Hydrolyzed Fish Collagen Serum from By-Product of Food Industry: Cosmetic Product Formulation and Facial Skin Evaluation

Abstract

Fish collagen is a good source of protein and essential amino acids. It has a wide range of applications in food manufacturing, pharmaceutics, cosmetics, etc. By-products obtained from fish processing industries are currently used as alternative sources for the production of collagen and gelatin. Hydrolyzed collagen derived from fish skin has properties that are beneficial in cosmetic products. The aim of this study was to: (1) formulate a cosmetic product containing hydrolyzed fish collagen obtained from a by-product of fish processing industries, and other effective ingredients such as vitamin C, vitamin E and vitamin B3, (2) evaluate the physicochemical properties and stability of the product, and (3) conduct a facial skin evaluation test wherein volunteers use the product and have their facial skin analyzed. The formulated serum was translucent with low turbidity and had a pH of 7.7, and a viscosity of 1333 cps. The stability of the formulation was good, without any significant change in appearance, pH and viscosity as tested by freeze–thaw cycle testing, and after storage at room temperature for a period of 6 months. Furthermore, the results from the Visia skin analysis instrument revealed that the product was able to enhance facial skin moisture, reduce skin pores and wrinkles, and brighten the skin significantly when regularly used for at least 2 weeks. In conclusion, it was observed that hydrolyzed fish collagen obtained from a by-product of fish processing industries could be a sustainable and suitable material utilized in the production of safe and useful cosmeceutical products.

1. Introduction

Large quantities of Asian seabass skin are expelled as a by-product of aquaculture production in Thailand [1]. Effective management of this by-product is a major challenge faced by the marine food industries, as they are yet to find a solution to dispose of the large volumes of this by-product or waste. Therefore, many studies conducted in the last 10 years have focused on using Asian seabass skin as a raw material for the production of collagen, gelatin and hydrolyzed collagen (HC) [1,2,3,4,5,6]. Fish skin is usually composed of collagen type I [7], which is the same type of collagen found in human skin, making it beneficial for use in skin nourishing products. Furthermore, the collagen obtained from Asian seabass skin contains peptides of amino acids such as proline, hydroxyproline and glycine [3].

In recent times, it has been noted that HC is a promising source of biologically active peptides for nutritional, pharmaceutical and cosmeceutical applications. Peptides in HC, derived from by-products of fish processing, are safe natural compounds with high biological activities [1,4,5]. HC obtained from Asian seabass skin has a variety of beneficial properties such as good wound-healing and antioxidant activities, fibroblast proliferation activities and collagen synthesis enhancement [4,5,6]. HC also acts as a natural humectant and moisturizer for the skin, which can be useful in cosmetic skin care products [7]. As humans age, there is also a reduction in collagen content in the skin. However, using products containing HC can provide additional collagen to the skin, thereby helping the skin retain more collagen even with age. This is why collagen is one of the most preferred ingredients in anti-aging cosmetics or cosmeceutical products. Nonetheless, it is also important to determine the source of fish from which the HC is obtained, and the process of production before formulation development [8]. Even though the effectiveness of cosmeceutical products using collagen has been extensively studied, with results of approximately 35 papers available in the Scopus database [7], there have been no studies where HC obtained from Asian seabass skin was used as an ingredient in cosmeceutical products. Therefore, this study can be valuable in obtaining results and research data about cosmetic or cosmeceutical products made from HC obtained from Asian seabass skin. Furthermore, since HC was obtained from by-products of marine food processing industries, it is sustainable, and it could also address the problem of large quantities of by-products being expelled by the industries.

Serum is a formulation that is a concentrate of effective ingredients, and it has good skin absorption properties [9]. It is a liquid with low viscosity and is usually clear or slightly turbid in appearance. It is also popular in cosmetics or cosmeceutical marketing products because it provides high customer satisfaction. Moreover, it can also help significantly improve facial skin conditions by brightening the skin, reducing wrinkles, etc.

