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J Cosmet Med 2024; 8(1): 18-33

Published online June 30, 2024

https://doi.org/10.25056/JCM.2024.8.1.18

Impact of coated phycocyanin and phycoerythrin on antioxidant and antimicrobial activity of soap, anti-acne face wash and hand sanitizer gel

Bahareh Nowruzi, Hossein Hashemi

Department of Biotechnology, Science and Research Branch, Islamic Azad University, Tehran, Iran

Correspondence to :
Bahareh Nowruzi
E-mail: bahareh.nowruzi@srbiau.ac.ir

Received: January 11, 2024; Revised: March 25, 2024; Accepted: March 25, 2024

© Korean Society of Korean Cosmetic Surgery & Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: The use of natural ingredients in skincare products has become a topic of interest in contemporary society. The adverse effects and environmental risks of synthetic compounds have prompted studies on the use of photosynthetic organisms as sustainable and ecofriendly sources of effective ingredients. Natural extracts have gained attention in the cosmetic industry, especially those derived from plants, algae, and cyanobacteria. Cyanobacteria have gained prominence in the cosmetic industry because of their low culture requirements, rapid growth rates, and capacity to produce different bioactive metabolites. As a result, cyanobacteria are economically viable and sustainable resources.
Objective: We aimed to determine the effect of coating with natural phycocyanin and phycoerythrin pigments from two cyanobacterial species, Spirulina platensis and Nostoc sp., on the properties of three cosmetic products: soap, anti-acne face wash, and hand sanitizer gel.
Methods: After cyanobacterial culture, the pigments were extracted, purified, and coated with the stabilizer, chitosan. Thereafter, cosmetic products were formulated with phycocyanin and phycoerythrin, and their viscosity, pH, stability, and antioxidant and antibacterial properties against bacteria were evaluated using the appropriate assays.
Results: The anti-acne wash gel and soap had better properties when coated with both pigments and phycocyanin alone, whereas the hand sanitizer gel had better properties when coated with phycoerythrin alone and both pigments.
Conclusion: Overall, three coated and stable cosmetic products can be produced using cyanobacterial pigments owing to their high fortifying capacity.

Keywords: nostoc sp., phycocyanin, phycoerythrin, skin care products, spirulina sp.

Approximately 2,000 years ago, cyanobacteria were used by Chinese as a means of survival during periods of malnutrition, with Nostoc as the specific species employed. Cyanobacterial biotechnology emerged in the 1950s and has since been utilized for various commercial purposes, such as improving the dietary value of food and animal feed, owing to its chemical makeup. Cyanobacteria are essential in aquaculture, and their byproducts have applications in the cosmetic industry [1,2]. Microalgal biomass is commonly used to produce valuable products. Microalgae-derived products are commonly used as high-protein dietary supplements for human nutrition, aquaculture, and nutraceuticals [3].

Cosmetics play a significant role in the daily routines of many individuals. The demand for skin care products has significantly increased owing to growing concerns regarding skin health and is associated with their aesthetically pleasing qualities. Ethical concerns regarding the use of animal-derived products and the potential harmful effects of artificial substances on humans, such as allergies and environmental risks, have led to increased studies on cosmetic ingredients derived from photosynthetic organisms [4].

One of the main natural sources of cosmetically beneficial metabolites is microalgae, which can expedite skin healing and repair, and exhibit anti-blemish and anti-inflammatory effects [5-7]. Nostoc, Spirulina (Arthrospira), and Aphanizomenon are the most studied cyanobacterial species owing to their high contents of calcium, beta-carotene, phosphorous, iron, biotin, folic acid, pantothenic acid, and vitamin B12 [8]. As a result, the extracts and bioactive compounds of these species are being studied to determine their potential for use in cosmetics to protect the skin and hair [9]. Beta-carotene from Desmonostoc muscorum, Leptolyngbya foveolarum, and Arthrospira platensis can regulate ultraviolet (UV)-A-induced gene expression in human keratinocytes [10] and modulate biological targets, such as NF-κB, COX-2, and matrix metalloproteinase-9, due to its anti-inflammatory activity [5]. Cyanobacterial phycobiliproteins (PBPs), such as phycocyanin (PC), have antioxidant, anti-inflammatory, and antiaging properties, and are used as cosmetic ingredients [7,11].

Cyanobacterial PBPs have potential commercial applications as natural constituents in cosmetics. PBPs are hydrophilic proteins that bind to phycobilins and photosynthetic pigments, and are primarily found in cyanobacteria and certain red algae. Metabolites with structural similarities to bilirubin have been previously found to possess effective quenching properties against various oxygen derivatives [12]. As a result, these metabolites, known as PBPs, are suggested to serve as promising antioxidant agents. Fluorescent protein-pigment complexes comprise three components: phycoerythrin (PE; red pigment), allophycocyanin (APC; bluish-green pigment), and PC (blue pigment) [13].

PC exhibits potent antioxidant and radical-scavenging effects, leading to the inhibition of cell proliferation and induction of apoptosis of cancer cells. Spirulina contains various phytopigments, including PC, gamma-linolenic acid, phycocyanobilin, and phycoerythrobilin [11]. These compounds have been recognized for their antioxidant, anti-melanogenic, antiwrinkle, and antiaging properties. These compounds are used as natural colorants in cosmetics, such as lipsticks, eyeliners, and eye shadows. Spirulina is commonly employed because of its anti-inflammatory, neuroprotective, and hepatoprotective properties [4,14]. PC reduces the levels of alanine amino transferase, aspartate amino transferase, and malondialdehyde in serum [15]. Certain cyanobacterial extracts contain peptides and proteins that have extensive applications in hair care products, such as lotions, shampoos, solutions for permanent hair waves, and hair coloring products [16]. The use of extracts from Chlorogloeopsis sp. and Spirulina in hair care products was found to lead to positive outcomes, such as increased gloss, improved ease of combing, and enhanced hair restoration and moisturization [16]. The Blue Green Algae Hair Rescue Conditioning Mask (Aubrey Organics Company, Ltd.) was found to effectively strengthen hair and prevent breakage and splitting [6].

Cyanobacteria active extracts, such as Spirulin extract Spiralin, have been developed and are currently being used in the cosmetic industry to protect the skin. These extracts, which are found in products, such as Skinicer Repair Cream and Spirularin, have regenerative effects on damaged skin cells and collagen, and protect against UV radiation. Phycobiliprotein C-phycocyanin (C-PC), which is derived from Aphanizomenon flos-aquae, has been used as a natural colorant alternative to synthetic pigments. This substitution is because of the appealing pink-purple hue that C-PC imparts to the end products [17]. As studies on the use of other cyanobacterial genera for cosmetic purposes are limited, further investigations should be performed in this area. This study sought to determine the effects of chitosan-coated PC and PE on the antioxidant and antimicrobial properties of soap, anti-acne face wash, and hand sanitizer gel.

Culture conditions for Nostoc sp. and Spirulina sp.

The cyanobacterial strains, Spirulina platensis and Nostoc sp., were isolated from Cyanobacteria culture collection (CCC) of herbarium ALBORZ at the Science and Research Branch, Islamic Azad University, Tehran. The strains were grown in modified Zarook and BG110, respectively, and illuminated (300 m-2 s-1) growth rooms at 28°C±2°C for 30 days [18,19].

Extraction and purification of analytical-grade PC and PE

PC and PE were extracted and purified as described by Nowruzi et al. [20]. Pigments were extracted from 500 ml of homogenized log-phase (14-day-old) culture following centrifugation at 4,000 rpm to obtain the pellet. The pellet was resuspended in 100 ml of 20 mM acetate buffer (pH 5.1) for PC and potassium phosphate buffer (pH 7.1) for PE. The extraction was conducted via repeated freezing (-20°C) and thawing (room temperature) for 4 days to obtain a dark purple cell biomass. Cell debris was removed via centrifugation at 5,000 rpm for 10 minutes to obtain the crude extract. Purification was performed as described by Afreen and Fatma [21]. Solid ammonium sulfate was slowly added to the crude extract under continuous stirring to achieve 65% saturation. The resulting solution was allowed to stand for 12 hours in a cold room and centrifuged at 4,500 × g for 10 minutes. The pellets were resuspended in a small volume of 50 mM acetic acid-sodium acetate buffer (pH 7.1) and dialyzed overnight. The extracts were recovered from the dialysis membrane and filtered through a 0.45 μm filter.

The absorption spectrum was determined by scanning the sample in the range of 300–750 nm using a Specord 200 spectrophotometer (Analytik Jena). The amounts of PC and PE were calculated based on their optical density (OD) values at 620 and 650 nm (for PC) and 565 nm (for PE) using the equations below. The purity of the pigments was determined at each step as the purity ratio (A620/A652) for PC and (A555/A280) for PE (Fig. 1) [22].

Fig. 1.Different stages of separation and purification of phycocyanin (A to D) and phycoerythrin (E to H) pigments. (A) Primary culture of Spirulina cyanobacteria. (B) Preparation of crude extract. (C) Dialysis. (D) Freeze drying and preparation of phycocyanin powder. (E) Primary culture of Nostoc cyanobacteria. (F) Preparation of crude extract. (G) Dialysis. (H) Freeze-drying and preparation of phycoerythrin powder.
PC(μgml1)=(OD 620 nm-0.7OD 650 nm)7.38 APC(μgml1)=(OD 650n m-0.19OD 620 nm)5.65 PE(μgml1)=(OD 565 nm-2.8 [PC]-1.34 [APC])12.7

Stabilization and coating of pigments with chitosan

PC and PE were encapsulated by combining the pigments with a water-soluble chitosan (WSC). Sodium tripolyphosphate was used as a cross-linking agent. A WSC solution was prepared by soaking 1 mg/ml of oligochitosan in distilled water. The resulting mixture was stored at 4°C for 24 hours to ensure complete hydration. Thereafter, 1 ml of PC and PE in deionized water was gradually added to 1 ml of WSC (1 mg/ml) at 25°C with agitation. Sodium tripolyphosphate (2 mg/ml) was added to 0.5-ml aliquots, and the pH was adjusted to 7 using 1% HCl. Polyethylene glycol (0.5 ml) was then added to the mixture [23].

Formulation of soap, anti-acne face wash, and hand sanitizer gel

A series of experimental trials on compositions were used to optimize the laboratory formulation of soap, anti-acne face wash, and hand sanitizer gel. The composition of the final product was optimized using the ingredients listed in Table 1. Four conditions were established to generate the three products: condition one without any pigment, condition two with the PC pigment (1.5 g), condition three with the PE pigment (1.5 g), and condition four with both the PC and PE pigments (1.5 g) (Fig. 2) [24-26].

Table 1 . Composition of anti-acne washing gel, soap and antibacterial hand sanitizer

ProductsIngredientsQuantity
Anti-acne washing gelCarbapol0.1 gr
Distilled water2.0 ml
Methylparaben0.1 mg
Propylenglycol0.1 mg
Tea0.1 gr
Phycocyanin and phycoerythrin1.5 gr
SoapWater2.0 ml
Ethanol5 ml
Cinnamon oil1 ml
Citronella oil1 ml
Melted glycerine soap9.0%
Stearic acid0.033 g
Phycocyanin and phycoerythrin1.5 gr
Antibacterial hand sanitizerCarbopol 9401 gr
EDTA0.1 gr
Distilled water2.0 ml
Glycerine5 gr
Perfume0.3%
Phycocyanin and phycoerythrin1.5 gr

Fig. 2.Preparation of (A) anti-acne gel; (a) without pigment, (b) with phycoerythrin pigment, (c) total of phycoerythrin and phycocyanin pigments, (d) with phycocyanin pigment. (B) Hand gel; (a) total of phycoerythrin and phycocyanin pigments, (b) with phycoerythrin pigment, (c) with phycocyanin pigment (d) without pigment. (C) Soap; (a) without pigment, (b) with phycocyanin pigment, (c) the sum of phycoerythrin and phycocyanin pigments, (d) with phycocyanin pigment.

Evaluations using the anti-acne face wash

Viscosity

The viscosity of the anti-acne face wash was measured using a digital Brookfield viscometer with a yarn number of 64, operating at 10 revolutions per min and a temperature of 25°C. After a specific volume of hand wash was placed in a beaker, the viscometer tip was submerged in the hand wash gel to measure its viscosity. The tests were conducted in triplicate [27].

pH

The pH of a 1% aqueous solution of the formulation was determined at a constant temperature of 25°C using a calibrated digital pH meter [28].

Physical evaluation

Visual inspection was conducted to assess the physical attributes, including color, appearance, and consistency, of the formulation [29].

Gel stability

The durability of the gels was assessed using freeze-thaw cycles. The gels were stored at 4, 25, 37, and 40°C for 7 days [30].

Gel homogeneity

Homogeneity was determined via visual inspection after placing the samples in a specific container. The appearance, mass, and density of the gels were then evaluated [31].

Antimicrobial assay using the anti-acne face wash

Turbidimetry was used to screen for antimicrobial activity. The sterile nutrient agar medium was prepared aseptically and spread onto a Petri plate. The faces of volunteers with noticeable acne were cleaned with distilled water and allowed to air-dry. A cotton swab was used to apply distilled water to the ruptured pimple to ensure coverage of the entire acne surface. The solution was then uniformly applied to the prepared medium. The microbial cultures were incubated at 37°C for 24 hours to achieve optimal growth. Six sterile cotton balls, each 1 cm in diameter, were immersed in the prepared formulations of the standard drug and distilled water for 5 minutes. A 50-ml nutrient broth was prepared and sterilized. Five milliliters of the broth was collected and used as the reference standard in one cell of the UV spectrophotometer. The remaining broth was inoculated with the organism and cultured on a Petri plate. Five milliliters of the inoculated broth was distributed into six sterile test tubes. The cotton balls were then suspended in each test tube and labeled accordingly. The samples were incubated at 37°C for 24 hours. Subsequently, the samples were extracted and subjected to absorbance measurements at 600 nm [32].

Evaluations using the soap

Moisture content

A soap sample weighing 10 g was immediately measured and recorded as the “wet weight of the sample.” The wet sample was dried using an appropriate drying apparatus at a temperature below 239°F (115°C) until a constant weight was achieved. The sample was subsequently cooled and reweighed, and its dry weight was recorded. The amount of moisture in the samples was determined using the following equation [33]:

%W=100 A-B/B×100

where %W=percentage of moisture in the sample, A=weight of the wet sample (g), and B=weight of the dry sample (g).

Soap viscosity

Forty milliliters of the soap solution was transferred to a 100-ml beaker. The viscometer tip was then submerged in the beaker, and the viscosity was measured using a Brookfield digital viscometer [34].

