result is in line with a study on A. hypogaea oil as an antiaging, in which 10% A. hypogaea oil has an antiaging effect. Another study reported that the antioxidant activity of 10% A. hypogaea shells was obtained IC50 of 380.18 μg/mL.

TableIV. Absorbance vs concentration of peel-off mask formulation with A. hypogaea kefir

TableV. %inhibition and IC50 of peel-off mask formulation with A. hypogaea kefir

Based on the physical properties of the peel-off A. hypogaea kefir mask, including the organoleptic test, spreadability, dry time, and pH test, it shows that the F1 and F2 formulas had good physical properties of peel-off masks. The best antioxidant activity was obtained in F2 with the addition of 2% A. hypogaea kefir, indicating an IC50 value of 1.865 ppm.

The authors would like to acknowledge the Universitas Lambung Mangkurat for funding this research (PNBP ULM), the Laboratory of Pharmacy Technology of Universitas Lambung Mangkurat for supporting the facilities used in the research, and those who helped carry out the research.

All authors have an equal contribution in carrying out this study.

None.

The author declares there is no conflict of interest.

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