The primary aim of this study was to investigate the possibility of using HC obtained from a by-product (Asian seabass skin) of marine food processing industries as an effective ingredient in a cosmeceutical formulation, which could potentially become a commercial product. The serum was formulated using HC powder and other effective ingredients such as vitamin C, vitamin E and vitamin B3. Thereafter, the physicochemical properties, DPPH antioxidant activity and stability of the serum were determined. The formulation was tested on healthy human volunteers for the evaluation of the overall appearance of facial skin, skin roughness, wrinkles and skin brightening. The results before and after the product usage were compared, and the satisfaction of volunteers who used the product was also recorded.

2. Materials and Methods

2.1. Chemicals

HC from Asian seabass skin was provided by the laboratory of Professor Dr. Soottawat Benjakul, Prince of Songkla University, Thailand. Disodium ethylenediamine tetraacetate (disodium EDTA), vitamin E, vitamin B3, polysorbate 20, propylene glycol, phenoxyethanol, sodium hydroxide and sodium carboxymethyl cellulose (1500–2500 cps), L-ascorbic acid were purchased from P.C. Drug Center Co., Ltd., Bangkok, Thailand. Ascorbyl Glucoside (AA2G™, Stable Vitamin C) was purchased from Chanjao Longevity Co., Ltd. Bangkok, Thailand. Perfume was purchased from Thai-China Flavours and Fragrances Industry Co., Ltd., Nonthaburi, Thailand. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich (Schnelldorf, Germany). Other analytical chemical reagents and solvents were bought from Labscan Asia Co., Ltd. (Bangkok, Thailand).

2.2. Preparation of HC Powder of Asian Seabass Skin and Characterization

Preparation and characterization of HC powder was carried out according to the method mentioned by Benjakul et al. [1]. The first step was the pretreatment of the skin, in which the frozen fish skin was thawed by running tap water, after which the remaining meat and non-collagenous proteins were removed following the method of Sae-Leaw et al. [2]. Fish skin was soaked in alkaline solution (0.10 M NaOH) at the ratio of 1:10 (w/v) (skin per alkaline solution). The alkaline-treated skin was further neutralized by washing with a mild acid solution (0.01 M HCl) until a neutral pH of wash water was obtained. The second step was two-step hydrolysis process, in which citric acid pretreated skin was hydrolyzed using 3% papain at 40 °C for 3 h, followed by 2% alcalase at 50 °C for 2 h. In the final step, HC solution obtained from the previous step was further spray-dried to obtain HC powder [1]. The α-amino group content of HC was determined and expressed in terms of L-leucine [10]. Molecular weight distribution was determined using a Sephadex G-25 gel filtration column (2.5 × 50 cm) (17-0032-01, GE Healthcare Bio Science AB, Uppsala, Sweden). Additionally, some physicochemical properties such as antioxidant activity and solubility were also determined [1].

2.3. Preparation of HC Serum

The serum containing HC was formulated by simply mixing all the ingredients in water. Ingredients such as disodium EDTA, HC, vitamin C, vitamin E, vitamin B3, polysorbate 20, propylene glycol, phenoxyethanol and perfume was added into water and stirred continuously until a clear solution was obtained. Subsequently, sodium carboxymethyl cellulose was added into water to increase the viscosity of formulation, and 10% (w/v) sodium hydroxide was used to adjust the pH of formulation in the final step. All of the ingredients and the amount of ingredients used to prepare the HC serum are shown in

Table 1. List of ingredients and amount of ingredients used to prepare the HC serum.

Ingredients	Amount (% w/w)
HC	2.5
Vitamin C	0.3
Vitamin E	0.5
Vitamin B3	1
Sodium carboxymethyl cellulose	0.5
Disodium EDTA	0.1
Polysorbate 20	7
Propylene glycol	20
Phenoxyethanol	0.5
10% w/v Sodium hydroxide	0.5
Perfume	0.8
Water	66.3

.