Total fatty matter

Five grams of soap was obtained and transferred to a 250-ml beaker. Hot water (100 ml) was added to facilitate complete dissolution of the soap. Nitric acid (40 ml; 0.5 N) was added to the solution until a mildly acidic pH was achieved. The mixture was then heated in a water bath until the fatty acids formed a distinct layer above the solution. The fatty acids were then cooled and separated on ice. Chloroform (50 ml) was added to the remaining solution, which was then transferred to a separating funnel. The solution was agitated and allowed to undergo phase separation. The content of the lower stratum was depleted and chloroform (50 ml) was added to the remaining solution in a separating funnel. The fatty acids dissolved in chloroform were separated and transferred to the collected fatty matter, according to the procedure described above. The lipid content was measured using a pre-weighed porcelain dish. The contents were allowed to evaporate, and the resulting residue was weighed. The percentage of fatty matter in soap samples was determined by calculating the weight difference [35].

pH

A 10% soap solution was prepared by dissolving 1 g of soap in 10 ml of distilled water in a volumetric flask. The pH was determined using a pre-calibrated digital pH meter [33].

Foam height

The soap sample (0.5 g) was dispersed in 25 ml of distilled water. The solution was then transferred to a measuring cylinder and diluted with water to a final volume of 50 ml. A total of 25 strokes were administered until the aqueous volume reached 50 ml. The height of the foam above the aqueous volume was then measured [33].

Foam retention

A 25-ml sample of a 1% soap solution was transferred to a 100-ml graduated measuring cylinder. The cylinder was manually covered and shaken 10 times. The foam volume was measured at 1-minute intervals over 4 minutes [33].

Estimation of alcohol-insoluble content

A soap sample (5 g) was dissolved in 50 ml of heated alcohol. The solution was filtered using filter paper coated with tar and 20 ml of warm ethanol. The resulting mixture was then dried at 105°C for 1 hour. The mass of the dried filter paper was measured as follows:

Alcohol-insoluble matter (%)=wt. of residue×100/wt. of sample [36].

High temperature stability

The liquid soap was subjected to temperatures of 25, 37, 40, and 50°C for one month. The stability of liquid soap was assessed during the observation period. A homogeneous liquid sample was classified as stable, whereas a sample with roughened crystals and precipitation was considered unstable [37].

Percentage free alkali

Approximately 5 g of each sample was placed in a conical flask and mixed with 50 ml of neutralized alcohol. The mixture was refluxed for 30 minutes in a water bath and then cooled before the addition of 1 ml of phenolphthalein solution. The solution was promptly titrated using 0.1 N HCl [37].

Saponification value

The quantity of potassium hydroxide, measured in milligrams, required for the complete saponification of 1 g of fat or oil was determined. The term “mean molecular weight of fatty acid” refers to the average molecular weight of fatty acids found in oil or fat (Schumtterer et al., 1983). Briefly, approximately 2 g of soap sample was placed in a conical flask to determine its saponification value. Subsequently, a solution of 0.5 M KOH was added to the flask. The mixture was heated to approximately 55°C in a hot water bath with continuous stirring. Subsequently, the temperature was increased by an additional 100°C, and boiling was continued for approximately 1 hour. Titration was conducted using phenolphthalein as an indicator and 0.5 M HCl. The observed endpoint was the disappearance of the pink color [38]. The saponification value was calculated as follows:

Saponification value=(Avg volume of KOH 28.056)Weight of oil (g)

Antimicrobial assay using soap

Antimicrobial evaluation was conducted to determine the biological functions of the optimized formulations. The diffusion technique on agar wells was employed to determine the effectiveness of the soap against E. coli, S. aureus, and P. aeruginosa. The formulations were added to individual cups made with sterile nutrient agar; these cups were previously inoculated with the test organisms. After the solutions were allowed to diffuse for 2 hours, the agar plates were incubated at 37°C for 24 hours. The zone of inhibition (ZOI) was measured around each cup and subsequently compared [39].

Evaluations using the hand sanitizer gel

Physical analysis

The pH values of the gels were measured using a pre-calibrated pH meter (Mettler Toledo). Viscosity was measured using a Brookfield digital viscometer. The prescribed quantity of washing gel was added to the glass, and the viscometer tip was submerged in the gel to measure the viscosity [26].

Antibacterial activity based on the agar well diffusion assay

Five bacterial species, two gram-positive species (Staphylococcus aureus and Enterococcus faecalis) and three gram-negative species (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi), were tested to determine the antibacterial efficiency of the gel. The cultures were preserved on tryptone soya agar at 40°C. The inoculum was prepared according to the guidelines outlined in Clinical and Laboratory Standards Institute (CLSI) M02-A12 [39]. Briefly, isolated colonies of each bacterial culture were selected from the agar plates and incubated for 18–24 hours. The colonies were inoculated in tryptone soy broth to create a suspension. The turbidity of the suspension was adjusted to achieve a colony-forming unit (CFU) concentration of 1.0 to 2.0×108 CFU/ml, according to the CLSI guidelines, using a UV-visible spectrophotometer at a wavelength of 600 nm. Thereafter, 0.1 ml of each bacterial culture suspension was inoculated on Mueller–Hinton agar plates and evenly distributed using a sterile spreader. Sterile borers were used to cut 6 mm wells; 50 µl of formulated gel and commercial brand gels was added to the wells. The positive and negative controls included 70% ethanol and dimethyl sulfoxide (DMSO), respectively. The plates were allowed to settle for 5 minutes and then incubated at 37°C for 18–24 hours. Following incubation, the inhibition zones surrounding the wells owing to each sanitizing gel were measured using an automatic colony counter in inhibition zone mode [40].

Determination of the minimal inhibitory concentration

The minimal inhibitory concentration (MIC) was determined using the macrodilution method in sterile test tubes following the guidelines outlined in CLSI 07-08 [41]. The prepared gel was diluted in Mueller–Hinton broth using a 1:2 dilution at each step. Dilution resulted in a series of concentrations ranging from 100 to 0.195%. The test-strain inoculum was prepared in three steps. First, a cell suspension of each bacterial strain was prepared according to the agar well diffusion assay, with a concentration of 1.0–2.0×108 CFU/ml. Second, the suspension was diluted 1:150 to achieve a cell density of 1×106 CFU/ml. Third, the cell suspension was diluted 1:2 to a final inoculum concentration of 5×105 CFU/ml. The test was conducted by adding 1 ml of the prepared inoculum to each tube containing 1 ml of the hand sanitizer in a dilution series within 15 minutes of inoculum preparation. The mixture was homogenized and 1 ml was transferred from the first tube to the second tube, creating a dilution series with a dilution factor of 1:2. A positive control was established by adding the broth to the tube. This process was repeated until the final tube content was 0.195%. The MIC of all pigment concentrations in the hand sanitizer were determined using a similar method. The tubes were incubated at 37°C for 16 to 20 hours. The MIC of the hand sanitizer was the lowest concentration at which growth of the test organism was completely inhibited without the use of any aid [40].

Determination of the minimal bactericidal concentration

The minimal bactericidal concentration (MBC) was determined by inoculating 0.1 ml of sample from each tube of the MIC experiment on Mueller–Hinton agar plates using the spread plate technique after incubation. The plates were incubated at 37°C for 18–24 hours. After incubation, the MBC was determined as the lowest concentration of hand sanitizer gel that effectively eradicated the tested bacterial strains. The absence of colony formation on Mueller-Hinton agar plates indicated that the sanitizer effectively killed the bacterial cells, rendering them nonviable for growth on nutrient media in the absence of antibiotics. MBC were determined for all concentrations of pigments in the hand sanitizers against the five bacterial strains [40].

Hand sanitizer gel stability test

Stability studies were conducted by storing the samples at various temperatures (25, 37, and 40°C) for one month. The samples were monitored to identify alterations in color, odor, and phase separation [26].

Antioxidant activity assay

2,2-diphenyl-1-picrylhydrazl (DPPH) was used to assess the antioxidant activity of the extracts and products derived from cyanobacterial strains. This activity was determined by measuring the ability of antioxidant compounds to neutralize DPPH free radicals in a laboratory setting. The test sample was prepared by combining 1 ml of A. paniculata extract with 2 ml of a 0.1 mM DPPH methanolic solution. The mixture was then incubated in the dark for 30 min to allow the reaction to proceed. A small quantity of this solution was transferred to a cuvette. The absorbance of the reaction mixture was measured at 517 nm using a spectrophotometer. The control was prepared by combining 2 ml of a 0.1-mM DPPH methanol solution with 1 ml of methanol. The absorbance of each extract was measured in triplicate. The optical density was converted to the percentage of free radical-scavenging activity (% inhibition) using the following formula [42]:

DPPH %=A control-A sampleA control×100

Statistical analysis

The results of each experiment were analyzed using one-way analysis of variance (ANOVA) with the statistical software package, SPSS version 24 (IBM Co.). A significance level of 95% was considered to indicate statistically significance. The Tukey’s test was performed to assess the significance of differences in mean values following detection of a significant variation (p≤0.05) using ANOVA. Three replicate measurements were conducted for each treatment and the data are presented as mean values±standard error of the mean [43].

Determination of the concentration and purity of the coated pigments

Based on the results in Table 2, PE exhibited maximum adsorption at 562 nm, with a peak at 617 nm, while PC exhibited maximum absorption at 621.9 nm. The purities of PE and PC were 0.845 and 0.401, respectively (Fig. 3).

Table 2 . The results of the concentration and purity of purified phycoerythrin and phycocyanin pigments

StepsPeakPE (μg/ml)Purity of PE (OD555/OD280)
PhycoerythrinCrude extract562.8–617.70.1120.845
StepsPeakPC (μg/ml)Purity of PC (A620/A280)
PhycocyaninCrude extract619/80.05750.401

PE, phycoerythrin; PC, phycocyanin.


Fig. 3.The results of spectrometry of pigment extracted and purified from (A) Nostoc sp. and (B) Spirulina sp.

Test results of the anti-acne face wash gel containing the PE and PC pigments

Viscosity

One-way ANOVA and Tukey’s test revealed no significant differences among the different treatments over 30 days. However, PC alone and the combination of PC and PE led to the highest viscosity compared to the control.

pH

One-way ANOVA and Tukey’s test revealed no significant differences between the different pigments over 30 days. The highest pH was observed in the control group.

Physical properties of the anti-acne face wash gel

Gels prepared with PE alone, PC alone, both PE and PC, and the control were pale pink, blue, green, and colorless, respectively. The anti-acne face wash gels prepared with the formulas were completely shiny and transparent. Application of the gels to the skin led to a very light and cooling sensation (Fig. 2).

Foamability of the anti-acne face wash gel

One-way ANOVA and Tukey’s test revealed no significant differences among the gels treated with the different pigments over 30 days.

Stability of the anti-acne face wash gel at different temperatures

All treated gels stored at 4°C for 30 days were assigned a full score of 5 for color, smell, and consistency. Based on the consistency results, no significant differences were found among the various treatments and the control at 25 and 37°C. On day 30, a significant decrease was observed with the control at 40°C compared to the pigments.

Based on the color measurements of the anti-acne face wash gel at 25°C, gels treated with the pigments had more color than those treated with the control over 30 days. In addition, starting from day 20, gels treated with the pigments displayed significantly more color than those treated with the control at 37°C and 40°C.

Based on the smell of the anti-acne face wash gels stored at 25°C, 37°C, and 40°C, no significant differences were found among the various treatment groups and the control group over 30 days.

Homogeneity of the anti-acne gel

Visual inspection of the anti-acne gels confirmed the homogeneity of all gels on all measurement days.

Determining the activity of the anti-acne face wash against acne-causing microorganisms

One-way ANOVA and Tukey’s test revealed no significant difference in the antimicrobial activity of gels prepared with PE and the control until day 30. However, a significant difference in the antimicrobial activity of gels prepared with PC and PE-PC pigments was observed on days 15 and 5, respectively. In addition, gels containing both PE and PC and PC alone had the highest killing percentage (Table 3).

Table 3 . The results of one-way analysis of variance and Tukey’s test of percentage of anti-acne face wash activity of gels prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during thirty days

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control34.234±0.901 (aA)30.476±0.952 (aA)28.125±0.000 (aA)24.731±1.075 (aA)22.917±1.042 (aA)20.430±1.075 (aA)19.355±0.000 (aA)
PE39.640±0.901 (aB)37.143±0.000 (aB)35.417±1.042 (aB)33.333±1.075 (aB)32.292±1.042 (aB)32.258±0.000 (aB)31.183±1.075 (aB)
PC41.441±0.901 (aB)39.048±0.952 (abBC)37.500±0.000 (aB)36.559±1.075 (abBC)36.458±1.042 (abBC)36.559±1.075 (bC)35.484±0.000 (bC)
PE+PC42.342±0.901 (aB)40.952±0.952 (bC)38.542±1.042 (bB)39.785±1.075 (bC)38.542±1.042 (bC)36.559±1.075 (bC)36.559±1.075 (bC)

Values are presented as mean±standard deviation.

PE, phycoerythrin; PC, phycocyanin.

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns.



Antioxidant activity of the anti-acne face wash gels determined using the photobleaching (FRAP) method

One-way ANOVA and Tukey’s test revealed that the highest antioxidant activity was observed on day 10 for the gels prepared with both PC and PE (Table 4).

Table 4 . The results of one-way analysis of variance and Tukey’s test measuring the antioxidant activity of gels prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 25 days

Day 1Day 5Day 10Day 15Day 20Day 25
Control0.83±0.001 (aA)0.77±0.004 (aA)0.75±0.003 (aA)0.68±0.002 (aA)0.59±0.003 (aA)0.42±0.003 (aA)
PE0.96±0.002 (bB)0.83±0.093 (Aa)0.90±0.003 (bB)0.89±0.003 (bB)0.84±0.003 (bB)0.74±0.004 (bB)
PC0.95±0.001 (bC)0.89±0.007 (aA)0.85±0.004 (bC)0.80±0.004 (bC)0.79±0.007 (bC)0.63±0.013 (bC)
PE+PC1.01±0.003 (bD)0.95±0.003 (aA)0.95±0.006 (bD)0.93±0.003(bD)0.89±0.002 (bD)0.80±0.002 (bD)

Values are presented as mean±standard deviation.

PE, phycoerythrin; PC, phycocyanin.

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns.



Results of the soap tests

Moisture content

One-way ANOVA and Tukey’s test revealed that the moisture content decreased significantly over 30 days. In addition, soaps coated with PE alone and the combination of the two pigments had higher contents on days 25 and 30 than those coated with the other treatments.

Total fat content and pH

One-way ANOVA and Tukey’s test revealed that the amount of fat and pH decreased significantly over 30 days. In addition, the amount of fat did not significantly differ among soaps prepared with different types of pigments.

Formation and height of soap foam

One-way variance analysis and Tukey’s test revealed that the foam height decreased significantly over 30 days, with no significant difference found between days 25 and 30. In addition, no significant difference in foam height was found between the soaps prepared with different types of pigments on days 25 and 30.

Soap foam shelf life

One-way ANOVA and Tukey’s test revealed that foam height decreased significantly over 30 days. In addition, the amount of foam retention did not significantly differ among soaps coated with the different pigments and control at different times.

Insoluble alcohol measurement

One-way ANOVA and Tukey’s test revealed a significant increase in the amount of insoluble alcohol in the soaps coated with the pigments compared to that treated with the control; however, no significant differences were observed between the different treatments.

Stability

One-way ANOVA and Tukey’s test revealed that the degree of color stability was not significantly different over the 30 days. In addition, no significant difference in color stability was observed between soaps coated with the different pigments and the control.

When soap stability was tested at 25°C and 30 days, no color changes were observed in stable soaps, and no phase separation occurred. A homogeneous sample was reported as a stable sample while a sediment-like sample was reported as an unstable sample. During the 30 days, all soaps received a score of 5 for stability.

Percentage of soap alkali

One-way ANOVA and Tukey’s test revealed no significant difference in the percentage of alkalinity between soaps coated with the different pigments and those treated with the control.

Determination of saponification rate

One-way ANOVA and Tukey’s test revealed that the amount of saponification in soaps coated with the different pigments was significantly different from that of soaps treated with the control, with significant decreases recorded.

Antibacterial properties of soap

One-way ANOVA and Tukey’s test results regarding the antibacterial properties of soaps treated with the control, PC, PE, and both PC and PE for 30 days are presented in Table 5. No significant differences in the antibacterial properties of soaps against E. coli were found over the 30 days, highlighting the stability of their antibacterial properties. On all days, soaps prepared with PC and the combination of both pigments had significant increases in their antibacterial activity against E. coli compared to those coated with the other treatments (Table 5). In terms of S. aureus, the antibacterial properties were not significantly different over the 30 days between the control soaps and soaps coated with PE. The highest antibacterial effects were obtained with soaps treated with the combination of the two pigments until day 20; however, no significant difference was observed compared with the control. Notably, the antibacterial effects against S. aureus decreased significantly from day 20 onwards (Table 5). In terms of P. aeruginosa, the antibacterial effects were not significantly different between the control soap and soaps containing PE and PC over the 30-day period. Initially, soaps containing both pigments had the highest antibacterial effects until day 10; however, these effects decreased significantly thereafter (Table 5).

Table 5 . The results of one-way analysis of variance and Tukey’s test to determine the antibacterial properties against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa bacteria of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
(a) Escherichia coli
Control7.33±0.333 (aA)7.33±0.333 (aA)7.00±0.000 (aA)7.00±0.000 (aA)6.67±0.333 (aA)6.67±0.333 (aA)6.33±0.333 (aA)
PE11.00±0.000 (bB)11.00±0.000 (bB)10.67±0.333 (bB)10.33±0.333 (bB)10.00±0.000 (bB)9.67±0.333 (bB)9.33±0.333 (bB)
PC12.67±0.333 (cC)12.33±0.333 (cC)12.33±0.333 (cC)12.33±0.333 (cC)12.00±0.000 (cC)11.67±0.333 (cC)11.33±0.333 (cC)
PE+PC13.33±0.333 (cC)13.33±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (cC)12.00±0.000 (cC)11.67±0.333 (cC)
(b) Staphylococcus aureus
Control8.00±0.000 (aA)8.00±0.000 (aA)7.67±0.333 (aA)7.67±0.333 (aA)7.33±0.333 (aA)7.00±0.000 (aA)6.67±0.333 (aA)
PE12.33±0.333 (aB)12.33±0.333 (aB)12.33±0.333 (aB)12.00±0.000 (aB)12.00±0.000 (aB)11.67±0.333 (aB)11.67±0.333 (aB)
PC13.00±0.000 (bcBC)13.00±0.000 (bcBC)12.67±0.333 (bB)12.67±0.333 (bcBC)12.33±0.33 (bcBC)12.00±0.000 (bB)12.00±0.000 (bB)
PE+PC13.67±0.333 (cC)13.67±0.333 (cC)13.67±0.333 (cB)13.33±0.333 (cC)13.33±0.333 (cC)12.67±0.333 (bB)12.67±0.333 (bB)
(c) Pseudomonas aeruginosa
Control7.67±0.333 (aA)7.67±0.333 (aA)8.00±0.000 (aA)7.67±0.333 (aA)7.33±0.333 (aA)7.33±0.333 (aA)6.67±0.333 (aA)
PE11.67±0.333 (bB)11.67±0.333 (bB)11.33±0.333 (bB)11.00±0.000 (bB)11.00±0.000 (bB)11.00±0.000 (bB)10.67±0.333 (Bb)
PC12.67±0.333 (bcBC)12.67±0.333 (bcBC)12.67±0.333 (bB)12.33±0.333 (cC)12.00±0.000 (bcBC)12.00±0 (bcBC)12.00±0.000 (cC)
PE+PC13.33±0.333 (bC)13.33±0.333 (bC)13.00±0.577 (bB)12.67±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (Cc)12.33±0.333 (cC)

Values are presented as mean±standard deviation.

PE, phycoerythrin; PC, phycocyanin.

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns.



Antioxidant activity of soap

One-way ANOVA and Tukey’s test revealed that the antioxidant activity significantly decreased over 30 days. However, the antioxidant activity of soaps coated with the different pigments was significantly increased compared to that of control soaps. In addition, no significant differences were found in the antioxidant properties of the treated soaps (Table 6).

Table 6 . The results of one-way variance analysis and Tukey’s test of antioxidant activity of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control2.18±0.01 (Aa)5.76±0.45 (Ab)6.39±0.21 (Ab)9.63±0.09 (Ac)11.37±0.14 (Ad)15.83±0.23 (Ae)18.48±0.56 (Af)
PE0.47±0.04 (Ba)0.68±0.01 (Bb)0.90±0.03 (Bc)1.36±0.03 (Bd)1.56±0.01 (Be)1.85±0.02 (Bf)2.43±0.02 (Bg)
PC0.45±0.04 (Ba)0.64±0.01 (Bab)0.80±0.04 (Bb)1.09±0.09 (Bc)1.32±0.04 (Bcd)1.51±0.01 (Bd)1.87±0.08 (Be)
PE+PC0.46±0.04 (Ba)0.67±0.01 (Bb)0.86±0.03 (Bc)1.25±0.01 (Bd)1.45±0.02 (Be)1.64±0.04 (Bf)2.15±0.07 (Bg)

Values are presented as mean±standard deviation.

PE, phycoerythrin; PC, phycocyanin.

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns.



Psychrophilic bacteria

One-way ANOVA and Tukey’s test revealed that cold-loving bacteria appeared in the control soap and soaps containing PC or PE from days 20, 25, and 30; however, these bacteria were not found in soaps containing both PC and PE. In addition, the number of cold-loving bacteria in the control soap was significantly higher than those in the pigment-coated soaps.

Colorimetry of soaps

One-way ANOVA and Tukey’s test revealed that soaps prepared with the PE pigment and both pigments had a significant level of brightness compared to those coated with the other treatments. The a*value did not significantly differ between the coated and control soaps during the evaluation period. However, soaps treated with PE had the highest a*values on all days. The b*value did not significantly differ between the coated and control soaps during the evaluation period. The lowest b*value was found for soaps containing PE, with a significant difference observed on day 30. Moreover, the amount of ΔE did not significantly differ between the coated and control soaps during the evaluation period. The ΔE value was lowest in soaps containing PC. Soaps coated with both pigments exhibited significantly differences in their ΔE value on day 30 (Table 7).

Table 7 . The results of one-way variance analysis and Tukey’s test to determine the ΔE value of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days

Control<br>day 5 to day 1Control<br>day 10 to day 1Control<br>day 15 to day 1Control<br>day 20 to day 1Control<br>day 25 to day 1Control<br>day 30 to day 1
Control1.51±0.082 (bA)1.89±0.269 (Aa)2.81±0.313 (bB)3.59±0.143 (bA)4.42±0.283 (cA)5.32±0.101 (bA)
PE1.49±0.062 (abAB)2.07±0.160 (aA)2.89±0.025 (bB)3.19±0.194 (ABab)3.68±0.140 (bcAB)4.67±0.274 (bA)
PC1.17±0.065 (abAB)1.35±0.187 (Aa)2.14±0.027 (ABab)2.90±0.176 (ABab)3.45±0.069 (abBC)2.84±0.174 (aB)
PE+PC1.11±0.128 (aB)1.28±0.264 (Aa)1.67±0.261 (Aa)2.50±0.209 (Ba)2.77±0.076 (aC)3.37±0.371 (aB)

Values are presented as mean±standard deviation.

PE, phycoerythrin; PC, phycocyanin.

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns.



Results of hand sanitizer gel tests

pH

One-way ANOVA and Tukey’s test revealed that the pH of the control gels decreased significantly. On day 30, the lowest pH was observed in gels coated with PC.

Viscosity

One-way ANOVA and Tukey’s test revealed that the viscosity decreased significantly over 30 days. On day 30, the highest viscosity was observed for gels coated with PE alone and both pigments, while the lowest amount was observed for control gels.

Antimicrobial activity of the hand sanitizer gel

One-way ANOVA and Tukey’s test revealed that gels coated with both pigments had the highest inhibition rate against S. aureus. Based on the inhibition diameters against E. faecalis and E. coli, no significant difference was found between the gels coated with the different pigments. However, gels coated with PE alone and both pigments exhibited the highest inhibitory activity against P. aeruginosa and S. typhi (Table 8).

Table 8 . The results of one-way analysis of variance and Tukey’s test of growth inhibition diameter of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments

Staphylococcus aureusEnterococcus faecalisEscherichia coliPseudomonas aeruginosaSalmonella typhi
Control7.00±0.00 (A)6.33±0.33 (A)6.00±0.00 (A)5.33±0.33 (A)5.00±0.58 (A)
PE9.67±0.33 (B)8.67±0.33 (B)7.67±0.33 (B)6.67±0.33 (B)6.33±0.33 (B)
PC8.67±0.33 (C)8.33±0.33 (B)7.33±0.33 (B)5.67±0.33 (A)5.33±0.33 (A)
PE+PC10.67±0.33 (D)8.67±0.33 (B)8.00±0.58 (B)7.33±0.33 (B)7.00±0.58 (B)

Values are presented as mean±standard deviation.

PE, phycoerythrin; PC, phycocyanin.

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns.



MIC of the hand sanitizer gel

One-way ANOVA and Tukey’s test revealed that gels coated with both pigments and PE alone had the lowest MIC against S. aureus, whereas gels coated with both pigments had the lowest MIC against E. faecalis, E. coli, P. aeruginosa, and S. typhi.

MBC of the hand sanitizer gel

One-way variance analysis and Tukey’s test revealed that gels coated with both pigments, PE alone, and PC alone had the lowest MBCs against S. aureus, P. aeruginosa, E. faecalis, and S. typhi, whereas gels coated with the PC and phycothyrin pigments had the lowest MBC against E. coli.

Hand sanitizer gel stability test

One-way ANOVA and Tukey’s test were performed to determine the stability of smell, color, and phase separation of gels treated with PE, PC, both PE and PC, and control. At 4, 25 (until day 15), and 40°C, the best score of 5 was assigned for smell, color, and phase separation. The stability of the smell and color decreased significantly over the 30 days. However, no significant difference was found between the gels prepared with the control and those coated with the pigments. Significant difference in phase separation was observed at 25, 37, and 40°C on days 25 and 30. However, no significant differences were observed between the control gels and pigment-coated gels. The phase separation and color on days 10 and 15 at 40°C and days 15 and 20 at 25°C also received a score of 5.

Antioxidant activity of hand sanitizer gel determined using the DPPH method

One-way ANOVA and Tukey’s test revealed that antioxidant activity decreased over 30 days. On the last day, gels coated with PE alone and both pigments had the highest antioxidant activity (Table 9).

Table 9 . The results of one-way variance analysis and Tukey’s test to determine the antioxidant activity of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments during 30 days

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control4.39±0.10 (Aa)5.21±0.10 (Aab)5.83±0.13 (Ab)7.38±0.16 (Ac)8.56±0.18 (Ad)11.57±0.33 (Ae)13.34±0.25 (Af)
PE2.22±0.01 (Ba)2.42±0.05 (Bb)2.45±0.01 (Bbc)2.47±0.01 (Bbc)2.50±0.02 (Bcb)2.53±0.02 (Bcb)2.56±0.02 (Bc)
PC3.45±0.08 (Ca)3.83±0.06 (Cab)4.23±0.09 (Cbc)4.36±0.05 (Ccd)4.75±0.09 (Cde)4.85±0.12 (Ce)5.18±0.14 (Ce)
PE+ PC2.39±0.04 (Ba)2.44±0.02 (Bab)2.47±0.0 2(Bab)2.48±0.02 (Bbac)2.55±0.01 (Bcb)2.59±0.04 (Bcb)2.63±0.05 (cB)

Values are presented as mean±standard deviation.

PE, phycoerythrin; PC, phycocyanin.

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns.



Foaming ability of the hand sanitizer gel

One-way ANOVA and Tukey’s test revealed no significant differences in foaming ability between the control and coated gels.

Lethality percentage of the hand sanitizer gel

One-way ANOVA and Tukey’s test revealed that the lethality rate decreased significantly over 30 days. The most significant difference was observed in gels coated with both pigments (Table 10).

Table 10 . The results of one-way variance analysis and Tukey’s test to determine the lethality percentage of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments during 30 days

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control73.66±0.41 (aA)71.89±0.40 (Aab)70.09±0.43 (Abc)68.40±0.43 (Acd)66.23±0.44 (Ad)63.60±0.44 (Ae)62.34±0.75 (Ae)
PE76.95±0.41 (BCa)75.10±0.40 (Bab)75.21±0.43 (Bab)73.16±0.43 (Bbc)71.93±0.44 (Bc)71.49±0.44 (BCcd)69.70±0.43 (BCd)
PC76.54±0.00 (Ba)75.10±0.40 (Bab)74.36±0.74 (Bab)73.16±0.43 (Bbc)71.49±0.44 (Bcd)70.61±0.44 (Bde)68.83±0.00 (Be)
PE+ PC78.19±0.41 (ACa)75.90±0.70 (Bb)75.64±0.00 (Bb)74.46±0.43 (Bbc)73.25±0.44 (Bcd)72.81±0.44 (ACcd)71.86±0.43 (ACd)

Values are presented as mean±standard deviation.

PE, phycoerythrin; PC, phycocyanin.

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns.