2.4. Characterization of Physical Properties of HC Serum

A visual assessment of the HC serum was carried out in terms of its color and appearance. The formulation’s pH was measured using a pH meter (Mettler Toledo pH510, Mettler-Toledo GmbH, Urdorf, Switzerland), and viscosity was determined using Brookfield viscometer DV-III Ultra-rheometer with an LV spindle, Brookfield engineering Laboratories, Inc., Middleboro, MA, USA. Both pH and viscosity were measured in triplicate at 25 °C.

2.5. Antioxidant Activity of HC Serum by DPPH Radical Scavenging Assay

Antioxidant activity of the HC serum was measured by DPPH radical scavenging assay [11]. The serum was diluted in 50% ethanol at the ratio of 1:5, after which it was centrifuged at 12,000× g at 25 °C for 25 min. Then, 100 µL of supernatant of the formulation was added into 100 µL of DPPH solution and mixed, which was then kept in the dark for 30 min for full reaction. A microplate reader (SPECTROstarNano, BMG LABTECH Gmbh, Ortenberg, Germany) was used to measure the absorbance at 517 nm. L-ascorbic acid was used as the positive control. The % inhibition was determined with absorbance using the following equation:

% inhibition = [(A control − A sample)/(A control)] × 100

(1)

where A control is the absorbance of 50% ethanol mixed with DPPH solution; A sample is the absorbance of the sample solution mixed with DPPH solution.

2.6. Stability Study of HC Serum

The stability of the formulation was studied at room temperature (30 ± 2°C) for 6 months, and accelerated freeze–thaw cycle test conditions. The accelerated freeze–thaw cycle testing was carried out by storing the HC serum at 4 °C for 24 h and 45 °C for 24 h, which was considered one cycle. Physicochemical properties of HC serum in terms of appearance, color, pH, viscosity and antioxidant activity were evaluated before and after 6 months of room temperature conditions, and 6 cycles of accelerated freeze–thaw cycle testing [11].

2.7. Microbiological Testing of HC Serum

HC serum was tested for microbial contamination to ensure safety and stability of product [12,13]. The acceptance criterion for microbiological quality was that the colony-forming unit (CFU) of total microorganisms (bacteria, yeasts and molds) count in 1 g of product must be less than 1000 CFU. Furthermore, specified microorganisms (Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, Clostridium spp.) were not to be found in the product.

Total aerobic microbial count (TAMC) and total combined yeast and mold count (TYMC): Samples (10 g) were diluted in soybean-casein digest broth (Difco Laboratories, Inc., Sparks, NV, USA) (90 mL), and shaken vigorously for at least 30 min. 1 mL of each diluted sample was pipetted into soybean-casein digest agar plate (Difco Laboratories, Inc., Sparks, NV, USA) for calculating TAMC, and into Sabouraud dextrose agar (SDA) plate (Difco Laboratories, Inc., Sparks, NV, USA) for calculating TYMC. Thereafter, all soybean-casein digest agar plates and SDA plates were incubated at 30 to 35 °C for 3 to 5 days, and 20 to 25 °C for 5 for 7 days, respectively [13]. The bacterial colony was counted and reported as CFU/g.

Pseudomonas aeruginosa, Staphylococcus aureus: Samples (10 g) were diluted in soybean-casein digest broth (90 mL), and filtered through a sterile filter membrane (EMD Millipore Corporation, Burlington, VT, USA). The filter was then placed in 100 mL of soybean-casein digest broth and this mixture was incubated at 30 to 35 °C for 24 h. After the incubation period, a portion of the soybean-casein digest broth was streaked on the surface of two Cetrimide agar plates (Difco Laboratories, Inc., Sparks, NV, USA) for P. aeruginosa test, and was streaked on the surface of two Baird-Parker agar (BPA) plates (Difco Laboratories, Inc., Sparks, NV, USA) for S. aureus test. All plates were incubated at 30 to 35 °C for 18 to 72 h and they were checked for the presence of any colony [13].