Natural ingredients have historically been used to synthesize cosmetic products, thereby predating the formalization of cosmetics [44]. The emergence of cosmeceuticals has further highlighted the potential of cyanobacteria as valuable sources of components owing to their ability to produce a wide range of compounds. Cyanobacteria produce various chemicals, such as fatty acids, polyphenols, peptides, polysaccharides, and pigments, which possess distinct potential for health-related applications [45]. Cyanobacteria contain three types of pigments: chlorophyll; carotenoids; and PBPs. These pigments exhibit a range of colors from blue to red and possess diverse physicochemical properties. Notably, pigments have potential applications in food, feed, nutraceuticals, and cosmetics [46]. Cyanobacterial pigments, such as carotenoids and PBPs, have bioactive properties, a range of colors, and cosmetic enhancement properties, such as the ability to moisturize and stabilize the skin. Owing to these attributes, cyanobacterial pigments are appealing in the natural cosmetic industry, which commonly uses the extract of these compounds [6,47]. Carotenoids are present in sunscreens, antiaging products, and antioxidant formulations because of their exceptional antioxidant properties. In cyanobacteria, these compounds are responsible for capturing excess energy from photosynthetic metabolism and protecting against harmful effects and cellular damage [48]. Prolonged exposure to sunlight (UV radiation and intense light) can cause cellular damage to human skin, enabling the potential extraction and use of these compounds for similar purposes [49]. PBPs have been linked to many biological activities, including anticancer, antiviral, antimicrobial, and antioxidant [50]. Therefore, PBPs can be used as functional additives. These compounds can serve as natural colorants, reducing the potential for skin toxicity, damage, and allergic reactions to synthetic dyes. Owing to their water solubility, hydrophilic pigments are suitable for use in skincare products, specifically in sera and lotions [46]. This study aimed to determine the effect of PC and PE coatings on the antioxidant and antimicrobial activities of different cosmetic products, including soap, anti-acne face wash, and hand sanitizer gel.

Based on the tests using the anti-acne face wash gel containing PE and PC, the amount of pigments used to determine viscosity (PX and PC-PE), pH (control treatment), antioxidant (PC-PE), and antimicrobial activity against acne-causing microorganisms (PC-PE) increased, with differences found among gels coated with the various pigments. In contrast, the pH and viscosity of the hand sanitizer gel decreased, and the lowest values were observed in the PC and control groups, respectively. The lowest antioxidant results were obtained for the hand sanitizer gel containing PE alone and both pigments compared to gels containing the other cosmetic products.

Data related to gel homogeneity revealed that the gel was homogenous on all days. The anti-acne face wash gels were completely shiny and transparent. Application of the gel to the skin led to a very light and cooling sensation. The highest lethality, based on the anti-acne face wash gel test, was observed for gels prepared using both PC and PE and PC alone. However, hand sanitizer gels coated with both pigments had the best killing rate.

Optimal cultivation conditions exist to maximize the production rate of secondary metabolites in extremophilic cyanobacteria. Zucchi and Necchi [51] examined the growth and pigment content of freshwater algal cultures. Their findings indicate that temperature primarily influenced pigment content, whereas variations in irradiance and photoperiod, alone and combined, accounted for a smaller portion of the observed differences. The responses varied among species, with PC being more concentrated than PE and PBPs being more concentrated than chlorophyll a. However, Rizzo et al. [52] did not observe any variation in total protein and penicillin-binding protein (PBP) production under different light conditions. Ma et al. [53] found a peak in PBP contents at light intensities below 90 μmol·m-2 s-1. Conversely, PC and APC levels increased with increasing light intensity, whereas PE levels decreased. The investigators also found that blue and red light led to the greatest increases in fresh weight, protein content, and PBP content, leading to higher dry matter, PC, and chlorophyll a levels.

PE has diverse applications in the pharmaceutical, antioxidant, and food industries [54,55]. However, the limited stability of PE poses a significant challenge to their widespread use. The sensitivity of PE to pH, salts, temperature, water, and light, and to in vitro processes, such as extraction, purification, storage, and utilization, hinders its practical application. The size and protein composition of the PE complex vary in response to changes in light and nutritional conditions. Low-intensity light promotes the synthesis of PE, leading to the elongation of rod structures. Various purification and characterization methods are available for the PEs derived from different strains of cyanobacteria and red algae [14].

Based on the stability analysis of the color, smell, and consistency of the anti-acne face wash and hand sanitizer gel, both products were assigned a full score of 5 owing to treatment with the pigments. The color measurements of the anti-acne face wash gel at 25, 37 and 40°C (from day 20) were the highest with the control. However, the smell of anti-acne face wash gels at 25, 37, and 40°C did not significantly differ among the various treatment and control groups over 30 days. The smell, color, and phase separation of the hand sanitizer gel received full scores of 5 at 4, 25 (until day 15), and 40°C. Similar to the two other cosmetic products (i.e., anti-acne face wash and hand sanitizer gel), soap also received a full score of 5. In addition, the foamability of the anti-acne face wash and hand sanitizer gels did not change significantly. The total fat content, pH, foam formation and height, shelf life, percentage of alkali, and antioxidant activity of soap revealed no significant differences, despite decrease over time. Although insoluble alcohol levels increased over 30 days, no significant differences were found.

Cyanobacteria possess inherent protective mechanisms against dehydration, which makes them potentially valuable moisturizing agents for cosmetics. Several studies have investigated the moisture uptake properties of cyanobacteria [56,57]. For example, applying microalgal can improve skin moisture and elasticity [4]. Additional studies on Nostoc commune revealed that cells containing extracellular polymeric substances (EPSs) exhibit significantly greater desiccation tolerance. This finding highlights the remarkable moisture absorption and water retention properties of these substances compared with those of urea and chitosan [58,59]. EPSs are large molecules with high molecular mass. These substances consist of hydrated sulfate groups, neutral sugars (such as glucose, galactose, mannose, fructose, ribose, xylose, arabinose, fucose, and rhamnose), non-carbohydrate components (such as phosphate, lactate, acetate, and glycerol), and various uronic acids (such as glucuronic and galacturonic acid). Polysaccharides are recognized for their notable ability to retain moisture due to strong interactions between water molecules and hydrophilic -OH groups. Environmental conditions influence the composition of EPSs in different species [60]. The cellular envelope of Chroococcidiopsis exhibits variations in composition in response to varying levels of water stress. This cyanobacterium contains various organic metabolites, including sporopollenin-like compounds, proteins, beta-linked polysaccharides, acid sulfate, and lipids. These metabolites are associated with reduced and regulated rates of water loss [61].

In this study, both the moisture content and saponification rate decreased. The highest reduction in moisture occurred on days 25 and 30 for soaps coated with PE alone and both PC and PE.

In terms of psychrophilic bacteria, the control group had the highest number of cold-loving bacteria.

Based on the colorimetry results, the values of L* (PE and PC-PE) and a (PE alone) indicated that soaps had the highest level of brightness. However, soap had the lowest b (PE) and ΔE (PC and PC-PE) values.

In terms of antibacterial properties, the level of E. coli (over 30 days), S. aureus (over 30 days in control soaps and PE-coated soap), and P. aeruginosa (control soaps and soaps coated with PE and PC over a 30-day period) did not significant differ among soaps. The highest inhibition rate against S. aureus was obtained with the hand sanitizer gel coated with both PC and PE; however, no significant difference was found between the inhibition diameters of E. faecalis and E. coli for gels coated with the pigments. Notably, gels coated with PE alone and both PC and PE caused the highest inhibition of P. aeruginosa and S. typhi.

The lowest MIC was obtained with the hand sanitizer gel coated with PE alone and both PC and PE for S. aureus and both PC and PE for other bacteria (E. faecalis, E. coli, P. aeruginosa, and S. typhi). The lowest MBC against S. aureus, P. aeruginosa, E. faecalis, and S. typhi, was obtained with gels coated with both pigments, PE alone, and PC alone; however, lowest MBC against E. coli was obtained with gels coated with the PC and phycothyrin pigments.

The growing emphasis on skin health, particularly in relation to aesthetics and aging, has led to a greater desire for new cosmetic products. These products are primarily derived from natural sources, have few adverse effects, and are environmentally sustainable. Cyanobacteria possess effective mechanisms to combat dissection, radiation, and oxidative stress through the production of specific compounds. Owing to this characteristic, cyanobacteria serve as a promising group of organisms for applications in the cosmetic/cosmeceutical industry. Cyanobacteria can be utilized in biotechnology to produce cost-effective cosmetic formulations coated with natural pigments, which enhances production efficiency. This study highlights the use of cyanobacterial pigments in cosmetics and the biotechnological potential of three cosmetic products: soap, anti-acne face wash, and hand sanitizer gels. These products are economically viable and sustainable options derived from the cosmetic industry.

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Article

Original Article

J Cosmet Med 2024; 8(1): 18-33

Published online June 30, 2024 https://doi.org/10.25056/JCM.2024.8.1.18

Copyright © Korean Society of Korean Cosmetic Surgery & Medicine.

Impact of coated phycocyanin and phycoerythrin on antioxidant and antimicrobial activity of soap, anti-acne face wash and hand sanitizer gel

Bahareh Nowruzi, Hossein Hashemi

Department of Biotechnology, Science and Research Branch, Islamic Azad University, Tehran, Iran

Correspondence to:Bahareh Nowruzi
E-mail: bahareh.nowruzi@srbiau.ac.ir

Received: January 11, 2024; Revised: March 25, 2024; Accepted: March 25, 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background: The use of natural ingredients in skincare products has become a topic of interest in contemporary society. The adverse effects and environmental risks of synthetic compounds have prompted studies on the use of photosynthetic organisms as sustainable and ecofriendly sources of effective ingredients. Natural extracts have gained attention in the cosmetic industry, especially those derived from plants, algae, and cyanobacteria. Cyanobacteria have gained prominence in the cosmetic industry because of their low culture requirements, rapid growth rates, and capacity to produce different bioactive metabolites. As a result, cyanobacteria are economically viable and sustainable resources.
Objective: We aimed to determine the effect of coating with natural phycocyanin and phycoerythrin pigments from two cyanobacterial species, Spirulina platensis and Nostoc sp., on the properties of three cosmetic products: soap, anti-acne face wash, and hand sanitizer gel.
Methods: After cyanobacterial culture, the pigments were extracted, purified, and coated with the stabilizer, chitosan. Thereafter, cosmetic products were formulated with phycocyanin and phycoerythrin, and their viscosity, pH, stability, and antioxidant and antibacterial properties against bacteria were evaluated using the appropriate assays.
Results: The anti-acne wash gel and soap had better properties when coated with both pigments and phycocyanin alone, whereas the hand sanitizer gel had better properties when coated with phycoerythrin alone and both pigments.
Conclusion: Overall, three coated and stable cosmetic products can be produced using cyanobacterial pigments owing to their high fortifying capacity.

Keywords: nostoc sp., phycocyanin, phycoerythrin, skin care products, spirulina sp.

Introduction

Approximately 2,000 years ago, cyanobacteria were used by Chinese as a means of survival during periods of malnutrition, with Nostoc as the specific species employed. Cyanobacterial biotechnology emerged in the 1950s and has since been utilized for various commercial purposes, such as improving the dietary value of food and animal feed, owing to its chemical makeup. Cyanobacteria are essential in aquaculture, and their byproducts have applications in the cosmetic industry [1,2]. Microalgal biomass is commonly used to produce valuable products. Microalgae-derived products are commonly used as high-protein dietary supplements for human nutrition, aquaculture, and nutraceuticals [3].

Cosmetics play a significant role in the daily routines of many individuals. The demand for skin care products has significantly increased owing to growing concerns regarding skin health and is associated with their aesthetically pleasing qualities. Ethical concerns regarding the use of animal-derived products and the potential harmful effects of artificial substances on humans, such as allergies and environmental risks, have led to increased studies on cosmetic ingredients derived from photosynthetic organisms [4].

One of the main natural sources of cosmetically beneficial metabolites is microalgae, which can expedite skin healing and repair, and exhibit anti-blemish and anti-inflammatory effects [5-7]. Nostoc, Spirulina (Arthrospira), and Aphanizomenon are the most studied cyanobacterial species owing to their high contents of calcium, beta-carotene, phosphorous, iron, biotin, folic acid, pantothenic acid, and vitamin B12 [8]. As a result, the extracts and bioactive compounds of these species are being studied to determine their potential for use in cosmetics to protect the skin and hair [9]. Beta-carotene from Desmonostoc muscorum, Leptolyngbya foveolarum, and Arthrospira platensis can regulate ultraviolet (UV)-A-induced gene expression in human keratinocytes [10] and modulate biological targets, such as NF-κB, COX-2, and matrix metalloproteinase-9, due to its anti-inflammatory activity [5]. Cyanobacterial phycobiliproteins (PBPs), such as phycocyanin (PC), have antioxidant, anti-inflammatory, and antiaging properties, and are used as cosmetic ingredients [7,11].

Cyanobacterial PBPs have potential commercial applications as natural constituents in cosmetics. PBPs are hydrophilic proteins that bind to phycobilins and photosynthetic pigments, and are primarily found in cyanobacteria and certain red algae. Metabolites with structural similarities to bilirubin have been previously found to possess effective quenching properties against various oxygen derivatives [12]. As a result, these metabolites, known as PBPs, are suggested to serve as promising antioxidant agents. Fluorescent protein-pigment complexes comprise three components: phycoerythrin (PE; red pigment), allophycocyanin (APC; bluish-green pigment), and PC (blue pigment) [13].

PC exhibits potent antioxidant and radical-scavenging effects, leading to the inhibition of cell proliferation and induction of apoptosis of cancer cells. Spirulina contains various phytopigments, including PC, gamma-linolenic acid, phycocyanobilin, and phycoerythrobilin [11]. These compounds have been recognized for their antioxidant, anti-melanogenic, antiwrinkle, and antiaging properties. These compounds are used as natural colorants in cosmetics, such as lipsticks, eyeliners, and eye shadows. Spirulina is commonly employed because of its anti-inflammatory, neuroprotective, and hepatoprotective properties [4,14]. PC reduces the levels of alanine amino transferase, aspartate amino transferase, and malondialdehyde in serum [15]. Certain cyanobacterial extracts contain peptides and proteins that have extensive applications in hair care products, such as lotions, shampoos, solutions for permanent hair waves, and hair coloring products [16]. The use of extracts from Chlorogloeopsis sp. and Spirulina in hair care products was found to lead to positive outcomes, such as increased gloss, improved ease of combing, and enhanced hair restoration and moisturization [16]. The Blue Green Algae Hair Rescue Conditioning Mask (Aubrey Organics Company, Ltd.) was found to effectively strengthen hair and prevent breakage and splitting [6].