Clostridium spp.: Samples (10 g) were diluted in thioglycollate medium (Difco Laboratories, Inc., Sparks, NV, USA) (90 mL), heated at 80 °C for one min, and then incubated at 30 to 35 °C for 48 h in a carbon dioxide incubator (Memmert Trading Co., Ltd., Shanghai, China). After the incubation period, samples were sub-cultured on the surface of reinforced clostridial agar medium plates under anaerobic conditions at 30 to 35 °C for 48 to 72 h, and they were checked for the presence of any colony [13].

Candida albicans: Samples (10 g) were diluted in 90 mL of Sabouraud dextrose broth (SDB), (Difco Laboratories, Inc., Sparks, NV, USA) and incubated at 30 to 35 °C for 3 to 5 days. After the incubation period, a portion of SDB was streaked on the surface of two SDA plates. All plates were incubated at 30 to 35 °C for 24 to 48 h, and they were checked for the presence of any colony [13].

2.8. Heavy Metals Testing of HC Serum

The analytical measurement of arsenic (As), Cadmium (Cd) and lead (Pb) in HC serum were determined by an Inductively Coupled Plasma-Mass Spectrometer (ICP-MS, Perkin Elmer, NexION2000, New York, NY, USA). Mercury (Hg) was determined by Direct Mercury Analyzer (NIC MA3000, Osaka, Japan). The test was conducted by Office of Scientific Instrument and Testing, Prince of Songkla University, Thailand, using certified stock solutions and optimized conditions, according to the recommendations of the manufacturer.

2.9. Facial Skin Evaluation of HC Serum

This study was approved by The Ethics Committee of Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand, before it was carried out. All volunteers signed informed consent for inclusion, before participating in the study. The clinical results and accompanying images used in this study were received with permission from the subjects who participated in the study. The criteria for inclusion were: 40 healthy male or female volunteers aged between 21 and 70 years, who are not participating in any other studies, willing to agree with the clinical testing protocols and sign the informed consent, and without the presence of any skin diseases. Furthermore, it was required of all the volunteers to not use any other facial skin care products on their faces, and female volunteers were not to be pregnant, or using birth control pills. Exclusion criteria were disappearance or discontinuation of volunteers, observance of moderate skin allergy or irritation, and having a history of allergies to natural ingredients or chemicals which are present in the HC serum formulation [11].

2.9.1. Skin Irritation Test

The HC serum was applied to the forearm area (size 2 × 2 inches) of subjects and observed for at least 24 h, to check for the presence of erythema, rash or swelling on the skin [11]. Thereafter, subjects were allowed to join facial skin evaluation test only if they passed the skin irritation test, without showing any symptoms of skin allergy or irritation.

2.9.2. Skin Testing by Visia Skin Analysis

The volunteers’ facial areas were cleaned, and the marked sites on the facial skin surface were measured using Visia skin analysis instrument (Guanzhou Newbelle Technology Co., Ltd., Guangdong, China) to find out various parameters such as skin roughness, wrinkles, spots, pores, sensitivity, UV spots and UV acne, and this record was set as a baseline of various parameters of volunteers on day zero. RGB and UV spectrum in Visia skin analysis were used to measure parameters such as amount of pigment, spots, facial acne, color, pores, wrinkles, etc. The volunteers participating in the study administered 1 mL of the serum containing HC on clean facial skin, and spread the product on the face evenly until it was absorbed into the skin. It was applied two times a day for 1 month. The facial skin of volunteers was checked using Visia skin analysis to obtain various parameters at 0, 2 and 4 weeks [11]. These parameters were summarized, and the values at baseline on day zero were compared with values at 2 and 4 weeks, to evaluate the effectiveness of HC serum.

2.9.3. Satisfaction Assessment Questionnaire

When the study period was concluded after 4 weeks, 10 questions were asked to the volunteers relevant to HC serum formulation about its appearance, color, texture, viscosity and odor. Furthermore, questions were also asked about the feeling of serum being absorbed into the skin, skin moisture, feeling of skin smoothness, firmness and overall preference. The levels of satisfaction were represented by scores: 5 (excellent), 4 (good), 3 (moderate), 2 (poor) and 1 (bad). The average scores were summarized and presented for the purpose of commercial product development and improvement in the future.