Cyanobacteria active extracts, such as Spirulin extract Spiralin, have been developed and are currently being used in the cosmetic industry to protect the skin. These extracts, which are found in products, such as Skinicer Repair Cream and Spirularin, have regenerative effects on damaged skin cells and collagen, and protect against UV radiation. Phycobiliprotein C-phycocyanin (C-PC), which is derived from Aphanizomenon flos-aquae, has been used as a natural colorant alternative to synthetic pigments. This substitution is because of the appealing pink-purple hue that C-PC imparts to the end products [17]. As studies on the use of other cyanobacterial genera for cosmetic purposes are limited, further investigations should be performed in this area. This study sought to determine the effects of chitosan-coated PC and PE on the antioxidant and antimicrobial properties of soap, anti-acne face wash, and hand sanitizer gel.

Materials and methods

Culture conditions for Nostoc sp. and Spirulina sp.

The cyanobacterial strains, Spirulina platensis and Nostoc sp., were isolated from Cyanobacteria culture collection (CCC) of herbarium ALBORZ at the Science and Research Branch, Islamic Azad University, Tehran. The strains were grown in modified Zarook and BG110, respectively, and illuminated (300 m-2 s-1) growth rooms at 28°C±2°C for 30 days [18,19].

Extraction and purification of analytical-grade PC and PE

PC and PE were extracted and purified as described by Nowruzi et al. [20]. Pigments were extracted from 500 ml of homogenized log-phase (14-day-old) culture following centrifugation at 4,000 rpm to obtain the pellet. The pellet was resuspended in 100 ml of 20 mM acetate buffer (pH 5.1) for PC and potassium phosphate buffer (pH 7.1) for PE. The extraction was conducted via repeated freezing (-20°C) and thawing (room temperature) for 4 days to obtain a dark purple cell biomass. Cell debris was removed via centrifugation at 5,000 rpm for 10 minutes to obtain the crude extract. Purification was performed as described by Afreen and Fatma [21]. Solid ammonium sulfate was slowly added to the crude extract under continuous stirring to achieve 65% saturation. The resulting solution was allowed to stand for 12 hours in a cold room and centrifuged at 4,500 × g for 10 minutes. The pellets were resuspended in a small volume of 50 mM acetic acid-sodium acetate buffer (pH 7.1) and dialyzed overnight. The extracts were recovered from the dialysis membrane and filtered through a 0.45 μm filter.

The absorption spectrum was determined by scanning the sample in the range of 300–750 nm using a Specord 200 spectrophotometer (Analytik Jena). The amounts of PC and PE were calculated based on their optical density (OD) values at 620 and 650 nm (for PC) and 565 nm (for PE) using the equations below. The purity of the pigments was determined at each step as the purity ratio (A620/A652) for PC and (A555/A280) for PE (Fig. 1) [22].

Figure 1. Different stages of separation and purification of phycocyanin (A to D) and phycoerythrin (E to H) pigments. (A) Primary culture of Spirulina cyanobacteria. (B) Preparation of crude extract. (C) Dialysis. (D) Freeze drying and preparation of phycocyanin powder. (E) Primary culture of Nostoc cyanobacteria. (F) Preparation of crude extract. (G) Dialysis. (H) Freeze-drying and preparation of phycoerythrin powder.
PC(μgml1)=(OD 620 nm-0.7OD 650 nm)7.38 APC(μgml1)=(OD 650n m-0.19OD 620 nm)5.65 PE(μgml1)=(OD 565 nm-2.8 [PC]-1.34 [APC])12.7

Stabilization and coating of pigments with chitosan

PC and PE were encapsulated by combining the pigments with a water-soluble chitosan (WSC). Sodium tripolyphosphate was used as a cross-linking agent. A WSC solution was prepared by soaking 1 mg/ml of oligochitosan in distilled water. The resulting mixture was stored at 4°C for 24 hours to ensure complete hydration. Thereafter, 1 ml of PC and PE in deionized water was gradually added to 1 ml of WSC (1 mg/ml) at 25°C with agitation. Sodium tripolyphosphate (2 mg/ml) was added to 0.5-ml aliquots, and the pH was adjusted to 7 using 1% HCl. Polyethylene glycol (0.5 ml) was then added to the mixture [23].

Formulation of soap, anti-acne face wash, and hand sanitizer gel

A series of experimental trials on compositions were used to optimize the laboratory formulation of soap, anti-acne face wash, and hand sanitizer gel. The composition of the final product was optimized using the ingredients listed in Table 1. Four conditions were established to generate the three products: condition one without any pigment, condition two with the PC pigment (1.5 g), condition three with the PE pigment (1.5 g), and condition four with both the PC and PE pigments (1.5 g) (Fig. 2) [24-26].

Table 1 . Composition of anti-acne washing gel, soap and antibacterial hand sanitizer.

ProductsIngredientsQuantity
Anti-acne washing gelCarbapol0.1 gr
Distilled water2.0 ml
Methylparaben0.1 mg
Propylenglycol0.1 mg
Tea0.1 gr
Phycocyanin and phycoerythrin1.5 gr
SoapWater2.0 ml
Ethanol5 ml
Cinnamon oil1 ml
Citronella oil1 ml
Melted glycerine soap9.0%
Stearic acid0.033 g
Phycocyanin and phycoerythrin1.5 gr
Antibacterial hand sanitizerCarbopol 9401 gr
EDTA0.1 gr
Distilled water2.0 ml
Glycerine5 gr
Perfume0.3%
Phycocyanin and phycoerythrin1.5 gr

Figure 2. Preparation of (A) anti-acne gel; (a) without pigment, (b) with phycoerythrin pigment, (c) total of phycoerythrin and phycocyanin pigments, (d) with phycocyanin pigment. (B) Hand gel; (a) total of phycoerythrin and phycocyanin pigments, (b) with phycoerythrin pigment, (c) with phycocyanin pigment (d) without pigment. (C) Soap; (a) without pigment, (b) with phycocyanin pigment, (c) the sum of phycoerythrin and phycocyanin pigments, (d) with phycocyanin pigment.

Evaluations using the anti-acne face wash

Viscosity

The viscosity of the anti-acne face wash was measured using a digital Brookfield viscometer with a yarn number of 64, operating at 10 revolutions per min and a temperature of 25°C. After a specific volume of hand wash was placed in a beaker, the viscometer tip was submerged in the hand wash gel to measure its viscosity. The tests were conducted in triplicate [27].

pH

The pH of a 1% aqueous solution of the formulation was determined at a constant temperature of 25°C using a calibrated digital pH meter [28].

Physical evaluation

Visual inspection was conducted to assess the physical attributes, including color, appearance, and consistency, of the formulation [29].

Gel stability

The durability of the gels was assessed using freeze-thaw cycles. The gels were stored at 4, 25, 37, and 40°C for 7 days [30].

Gel homogeneity

Homogeneity was determined via visual inspection after placing the samples in a specific container. The appearance, mass, and density of the gels were then evaluated [31].

Antimicrobial assay using the anti-acne face wash

Turbidimetry was used to screen for antimicrobial activity. The sterile nutrient agar medium was prepared aseptically and spread onto a Petri plate. The faces of volunteers with noticeable acne were cleaned with distilled water and allowed to air-dry. A cotton swab was used to apply distilled water to the ruptured pimple to ensure coverage of the entire acne surface. The solution was then uniformly applied to the prepared medium. The microbial cultures were incubated at 37°C for 24 hours to achieve optimal growth. Six sterile cotton balls, each 1 cm in diameter, were immersed in the prepared formulations of the standard drug and distilled water for 5 minutes. A 50-ml nutrient broth was prepared and sterilized. Five milliliters of the broth was collected and used as the reference standard in one cell of the UV spectrophotometer. The remaining broth was inoculated with the organism and cultured on a Petri plate. Five milliliters of the inoculated broth was distributed into six sterile test tubes. The cotton balls were then suspended in each test tube and labeled accordingly. The samples were incubated at 37°C for 24 hours. Subsequently, the samples were extracted and subjected to absorbance measurements at 600 nm [32].

Evaluations using the soap

Moisture content

A soap sample weighing 10 g was immediately measured and recorded as the “wet weight of the sample.” The wet sample was dried using an appropriate drying apparatus at a temperature below 239°F (115°C) until a constant weight was achieved. The sample was subsequently cooled and reweighed, and its dry weight was recorded. The amount of moisture in the samples was determined using the following equation [33]:

%W=100 A-B/B×100

where %W=percentage of moisture in the sample, A=weight of the wet sample (g), and B=weight of the dry sample (g).

Soap viscosity

Forty milliliters of the soap solution was transferred to a 100-ml beaker. The viscometer tip was then submerged in the beaker, and the viscosity was measured using a Brookfield digital viscometer [34].

Total fatty matter

Five grams of soap was obtained and transferred to a 250-ml beaker. Hot water (100 ml) was added to facilitate complete dissolution of the soap. Nitric acid (40 ml; 0.5 N) was added to the solution until a mildly acidic pH was achieved. The mixture was then heated in a water bath until the fatty acids formed a distinct layer above the solution. The fatty acids were then cooled and separated on ice. Chloroform (50 ml) was added to the remaining solution, which was then transferred to a separating funnel. The solution was agitated and allowed to undergo phase separation. The content of the lower stratum was depleted and chloroform (50 ml) was added to the remaining solution in a separating funnel. The fatty acids dissolved in chloroform were separated and transferred to the collected fatty matter, according to the procedure described above. The lipid content was measured using a pre-weighed porcelain dish. The contents were allowed to evaporate, and the resulting residue was weighed. The percentage of fatty matter in soap samples was determined by calculating the weight difference [35].

pH

A 10% soap solution was prepared by dissolving 1 g of soap in 10 ml of distilled water in a volumetric flask. The pH was determined using a pre-calibrated digital pH meter [33].

Foam height

The soap sample (0.5 g) was dispersed in 25 ml of distilled water. The solution was then transferred to a measuring cylinder and diluted with water to a final volume of 50 ml. A total of 25 strokes were administered until the aqueous volume reached 50 ml. The height of the foam above the aqueous volume was then measured [33].

Foam retention

A 25-ml sample of a 1% soap solution was transferred to a 100-ml graduated measuring cylinder. The cylinder was manually covered and shaken 10 times. The foam volume was measured at 1-minute intervals over 4 minutes [33].

Estimation of alcohol-insoluble content

A soap sample (5 g) was dissolved in 50 ml of heated alcohol. The solution was filtered using filter paper coated with tar and 20 ml of warm ethanol. The resulting mixture was then dried at 105°C for 1 hour. The mass of the dried filter paper was measured as follows:

Alcohol-insoluble matter (%)=wt. of residue×100/wt. of sample [36].

High temperature stability

The liquid soap was subjected to temperatures of 25, 37, 40, and 50°C for one month. The stability of liquid soap was assessed during the observation period. A homogeneous liquid sample was classified as stable, whereas a sample with roughened crystals and precipitation was considered unstable [37].

Percentage free alkali

Approximately 5 g of each sample was placed in a conical flask and mixed with 50 ml of neutralized alcohol. The mixture was refluxed for 30 minutes in a water bath and then cooled before the addition of 1 ml of phenolphthalein solution. The solution was promptly titrated using 0.1 N HCl [37].

Saponification value

The quantity of potassium hydroxide, measured in milligrams, required for the complete saponification of 1 g of fat or oil was determined. The term “mean molecular weight of fatty acid” refers to the average molecular weight of fatty acids found in oil or fat (Schumtterer et al., 1983). Briefly, approximately 2 g of soap sample was placed in a conical flask to determine its saponification value. Subsequently, a solution of 0.5 M KOH was added to the flask. The mixture was heated to approximately 55°C in a hot water bath with continuous stirring. Subsequently, the temperature was increased by an additional 100°C, and boiling was continued for approximately 1 hour. Titration was conducted using phenolphthalein as an indicator and 0.5 M HCl. The observed endpoint was the disappearance of the pink color [38]. The saponification value was calculated as follows:

Saponification value=(Avg volume of KOH 28.056)Weight of oil (g)

Antimicrobial assay using soap

Antimicrobial evaluation was conducted to determine the biological functions of the optimized formulations. The diffusion technique on agar wells was employed to determine the effectiveness of the soap against E. coli, S. aureus, and P. aeruginosa. The formulations were added to individual cups made with sterile nutrient agar; these cups were previously inoculated with the test organisms. After the solutions were allowed to diffuse for 2 hours, the agar plates were incubated at 37°C for 24 hours. The zone of inhibition (ZOI) was measured around each cup and subsequently compared [39].

Evaluations using the hand sanitizer gel

Physical analysis

The pH values of the gels were measured using a pre-calibrated pH meter (Mettler Toledo). Viscosity was measured using a Brookfield digital viscometer. The prescribed quantity of washing gel was added to the glass, and the viscometer tip was submerged in the gel to measure the viscosity [26].

Antibacterial activity based on the agar well diffusion assay

Five bacterial species, two gram-positive species (Staphylococcus aureus and Enterococcus faecalis) and three gram-negative species (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi), were tested to determine the antibacterial efficiency of the gel. The cultures were preserved on tryptone soya agar at 40°C. The inoculum was prepared according to the guidelines outlined in Clinical and Laboratory Standards Institute (CLSI) M02-A12 [39]. Briefly, isolated colonies of each bacterial culture were selected from the agar plates and incubated for 18–24 hours. The colonies were inoculated in tryptone soy broth to create a suspension. The turbidity of the suspension was adjusted to achieve a colony-forming unit (CFU) concentration of 1.0 to 2.0×108 CFU/ml, according to the CLSI guidelines, using a UV-visible spectrophotometer at a wavelength of 600 nm. Thereafter, 0.1 ml of each bacterial culture suspension was inoculated on Mueller–Hinton agar plates and evenly distributed using a sterile spreader. Sterile borers were used to cut 6 mm wells; 50 µl of formulated gel and commercial brand gels was added to the wells. The positive and negative controls included 70% ethanol and dimethyl sulfoxide (DMSO), respectively. The plates were allowed to settle for 5 minutes and then incubated at 37°C for 18–24 hours. Following incubation, the inhibition zones surrounding the wells owing to each sanitizing gel were measured using an automatic colony counter in inhibition zone mode [40].

Determination of the minimal inhibitory concentration

The minimal inhibitory concentration (MIC) was determined using the macrodilution method in sterile test tubes following the guidelines outlined in CLSI 07-08 [41]. The prepared gel was diluted in Mueller–Hinton broth using a 1:2 dilution at each step. Dilution resulted in a series of concentrations ranging from 100 to 0.195%. The test-strain inoculum was prepared in three steps. First, a cell suspension of each bacterial strain was prepared according to the agar well diffusion assay, with a concentration of 1.0–2.0×108 CFU/ml. Second, the suspension was diluted 1:150 to achieve a cell density of 1×106 CFU/ml. Third, the cell suspension was diluted 1:2 to a final inoculum concentration of 5×105 CFU/ml. The test was conducted by adding 1 ml of the prepared inoculum to each tube containing 1 ml of the hand sanitizer in a dilution series within 15 minutes of inoculum preparation. The mixture was homogenized and 1 ml was transferred from the first tube to the second tube, creating a dilution series with a dilution factor of 1:2. A positive control was established by adding the broth to the tube. This process was repeated until the final tube content was 0.195%. The MIC of all pigment concentrations in the hand sanitizer were determined using a similar method. The tubes were incubated at 37°C for 16 to 20 hours. The MIC of the hand sanitizer was the lowest concentration at which growth of the test organism was completely inhibited without the use of any aid [40].

Determination of the minimal bactericidal concentration

The minimal bactericidal concentration (MBC) was determined by inoculating 0.1 ml of sample from each tube of the MIC experiment on Mueller–Hinton agar plates using the spread plate technique after incubation. The plates were incubated at 37°C for 18–24 hours. After incubation, the MBC was determined as the lowest concentration of hand sanitizer gel that effectively eradicated the tested bacterial strains. The absence of colony formation on Mueller-Hinton agar plates indicated that the sanitizer effectively killed the bacterial cells, rendering them nonviable for growth on nutrient media in the absence of antibiotics. MBC were determined for all concentrations of pigments in the hand sanitizers against the five bacterial strains [40].

Hand sanitizer gel stability test

Stability studies were conducted by storing the samples at various temperatures (25, 37, and 40°C) for one month. The samples were monitored to identify alterations in color, odor, and phase separation [26].

Antioxidant activity assay

2,2-diphenyl-1-picrylhydrazl (DPPH) was used to assess the antioxidant activity of the extracts and products derived from cyanobacterial strains. This activity was determined by measuring the ability of antioxidant compounds to neutralize DPPH free radicals in a laboratory setting. The test sample was prepared by combining 1 ml of A. paniculata extract with 2 ml of a 0.1 mM DPPH methanolic solution. The mixture was then incubated in the dark for 30 min to allow the reaction to proceed. A small quantity of this solution was transferred to a cuvette. The absorbance of the reaction mixture was measured at 517 nm using a spectrophotometer. The control was prepared by combining 2 ml of a 0.1-mM DPPH methanol solution with 1 ml of methanol. The absorbance of each extract was measured in triplicate. The optical density was converted to the percentage of free radical-scavenging activity (% inhibition) using the following formula [42]:

DPPH %=A control-A sampleA control×100

Statistical analysis

The results of each experiment were analyzed using one-way analysis of variance (ANOVA) with the statistical software package, SPSS version 24 (IBM Co.). A significance level of 95% was considered to indicate statistically significance. The Tukey’s test was performed to assess the significance of differences in mean values following detection of a significant variation (p≤0.05) using ANOVA. Three replicate measurements were conducted for each treatment and the data are presented as mean values±standard error of the mean [43].

Results

Determination of the concentration and purity of the coated pigments

Based on the results in Table 2, PE exhibited maximum adsorption at 562 nm, with a peak at 617 nm, while PC exhibited maximum absorption at 621.9 nm. The purities of PE and PC were 0.845 and 0.401, respectively (Fig. 3).

Table 2 . The results of the concentration and purity of purified phycoerythrin and phycocyanin pigments.

StepsPeakPE (μg/ml)Purity of PE (OD555/OD280)
PhycoerythrinCrude extract562.8–617.70.1120.845
StepsPeakPC (μg/ml)Purity of PC (A620/A280)
PhycocyaninCrude extract619/80.05750.401

PE, phycoerythrin; PC, phycocyanin..


Figure 3. The results of spectrometry of pigment extracted and purified from (A) Nostoc sp. and (B) Spirulina sp.

Test results of the anti-acne face wash gel containing the PE and PC pigments

Viscosity

One-way ANOVA and Tukey’s test revealed no significant differences among the different treatments over 30 days. However, PC alone and the combination of PC and PE led to the highest viscosity compared to the control.

pH

One-way ANOVA and Tukey’s test revealed no significant differences between the different pigments over 30 days. The highest pH was observed in the control group.

Physical properties of the anti-acne face wash gel

Gels prepared with PE alone, PC alone, both PE and PC, and the control were pale pink, blue, green, and colorless, respectively. The anti-acne face wash gels prepared with the formulas were completely shiny and transparent. Application of the gels to the skin led to a very light and cooling sensation (Fig. 2).

Foamability of the anti-acne face wash gel

One-way ANOVA and Tukey’s test revealed no significant differences among the gels treated with the different pigments over 30 days.

Stability of the anti-acne face wash gel at different temperatures

All treated gels stored at 4°C for 30 days were assigned a full score of 5 for color, smell, and consistency. Based on the consistency results, no significant differences were found among the various treatments and the control at 25 and 37°C. On day 30, a significant decrease was observed with the control at 40°C compared to the pigments.

Based on the color measurements of the anti-acne face wash gel at 25°C, gels treated with the pigments had more color than those treated with the control over 30 days. In addition, starting from day 20, gels treated with the pigments displayed significantly more color than those treated with the control at 37°C and 40°C.

Based on the smell of the anti-acne face wash gels stored at 25°C, 37°C, and 40°C, no significant differences were found among the various treatment groups and the control group over 30 days.

Homogeneity of the anti-acne gel

Visual inspection of the anti-acne gels confirmed the homogeneity of all gels on all measurement days.

Determining the activity of the anti-acne face wash against acne-causing microorganisms

One-way ANOVA and Tukey’s test revealed no significant difference in the antimicrobial activity of gels prepared with PE and the control until day 30. However, a significant difference in the antimicrobial activity of gels prepared with PC and PE-PC pigments was observed on days 15 and 5, respectively. In addition, gels containing both PE and PC and PC alone had the highest killing percentage (Table 3).

Table 3 . The results of one-way analysis of variance and Tukey’s test of percentage of anti-acne face wash activity of gels prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during thirty days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control34.234±0.901 (aA)30.476±0.952 (aA)28.125±0.000 (aA)24.731±1.075 (aA)22.917±1.042 (aA)20.430±1.075 (aA)19.355±0.000 (aA)
PE39.640±0.901 (aB)37.143±0.000 (aB)35.417±1.042 (aB)33.333±1.075 (aB)32.292±1.042 (aB)32.258±0.000 (aB)31.183±1.075 (aB)
PC41.441±0.901 (aB)39.048±0.952 (abBC)37.500±0.000 (aB)36.559±1.075 (abBC)36.458±1.042 (abBC)36.559±1.075 (bC)35.484±0.000 (bC)
PE+PC42.342±0.901 (aB)40.952±0.952 (bC)38.542±1.042 (bB)39.785±1.075 (bC)38.542±1.042 (bC)36.559±1.075 (bC)36.559±1.075 (bC)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..



Antioxidant activity of the anti-acne face wash gels determined using the photobleaching (FRAP) method

One-way ANOVA and Tukey’s test revealed that the highest antioxidant activity was observed on day 10 for the gels prepared with both PC and PE (Table 4).

Table 4 . The results of one-way analysis of variance and Tukey’s test measuring the antioxidant activity of gels prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 25 days.

Day 1Day 5Day 10Day 15Day 20Day 25
Control0.83±0.001 (aA)0.77±0.004 (aA)0.75±0.003 (aA)0.68±0.002 (aA)0.59±0.003 (aA)0.42±0.003 (aA)
PE0.96±0.002 (bB)0.83±0.093 (Aa)0.90±0.003 (bB)0.89±0.003 (bB)0.84±0.003 (bB)0.74±0.004 (bB)
PC0.95±0.001 (bC)0.89±0.007 (aA)0.85±0.004 (bC)0.80±0.004 (bC)0.79±0.007 (bC)0.63±0.013 (bC)
PE+PC1.01±0.003 (bD)0.95±0.003 (aA)0.95±0.006 (bD)0.93±0.003(bD)0.89±0.002 (bD)0.80±0.002 (bD)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..



Results of the soap tests

Moisture content

One-way ANOVA and Tukey’s test revealed that the moisture content decreased significantly over 30 days. In addition, soaps coated with PE alone and the combination of the two pigments had higher contents on days 25 and 30 than those coated with the other treatments.

Total fat content and pH

One-way ANOVA and Tukey’s test revealed that the amount of fat and pH decreased significantly over 30 days. In addition, the amount of fat did not significantly differ among soaps prepared with different types of pigments.

Formation and height of soap foam

One-way variance analysis and Tukey’s test revealed that the foam height decreased significantly over 30 days, with no significant difference found between days 25 and 30. In addition, no significant difference in foam height was found between the soaps prepared with different types of pigments on days 25 and 30.

Soap foam shelf life

One-way ANOVA and Tukey’s test revealed that foam height decreased significantly over 30 days. In addition, the amount of foam retention did not significantly differ among soaps coated with the different pigments and control at different times.

Insoluble alcohol measurement

One-way ANOVA and Tukey’s test revealed a significant increase in the amount of insoluble alcohol in the soaps coated with the pigments compared to that treated with the control; however, no significant differences were observed between the different treatments.

Stability

One-way ANOVA and Tukey’s test revealed that the degree of color stability was not significantly different over the 30 days. In addition, no significant difference in color stability was observed between soaps coated with the different pigments and the control.

When soap stability was tested at 25°C and 30 days, no color changes were observed in stable soaps, and no phase separation occurred. A homogeneous sample was reported as a stable sample while a sediment-like sample was reported as an unstable sample. During the 30 days, all soaps received a score of 5 for stability.

Percentage of soap alkali

One-way ANOVA and Tukey’s test revealed no significant difference in the percentage of alkalinity between soaps coated with the different pigments and those treated with the control.

Determination of saponification rate

One-way ANOVA and Tukey’s test revealed that the amount of saponification in soaps coated with the different pigments was significantly different from that of soaps treated with the control, with significant decreases recorded.

Antibacterial properties of soap

One-way ANOVA and Tukey’s test results regarding the antibacterial properties of soaps treated with the control, PC, PE, and both PC and PE for 30 days are presented in Table 5. No significant differences in the antibacterial properties of soaps against E. coli were found over the 30 days, highlighting the stability of their antibacterial properties. On all days, soaps prepared with PC and the combination of both pigments had significant increases in their antibacterial activity against E. coli compared to those coated with the other treatments (Table 5). In terms of S. aureus, the antibacterial properties were not significantly different over the 30 days between the control soaps and soaps coated with PE. The highest antibacterial effects were obtained with soaps treated with the combination of the two pigments until day 20; however, no significant difference was observed compared with the control. Notably, the antibacterial effects against S. aureus decreased significantly from day 20 onwards (Table 5). In terms of P. aeruginosa, the antibacterial effects were not significantly different between the control soap and soaps containing PE and PC over the 30-day period. Initially, soaps containing both pigments had the highest antibacterial effects until day 10; however, these effects decreased significantly thereafter (Table 5).

Table 5 . The results of one-way analysis of variance and Tukey’s test to determine the antibacterial properties against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa bacteria of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
(a) Escherichia coli
Control7.33±0.333 (aA)7.33±0.333 (aA)7.00±0.000 (aA)7.00±0.000 (aA)6.67±0.333 (aA)6.67±0.333 (aA)6.33±0.333 (aA)
PE11.00±0.000 (bB)11.00±0.000 (bB)10.67±0.333 (bB)10.33±0.333 (bB)10.00±0.000 (bB)9.67±0.333 (bB)9.33±0.333 (bB)
PC12.67±0.333 (cC)12.33±0.333 (cC)12.33±0.333 (cC)12.33±0.333 (cC)12.00±0.000 (cC)11.67±0.333 (cC)11.33±0.333 (cC)
PE+PC13.33±0.333 (cC)13.33±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (cC)12.00±0.000 (cC)11.67±0.333 (cC)
(b) Staphylococcus aureus
Control8.00±0.000 (aA)8.00±0.000 (aA)7.67±0.333 (aA)7.67±0.333 (aA)7.33±0.333 (aA)7.00±0.000 (aA)6.67±0.333 (aA)
PE12.33±0.333 (aB)12.33±0.333 (aB)12.33±0.333 (aB)12.00±0.000 (aB)12.00±0.000 (aB)11.67±0.333 (aB)11.67±0.333 (aB)
PC13.00±0.000 (bcBC)13.00±0.000 (bcBC)12.67±0.333 (bB)12.67±0.333 (bcBC)12.33±0.33 (bcBC)12.00±0.000 (bB)12.00±0.000 (bB)
PE+PC13.67±0.333 (cC)13.67±0.333 (cC)13.67±0.333 (cB)13.33±0.333 (cC)13.33±0.333 (cC)12.67±0.333 (bB)12.67±0.333 (bB)
(c) Pseudomonas aeruginosa
Control7.67±0.333 (aA)7.67±0.333 (aA)8.00±0.000 (aA)7.67±0.333 (aA)7.33±0.333 (aA)7.33±0.333 (aA)6.67±0.333 (aA)
PE11.67±0.333 (bB)11.67±0.333 (bB)11.33±0.333 (bB)11.00±0.000 (bB)11.00±0.000 (bB)11.00±0.000 (bB)10.67±0.333 (Bb)
PC12.67±0.333 (bcBC)12.67±0.333 (bcBC)12.67±0.333 (bB)12.33±0.333 (cC)12.00±0.000 (bcBC)12.00±0 (bcBC)12.00±0.000 (cC)
PE+PC13.33±0.333 (bC)13.33±0.333 (bC)13.00±0.577 (bB)12.67±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (Cc)12.33±0.333 (cC)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..



Antioxidant activity of soap

One-way ANOVA and Tukey’s test revealed that the antioxidant activity significantly decreased over 30 days. However, the antioxidant activity of soaps coated with the different pigments was significantly increased compared to that of control soaps. In addition, no significant differences were found in the antioxidant properties of the treated soaps (Table 6).

Table 6 . The results of one-way variance analysis and Tukey’s test of antioxidant activity of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control2.18±0.01 (Aa)5.76±0.45 (Ab)6.39±0.21 (Ab)9.63±0.09 (Ac)11.37±0.14 (Ad)15.83±0.23 (Ae)18.48±0.56 (Af)
PE0.47±0.04 (Ba)0.68±0.01 (Bb)0.90±0.03 (Bc)1.36±0.03 (Bd)1.56±0.01 (Be)1.85±0.02 (Bf)2.43±0.02 (Bg)
PC0.45±0.04 (Ba)0.64±0.01 (Bab)0.80±0.04 (Bb)1.09±0.09 (Bc)1.32±0.04 (Bcd)1.51±0.01 (Bd)1.87±0.08 (Be)
PE+PC0.46±0.04 (Ba)0.67±0.01 (Bb)0.86±0.03 (Bc)1.25±0.01 (Bd)1.45±0.02 (Be)1.64±0.04 (Bf)2.15±0.07 (Bg)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..