2.10. Data Analysis

All experiment data were gathered in triplicate, and expressed as mean ± standard deviation (SD). Student t-test was used to analyze the level of significance at 95% (p < 0.05).

3. Results

3.1. HC Powder of Asian Seabass Skin and Physicochemical Properties

The HC powder was grayish white in appearance, as shown in Figure 1. The α-amino group content of the HC was 2.00 ± 0.02 mmol/g sample. Peptide with a molecular weight of 4710 Da was the dominant peptide, while those having molecular weights of 11,820, 1610 and 220 Da were minor peptides. The solubility of the HC powder was in the range of 2.7–3.0 mg/mL in water, and solubility was at its highest when the pH of the water was around 7. The DPPH antioxidant activity of the HC powder was 0.73 ± 0.02 µmol trolox equivalents/g sample [1].

3.2. HC Serum and Physicochemical Properties

The serum containing HC was pale yellow and clear in appearance, as shown in Figure 2, and the pH, viscosity and DPPH antioxidant activity of the HC serum is exhibited in

Table 2. Physicochemical properties of HC serum.

pH	Viscosity (cps) *	DPPH (%)
7.77 ± 0.02	1333 ± 144	HC serum (100 µg/mL) 90.04 ± 1.00
		Ascorbic acid (10 µg/mL) 98.13 ± 0.74

* cps = centipoises, values are presented as mean ± SD (n = 3).

.

The pH of the HC serum was adjusted to approximately 7, with 10% w/v of sodium hydroxide, because the solubility of the HC powder was highest in water when the pH was around 7, as mentioned in the previous section. The serum formulation was prepared with 2.5% w/w of HC powder; therefore, having a suitable pH of water helps the HC powder to solubilize completely, and appear as a clear formulation. Other ingredients were also added to the serum such as polysorbate 20, which is a solubilizing agent that mixes oily ingredients (vitamin E and perfume) homogeneously, and phenoxyethanol, which is a preservative that keeps the formulation stable and safe from microbial contamination. The antioxidant activity of the HC serum was high, similar to the positive control (L-ascorbic acid), which indicated that the serum was effective as a cosmeceutical product.

3.3. Stability of HC Serum

The physicochemical properties of the HC serum under freeze–thaw cycle testing and room temperature conditions are shown in

Table 3. Physicochemical properties of HC serum under freeze–thaw cycle testing and room temperature conditions.

Physicochemical Properties	Freeze–Thaw Cycle	Room Temperature
Color	Clear pale yellow	Clear pale yellow
pH	7.68 ± 0.03 *	7.74 ± 0.04
Viscosity (cps)	1250 ± 72	1291 ± 0
% DPPH (HC serum 100 µg/mL)	86.51 ± 5.64	88.78 ± 0.95

Values are presented as mean ± SD (n = 3), * statistically significant (p < 0.05).

. The color of the HC serum formulation remained the same, but there were slight changes in the viscosity and pH. Furthermore, antioxidant activity was a bit lower when compared to freshly prepared serum. Freeze–thaw cycle testing for 6 cycles displayed more negative effects than storage at room temperature for 6 months. Therefore, the HC serum was considered a stable and effective cosmeceutical product when it was kept at temperatures not exceeding 45 °C.

3.4. Microbiological Amount in HC Serum

The microbial amount in HC serum was evaluated and the count of all microorganisms (bacteria, yeasts and molds) in the HC serum was found to be 50 CFU/g. Moreover, Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans and Clostridium spp. were not found in the HC serum, which indicated that the serum was safe to be used as a cosmetic product for the nourishment of facial skin.

3.5. Heavy Metals Amount in HC Serum

The only heavy metal found in the formulation was Arsenic (<0.125 ppm). Cd, Hg and Pb were not found. The results are shown in

Table 4. Heavy metals content in HC serum and criteria of safety of heavy metal content in cosmetic products.