Psychrophilic bacteria

One-way ANOVA and Tukey’s test revealed that cold-loving bacteria appeared in the control soap and soaps containing PC or PE from days 20, 25, and 30; however, these bacteria were not found in soaps containing both PC and PE. In addition, the number of cold-loving bacteria in the control soap was significantly higher than those in the pigment-coated soaps.

Colorimetry of soaps

One-way ANOVA and Tukey’s test revealed that soaps prepared with the PE pigment and both pigments had a significant level of brightness compared to those coated with the other treatments. The a*value did not significantly differ between the coated and control soaps during the evaluation period. However, soaps treated with PE had the highest a*values on all days. The b*value did not significantly differ between the coated and control soaps during the evaluation period. The lowest b*value was found for soaps containing PE, with a significant difference observed on day 30. Moreover, the amount of ΔE did not significantly differ between the coated and control soaps during the evaluation period. The ΔE value was lowest in soaps containing PC. Soaps coated with both pigments exhibited significantly differences in their ΔE value on day 30 (Table 7).

Table 7 . The results of one-way variance analysis and Tukey’s test to determine the ΔE value of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days.

Control
day 5 to day 1
Control
day 10 to day 1
Control
day 15 to day 1
Control
day 20 to day 1
Control
day 25 to day 1
Control
day 30 to day 1
Control1.51±0.082 (bA)1.89±0.269 (Aa)2.81±0.313 (bB)3.59±0.143 (bA)4.42±0.283 (cA)5.32±0.101 (bA)
PE1.49±0.062 (abAB)2.07±0.160 (aA)2.89±0.025 (bB)3.19±0.194 (ABab)3.68±0.140 (bcAB)4.67±0.274 (bA)
PC1.17±0.065 (abAB)1.35±0.187 (Aa)2.14±0.027 (ABab)2.90±0.176 (ABab)3.45±0.069 (abBC)2.84±0.174 (aB)
PE+PC1.11±0.128 (aB)1.28±0.264 (Aa)1.67±0.261 (Aa)2.50±0.209 (Ba)2.77±0.076 (aC)3.37±0.371 (aB)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..



Results of hand sanitizer gel tests

pH

One-way ANOVA and Tukey’s test revealed that the pH of the control gels decreased significantly. On day 30, the lowest pH was observed in gels coated with PC.

Viscosity

One-way ANOVA and Tukey’s test revealed that the viscosity decreased significantly over 30 days. On day 30, the highest viscosity was observed for gels coated with PE alone and both pigments, while the lowest amount was observed for control gels.

Antimicrobial activity of the hand sanitizer gel

One-way ANOVA and Tukey’s test revealed that gels coated with both pigments had the highest inhibition rate against S. aureus. Based on the inhibition diameters against E. faecalis and E. coli, no significant difference was found between the gels coated with the different pigments. However, gels coated with PE alone and both pigments exhibited the highest inhibitory activity against P. aeruginosa and S. typhi (Table 8).

Table 8 . The results of one-way analysis of variance and Tukey’s test of growth inhibition diameter of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments.

Staphylococcus aureusEnterococcus faecalisEscherichia coliPseudomonas aeruginosaSalmonella typhi
Control7.00±0.00 (A)6.33±0.33 (A)6.00±0.00 (A)5.33±0.33 (A)5.00±0.58 (A)
PE9.67±0.33 (B)8.67±0.33 (B)7.67±0.33 (B)6.67±0.33 (B)6.33±0.33 (B)
PC8.67±0.33 (C)8.33±0.33 (B)7.33±0.33 (B)5.67±0.33 (A)5.33±0.33 (A)
PE+PC10.67±0.33 (D)8.67±0.33 (B)8.00±0.58 (B)7.33±0.33 (B)7.00±0.58 (B)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..



MIC of the hand sanitizer gel

One-way ANOVA and Tukey’s test revealed that gels coated with both pigments and PE alone had the lowest MIC against S. aureus, whereas gels coated with both pigments had the lowest MIC against E. faecalis, E. coli, P. aeruginosa, and S. typhi.

MBC of the hand sanitizer gel

One-way variance analysis and Tukey’s test revealed that gels coated with both pigments, PE alone, and PC alone had the lowest MBCs against S. aureus, P. aeruginosa, E. faecalis, and S. typhi, whereas gels coated with the PC and phycothyrin pigments had the lowest MBC against E. coli.

Hand sanitizer gel stability test

One-way ANOVA and Tukey’s test were performed to determine the stability of smell, color, and phase separation of gels treated with PE, PC, both PE and PC, and control. At 4, 25 (until day 15), and 40°C, the best score of 5 was assigned for smell, color, and phase separation. The stability of the smell and color decreased significantly over the 30 days. However, no significant difference was found between the gels prepared with the control and those coated with the pigments. Significant difference in phase separation was observed at 25, 37, and 40°C on days 25 and 30. However, no significant differences were observed between the control gels and pigment-coated gels. The phase separation and color on days 10 and 15 at 40°C and days 15 and 20 at 25°C also received a score of 5.

Antioxidant activity of hand sanitizer gel determined using the DPPH method

One-way ANOVA and Tukey’s test revealed that antioxidant activity decreased over 30 days. On the last day, gels coated with PE alone and both pigments had the highest antioxidant activity (Table 9).

Table 9 . The results of one-way variance analysis and Tukey’s test to determine the antioxidant activity of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments during 30 days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control4.39±0.10 (Aa)5.21±0.10 (Aab)5.83±0.13 (Ab)7.38±0.16 (Ac)8.56±0.18 (Ad)11.57±0.33 (Ae)13.34±0.25 (Af)
PE2.22±0.01 (Ba)2.42±0.05 (Bb)2.45±0.01 (Bbc)2.47±0.01 (Bbc)2.50±0.02 (Bcb)2.53±0.02 (Bcb)2.56±0.02 (Bc)
PC3.45±0.08 (Ca)3.83±0.06 (Cab)4.23±0.09 (Cbc)4.36±0.05 (Ccd)4.75±0.09 (Cde)4.85±0.12 (Ce)5.18±0.14 (Ce)
PE+ PC2.39±0.04 (Ba)2.44±0.02 (Bab)2.47±0.0 2(Bab)2.48±0.02 (Bbac)2.55±0.01 (Bcb)2.59±0.04 (Bcb)2.63±0.05 (cB)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..



Foaming ability of the hand sanitizer gel

One-way ANOVA and Tukey’s test revealed no significant differences in foaming ability between the control and coated gels.

Lethality percentage of the hand sanitizer gel

One-way ANOVA and Tukey’s test revealed that the lethality rate decreased significantly over 30 days. The most significant difference was observed in gels coated with both pigments (Table 10).

Table 10 . The results of one-way variance analysis and Tukey’s test to determine the lethality percentage of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments during 30 days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control73.66±0.41 (aA)71.89±0.40 (Aab)70.09±0.43 (Abc)68.40±0.43 (Acd)66.23±0.44 (Ad)63.60±0.44 (Ae)62.34±0.75 (Ae)
PE76.95±0.41 (BCa)75.10±0.40 (Bab)75.21±0.43 (Bab)73.16±0.43 (Bbc)71.93±0.44 (Bc)71.49±0.44 (BCcd)69.70±0.43 (BCd)
PC76.54±0.00 (Ba)75.10±0.40 (Bab)74.36±0.74 (Bab)73.16±0.43 (Bbc)71.49±0.44 (Bcd)70.61±0.44 (Bde)68.83±0.00 (Be)
PE+ PC78.19±0.41 (ACa)75.90±0.70 (Bb)75.64±0.00 (Bb)74.46±0.43 (Bbc)73.25±0.44 (Bcd)72.81±0.44 (ACcd)71.86±0.43 (ACd)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


Discussion

Natural ingredients have historically been used to synthesize cosmetic products, thereby predating the formalization of cosmetics [44]. The emergence of cosmeceuticals has further highlighted the potential of cyanobacteria as valuable sources of components owing to their ability to produce a wide range of compounds. Cyanobacteria produce various chemicals, such as fatty acids, polyphenols, peptides, polysaccharides, and pigments, which possess distinct potential for health-related applications [45]. Cyanobacteria contain three types of pigments: chlorophyll; carotenoids; and PBPs. These pigments exhibit a range of colors from blue to red and possess diverse physicochemical properties. Notably, pigments have potential applications in food, feed, nutraceuticals, and cosmetics [46]. Cyanobacterial pigments, such as carotenoids and PBPs, have bioactive properties, a range of colors, and cosmetic enhancement properties, such as the ability to moisturize and stabilize the skin. Owing to these attributes, cyanobacterial pigments are appealing in the natural cosmetic industry, which commonly uses the extract of these compounds [6,47]. Carotenoids are present in sunscreens, antiaging products, and antioxidant formulations because of their exceptional antioxidant properties. In cyanobacteria, these compounds are responsible for capturing excess energy from photosynthetic metabolism and protecting against harmful effects and cellular damage [48]. Prolonged exposure to sunlight (UV radiation and intense light) can cause cellular damage to human skin, enabling the potential extraction and use of these compounds for similar purposes [49]. PBPs have been linked to many biological activities, including anticancer, antiviral, antimicrobial, and antioxidant [50]. Therefore, PBPs can be used as functional additives. These compounds can serve as natural colorants, reducing the potential for skin toxicity, damage, and allergic reactions to synthetic dyes. Owing to their water solubility, hydrophilic pigments are suitable for use in skincare products, specifically in sera and lotions [46]. This study aimed to determine the effect of PC and PE coatings on the antioxidant and antimicrobial activities of different cosmetic products, including soap, anti-acne face wash, and hand sanitizer gel.

Based on the tests using the anti-acne face wash gel containing PE and PC, the amount of pigments used to determine viscosity (PX and PC-PE), pH (control treatment), antioxidant (PC-PE), and antimicrobial activity against acne-causing microorganisms (PC-PE) increased, with differences found among gels coated with the various pigments. In contrast, the pH and viscosity of the hand sanitizer gel decreased, and the lowest values were observed in the PC and control groups, respectively. The lowest antioxidant results were obtained for the hand sanitizer gel containing PE alone and both pigments compared to gels containing the other cosmetic products.

Data related to gel homogeneity revealed that the gel was homogenous on all days. The anti-acne face wash gels were completely shiny and transparent. Application of the gel to the skin led to a very light and cooling sensation. The highest lethality, based on the anti-acne face wash gel test, was observed for gels prepared using both PC and PE and PC alone. However, hand sanitizer gels coated with both pigments had the best killing rate.

Optimal cultivation conditions exist to maximize the production rate of secondary metabolites in extremophilic cyanobacteria. Zucchi and Necchi [51] examined the growth and pigment content of freshwater algal cultures. Their findings indicate that temperature primarily influenced pigment content, whereas variations in irradiance and photoperiod, alone and combined, accounted for a smaller portion of the observed differences. The responses varied among species, with PC being more concentrated than PE and PBPs being more concentrated than chlorophyll a. However, Rizzo et al. [52] did not observe any variation in total protein and penicillin-binding protein (PBP) production under different light conditions. Ma et al. [53] found a peak in PBP contents at light intensities below 90 μmol·m-2 s-1. Conversely, PC and APC levels increased with increasing light intensity, whereas PE levels decreased. The investigators also found that blue and red light led to the greatest increases in fresh weight, protein content, and PBP content, leading to higher dry matter, PC, and chlorophyll a levels.

PE has diverse applications in the pharmaceutical, antioxidant, and food industries [54,55]. However, the limited stability of PE poses a significant challenge to their widespread use. The sensitivity of PE to pH, salts, temperature, water, and light, and to in vitro processes, such as extraction, purification, storage, and utilization, hinders its practical application. The size and protein composition of the PE complex vary in response to changes in light and nutritional conditions. Low-intensity light promotes the synthesis of PE, leading to the elongation of rod structures. Various purification and characterization methods are available for the PEs derived from different strains of cyanobacteria and red algae [14].

Based on the stability analysis of the color, smell, and consistency of the anti-acne face wash and hand sanitizer gel, both products were assigned a full score of 5 owing to treatment with the pigments. The color measurements of the anti-acne face wash gel at 25, 37 and 40°C (from day 20) were the highest with the control. However, the smell of anti-acne face wash gels at 25, 37, and 40°C did not significantly differ among the various treatment and control groups over 30 days. The smell, color, and phase separation of the hand sanitizer gel received full scores of 5 at 4, 25 (until day 15), and 40°C. Similar to the two other cosmetic products (i.e., anti-acne face wash and hand sanitizer gel), soap also received a full score of 5. In addition, the foamability of the anti-acne face wash and hand sanitizer gels did not change significantly. The total fat content, pH, foam formation and height, shelf life, percentage of alkali, and antioxidant activity of soap revealed no significant differences, despite decrease over time. Although insoluble alcohol levels increased over 30 days, no significant differences were found.

Cyanobacteria possess inherent protective mechanisms against dehydration, which makes them potentially valuable moisturizing agents for cosmetics. Several studies have investigated the moisture uptake properties of cyanobacteria [56,57]. For example, applying microalgal can improve skin moisture and elasticity [4]. Additional studies on Nostoc commune revealed that cells containing extracellular polymeric substances (EPSs) exhibit significantly greater desiccation tolerance. This finding highlights the remarkable moisture absorption and water retention properties of these substances compared with those of urea and chitosan [58,59]. EPSs are large molecules with high molecular mass. These substances consist of hydrated sulfate groups, neutral sugars (such as glucose, galactose, mannose, fructose, ribose, xylose, arabinose, fucose, and rhamnose), non-carbohydrate components (such as phosphate, lactate, acetate, and glycerol), and various uronic acids (such as glucuronic and galacturonic acid). Polysaccharides are recognized for their notable ability to retain moisture due to strong interactions between water molecules and hydrophilic -OH groups. Environmental conditions influence the composition of EPSs in different species [60]. The cellular envelope of Chroococcidiopsis exhibits variations in composition in response to varying levels of water stress. This cyanobacterium contains various organic metabolites, including sporopollenin-like compounds, proteins, beta-linked polysaccharides, acid sulfate, and lipids. These metabolites are associated with reduced and regulated rates of water loss [61].

In this study, both the moisture content and saponification rate decreased. The highest reduction in moisture occurred on days 25 and 30 for soaps coated with PE alone and both PC and PE.

In terms of psychrophilic bacteria, the control group had the highest number of cold-loving bacteria.

Based on the colorimetry results, the values of L* (PE and PC-PE) and a (PE alone) indicated that soaps had the highest level of brightness. However, soap had the lowest b (PE) and ΔE (PC and PC-PE) values.

In terms of antibacterial properties, the level of E. coli (over 30 days), S. aureus (over 30 days in control soaps and PE-coated soap), and P. aeruginosa (control soaps and soaps coated with PE and PC over a 30-day period) did not significant differ among soaps. The highest inhibition rate against S. aureus was obtained with the hand sanitizer gel coated with both PC and PE; however, no significant difference was found between the inhibition diameters of E. faecalis and E. coli for gels coated with the pigments. Notably, gels coated with PE alone and both PC and PE caused the highest inhibition of P. aeruginosa and S. typhi.

The lowest MIC was obtained with the hand sanitizer gel coated with PE alone and both PC and PE for S. aureus and both PC and PE for other bacteria (E. faecalis, E. coli, P. aeruginosa, and S. typhi). The lowest MBC against S. aureus, P. aeruginosa, E. faecalis, and S. typhi, was obtained with gels coated with both pigments, PE alone, and PC alone; however, lowest MBC against E. coli was obtained with gels coated with the PC and phycothyrin pigments.

The growing emphasis on skin health, particularly in relation to aesthetics and aging, has led to a greater desire for new cosmetic products. These products are primarily derived from natural sources, have few adverse effects, and are environmentally sustainable. Cyanobacteria possess effective mechanisms to combat dissection, radiation, and oxidative stress through the production of specific compounds. Owing to this characteristic, cyanobacteria serve as a promising group of organisms for applications in the cosmetic/cosmeceutical industry. Cyanobacteria can be utilized in biotechnology to produce cost-effective cosmetic formulations coated with natural pigments, which enhances production efficiency. This study highlights the use of cyanobacterial pigments in cosmetics and the biotechnological potential of three cosmetic products: soap, anti-acne face wash, and hand sanitizer gels. These products are economically viable and sustainable options derived from the cosmetic industry.

Conflicts of interest

The authors have nothing to disclose.

Fig 1.

Figure 1.Different stages of separation and purification of phycocyanin (A to D) and phycoerythrin (E to H) pigments. (A) Primary culture of Spirulina cyanobacteria. (B) Preparation of crude extract. (C) Dialysis. (D) Freeze drying and preparation of phycocyanin powder. (E) Primary culture of Nostoc cyanobacteria. (F) Preparation of crude extract. (G) Dialysis. (H) Freeze-drying and preparation of phycoerythrin powder.
Journal of Cosmetic Medicine 2024; 8: 18-33https://doi.org/10.25056/JCM.2024.8.1.18

Fig 2.

Figure 2.Preparation of (A) anti-acne gel; (a) without pigment, (b) with phycoerythrin pigment, (c) total of phycoerythrin and phycocyanin pigments, (d) with phycocyanin pigment. (B) Hand gel; (a) total of phycoerythrin and phycocyanin pigments, (b) with phycoerythrin pigment, (c) with phycocyanin pigment (d) without pigment. (C) Soap; (a) without pigment, (b) with phycocyanin pigment, (c) the sum of phycoerythrin and phycocyanin pigments, (d) with phycocyanin pigment.
Journal of Cosmetic Medicine 2024; 8: 18-33https://doi.org/10.25056/JCM.2024.8.1.18

Fig 3.

Figure 3.The results of spectrometry of pigment extracted and purified from (A) Nostoc sp. and (B) Spirulina sp.
Journal of Cosmetic Medicine 2024; 8: 18-33https://doi.org/10.25056/JCM.2024.8.1.18

Table 1 . Composition of anti-acne washing gel, soap and antibacterial hand sanitizer.

ProductsIngredientsQuantity
Anti-acne washing gelCarbapol0.1 gr
Distilled water2.0 ml
Methylparaben0.1 mg
Propylenglycol0.1 mg
Tea0.1 gr
Phycocyanin and phycoerythrin1.5 gr
SoapWater2.0 ml
Ethanol5 ml
Cinnamon oil1 ml
Citronella oil1 ml
Melted glycerine soap9.0%
Stearic acid0.033 g
Phycocyanin and phycoerythrin1.5 gr
Antibacterial hand sanitizerCarbopol 9401 gr
EDTA0.1 gr
Distilled water2.0 ml
Glycerine5 gr
Perfume0.3%
Phycocyanin and phycoerythrin1.5 gr

Table 2 . The results of the concentration and purity of purified phycoerythrin and phycocyanin pigments.

StepsPeakPE (μg/ml)Purity of PE (OD555/OD280)
PhycoerythrinCrude extract562.8–617.70.1120.845
StepsPeakPC (μg/ml)Purity of PC (A620/A280)
PhycocyaninCrude extract619/80.05750.401

PE, phycoerythrin; PC, phycocyanin..


Table 3 . The results of one-way analysis of variance and Tukey’s test of percentage of anti-acne face wash activity of gels prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during thirty days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control34.234±0.901 (aA)30.476±0.952 (aA)28.125±0.000 (aA)24.731±1.075 (aA)22.917±1.042 (aA)20.430±1.075 (aA)19.355±0.000 (aA)
PE39.640±0.901 (aB)37.143±0.000 (aB)35.417±1.042 (aB)33.333±1.075 (aB)32.292±1.042 (aB)32.258±0.000 (aB)31.183±1.075 (aB)
PC41.441±0.901 (aB)39.048±0.952 (abBC)37.500±0.000 (aB)36.559±1.075 (abBC)36.458±1.042 (abBC)36.559±1.075 (bC)35.484±0.000 (bC)
PE+PC42.342±0.901 (aB)40.952±0.952 (bC)38.542±1.042 (bB)39.785±1.075 (bC)38.542±1.042 (bC)36.559±1.075 (bC)36.559±1.075 (bC)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


Table 4 . The results of one-way analysis of variance and Tukey’s test measuring the antioxidant activity of gels prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 25 days.

Day 1Day 5Day 10Day 15Day 20Day 25
Control0.83±0.001 (aA)0.77±0.004 (aA)0.75±0.003 (aA)0.68±0.002 (aA)0.59±0.003 (aA)0.42±0.003 (aA)
PE0.96±0.002 (bB)0.83±0.093 (Aa)0.90±0.003 (bB)0.89±0.003 (bB)0.84±0.003 (bB)0.74±0.004 (bB)
PC0.95±0.001 (bC)0.89±0.007 (aA)0.85±0.004 (bC)0.80±0.004 (bC)0.79±0.007 (bC)0.63±0.013 (bC)
PE+PC1.01±0.003 (bD)0.95±0.003 (aA)0.95±0.006 (bD)0.93±0.003(bD)0.89±0.002 (bD)0.80±0.002 (bD)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


Table 5 . The results of one-way analysis of variance and Tukey’s test to determine the antibacterial properties against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa bacteria of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
(a) Escherichia coli
Control7.33±0.333 (aA)7.33±0.333 (aA)7.00±0.000 (aA)7.00±0.000 (aA)6.67±0.333 (aA)6.67±0.333 (aA)6.33±0.333 (aA)
PE11.00±0.000 (bB)11.00±0.000 (bB)10.67±0.333 (bB)10.33±0.333 (bB)10.00±0.000 (bB)9.67±0.333 (bB)9.33±0.333 (bB)
PC12.67±0.333 (cC)12.33±0.333 (cC)12.33±0.333 (cC)12.33±0.333 (cC)12.00±0.000 (cC)11.67±0.333 (cC)11.33±0.333 (cC)
PE+PC13.33±0.333 (cC)13.33±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (cC)12.00±0.000 (cC)11.67±0.333 (cC)
(b) Staphylococcus aureus
Control8.00±0.000 (aA)8.00±0.000 (aA)7.67±0.333 (aA)7.67±0.333 (aA)7.33±0.333 (aA)7.00±0.000 (aA)6.67±0.333 (aA)
PE12.33±0.333 (aB)12.33±0.333 (aB)12.33±0.333 (aB)12.00±0.000 (aB)12.00±0.000 (aB)11.67±0.333 (aB)11.67±0.333 (aB)
PC13.00±0.000 (bcBC)13.00±0.000 (bcBC)12.67±0.333 (bB)12.67±0.333 (bcBC)12.33±0.33 (bcBC)12.00±0.000 (bB)12.00±0.000 (bB)
PE+PC13.67±0.333 (cC)13.67±0.333 (cC)13.67±0.333 (cB)13.33±0.333 (cC)13.33±0.333 (cC)12.67±0.333 (bB)12.67±0.333 (bB)
(c) Pseudomonas aeruginosa
Control7.67±0.333 (aA)7.67±0.333 (aA)8.00±0.000 (aA)7.67±0.333 (aA)7.33±0.333 (aA)7.33±0.333 (aA)6.67±0.333 (aA)
PE11.67±0.333 (bB)11.67±0.333 (bB)11.33±0.333 (bB)11.00±0.000 (bB)11.00±0.000 (bB)11.00±0.000 (bB)10.67±0.333 (Bb)
PC12.67±0.333 (bcBC)12.67±0.333 (bcBC)12.67±0.333 (bB)12.33±0.333 (cC)12.00±0.000 (bcBC)12.00±0 (bcBC)12.00±0.000 (cC)
PE+PC13.33±0.333 (bC)13.33±0.333 (bC)13.00±0.577 (bB)12.67±0.333 (cC)12.67±0.333 (cC)12.67±0.333 (Cc)12.33±0.333 (cC)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


Table 6 . The results of one-way variance analysis and Tukey’s test of antioxidant activity of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control2.18±0.01 (Aa)5.76±0.45 (Ab)6.39±0.21 (Ab)9.63±0.09 (Ac)11.37±0.14 (Ad)15.83±0.23 (Ae)18.48±0.56 (Af)
PE0.47±0.04 (Ba)0.68±0.01 (Bb)0.90±0.03 (Bc)1.36±0.03 (Bd)1.56±0.01 (Be)1.85±0.02 (Bf)2.43±0.02 (Bg)
PC0.45±0.04 (Ba)0.64±0.01 (Bab)0.80±0.04 (Bb)1.09±0.09 (Bc)1.32±0.04 (Bcd)1.51±0.01 (Bd)1.87±0.08 (Be)
PE+PC0.46±0.04 (Ba)0.67±0.01 (Bb)0.86±0.03 (Bc)1.25±0.01 (Bd)1.45±0.02 (Be)1.64±0.04 (Bf)2.15±0.07 (Bg)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


Table 7 . The results of one-way variance analysis and Tukey’s test to determine the ΔE value of soaps prepared with four different conditions of control, phycocyanin, phycoerythrin and both pigments during 30 days.

Control
day 5 to day 1
Control
day 10 to day 1
Control
day 15 to day 1
Control
day 20 to day 1
Control
day 25 to day 1
Control
day 30 to day 1
Control1.51±0.082 (bA)1.89±0.269 (Aa)2.81±0.313 (bB)3.59±0.143 (bA)4.42±0.283 (cA)5.32±0.101 (bA)
PE1.49±0.062 (abAB)2.07±0.160 (aA)2.89±0.025 (bB)3.19±0.194 (ABab)3.68±0.140 (bcAB)4.67±0.274 (bA)
PC1.17±0.065 (abAB)1.35±0.187 (Aa)2.14±0.027 (ABab)2.90±0.176 (ABab)3.45±0.069 (abBC)2.84±0.174 (aB)
PE+PC1.11±0.128 (aB)1.28±0.264 (Aa)1.67±0.261 (Aa)2.50±0.209 (Ba)2.77±0.076 (aC)3.37±0.371 (aB)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


Table 8 . The results of one-way analysis of variance and Tukey’s test of growth inhibition diameter of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments.

Staphylococcus aureusEnterococcus faecalisEscherichia coliPseudomonas aeruginosaSalmonella typhi
Control7.00±0.00 (A)6.33±0.33 (A)6.00±0.00 (A)5.33±0.33 (A)5.00±0.58 (A)
PE9.67±0.33 (B)8.67±0.33 (B)7.67±0.33 (B)6.67±0.33 (B)6.33±0.33 (B)
PC8.67±0.33 (C)8.33±0.33 (B)7.33±0.33 (B)5.67±0.33 (A)5.33±0.33 (A)
PE+PC10.67±0.33 (D)8.67±0.33 (B)8.00±0.58 (B)7.33±0.33 (B)7.00±0.58 (B)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


Table 9 . The results of one-way variance analysis and Tukey’s test to determine the antioxidant activity of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments during 30 days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control4.39±0.10 (Aa)5.21±0.10 (Aab)5.83±0.13 (Ab)7.38±0.16 (Ac)8.56±0.18 (Ad)11.57±0.33 (Ae)13.34±0.25 (Af)
PE2.22±0.01 (Ba)2.42±0.05 (Bb)2.45±0.01 (Bbc)2.47±0.01 (Bbc)2.50±0.02 (Bcb)2.53±0.02 (Bcb)2.56±0.02 (Bc)
PC3.45±0.08 (Ca)3.83±0.06 (Cab)4.23±0.09 (Cbc)4.36±0.05 (Ccd)4.75±0.09 (Cde)4.85±0.12 (Ce)5.18±0.14 (Ce)
PE+ PC2.39±0.04 (Ba)2.44±0.02 (Bab)2.47±0.0 2(Bab)2.48±0.02 (Bbac)2.55±0.01 (Bcb)2.59±0.04 (Bcb)2.63±0.05 (cB)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


Table 10 . The results of one-way variance analysis and Tukey’s test to determine the lethality percentage of hand sanitizer gels prepared with four different control conditions, phycocyanin, phycoerythrin and both pigments during 30 days.

Day 1Day 5Day 10Day 15Day 20Day 25Day 30
Control73.66±0.41 (aA)71.89±0.40 (Aab)70.09±0.43 (Abc)68.40±0.43 (Acd)66.23±0.44 (Ad)63.60±0.44 (Ae)62.34±0.75 (Ae)
PE76.95±0.41 (BCa)75.10±0.40 (Bab)75.21±0.43 (Bab)73.16±0.43 (Bbc)71.93±0.44 (Bc)71.49±0.44 (BCcd)69.70±0.43 (BCd)
PC76.54±0.00 (Ba)75.10±0.40 (Bab)74.36±0.74 (Bab)73.16±0.43 (Bbc)71.49±0.44 (Bcd)70.61±0.44 (Bde)68.83±0.00 (Be)
PE+ PC78.19±0.41 (ACa)75.90±0.70 (Bb)75.64±0.00 (Bb)74.46±0.43 (Bbc)73.25±0.44 (Bcd)72.81±0.44 (ACcd)71.86±0.43 (ACd)

Values are presented as mean±standard deviation..

PE, phycoerythrin; PC, phycocyanin..

Lowercase letters indicate significant differences in rows and uppercase letters indicate significant differences in columns..


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