Heavy Metals	MDL/LOQ (mg/kg) *	Amount (ppm)	Criteria (ppm)
Mercury (Hg)	0.001	Not found	≤ 1
Arsenic (As)	0.125	< 0.125	≤ 5
Cadmium (Cd)	0.01	Not found	≤ 3
Lead (Pb)	0.0425	Not found	≤ 20

* MDL/LOQ = the method detection limit/limit of quantification.

, which also displays the safety criteria for heavy metal content in cosmetic products [14]. Furthermore, the limit of detection for various metals is also shown in the table. Therefore, the HC serum was a safe cosmetic product according to the heavy metal content criteria.

3.6. Facial Skin Evaluation

The facial skin evaluation of all 40 volunteers (5 male and 35 female) was completed without any adverse effects occurring during the entire period of the study. The volunteers were categorized into the following age ranges: 21–30 (2%), 41–50 (27%), 51–60 (66%) and 61–70 (5%). The images and facial skin data of all the volunteers were captured and collected by Visia skin analysis on day 0 (baseline), and after using the HC serum for 2 weeks and 4 weeks. All the parameters such as wrinkles, skin roughness, spots, sensitivity, pores, UV spots and UV acne were represented as percentage values. A skin moisture analyzer was utilized to determine the moisture content in the skin. The skin parameters of all the volunteers at 0, 2 and 4 weeks after usage of the HC serum are represented in a horizontal bar chart as shown in Figure 3, where the values are expressed as mean ± SD. The results indicated a significant improvement in numerous skin parameters such as a decrease in pores, wrinkles and UV acne, and an increase in skin moisture at 2 and 4 weeks (p < 0.05). Moreover, skin parameters such as roughness, sensitivity, spots and UV spots did not change significantly compared to the baseline, even after using the HC serum for 4 weeks. A comprehensive look at some of the parameters shows that: skin moisture significantly increased from 35.00 ± 4.85% at baseline to 40.05 ± 5.01% and 39.85 ± 5.15% at 2 and 4 weeks, respectively; skin pores reduced from 68.15 ± 21.85% at baseline to 61.95 ± 20.26% and 62.50 ± 20.30% at 2 and 4 weeks, respectively; wrinkles reduced from 65.30 ± 12.82% at baseline to 60.45 ± 9.13 and 60.87 ± 9.01% at 2 and 4 weeks, respectively; UV acne reduced from 26.25 ± 16.05% at baseline to 21.02 ± 16.39% and 20.05 ± 16.35% at 2 and 4 weeks, respectively.

Facial skin images of the volunteers were captured before using the HC serum at the baseline profile (0 week), and after using the HC serum for 2 and 4 weeks, as shown in Figure 4. From these images, it could be noticed that there was an enhancement in skin brightness and wrinkle reduction at 2 and 4 weeks. The skin pores and UV acne of the volunteers were also measured by Visia skin analysis, as shown in Figure 5 and Figure 6, respectively. All the images clearly show a reduction in skin pores and UV acne from 2 weeks to 4 weeks after using HC serum. Furthermore, the HC serum was also able to increase skin moisture without any adverse effects or irritation in the volunteers. These results showed that the HC serum had great effectiveness as a cosmeceutical product, since a significant improvement in 4 parameters such as pores, wrinkles and UV acne, and skin moisture could be observed after 2 weeks of using the product.

3.7. Satisfaction Questionnaire of HC Serum from Volunteers

All the volunteers responded to 10 topics in the questionnaire on the HC serum which included appearance, texture, viscosity, color, odor, absorption, moisture, smoothness, firmness and overall satisfaction (Figure 7). The average scores of all topics were higher than 4.5, out of which moisture and overall satisfaction of product had the highest average score (4.85 ± 0.36). However, the viscosity of the HC serum received the lowest average score since the volunteers felt that the serum had a higher viscosity than their preference. Volunteers’ impressions indicated that they noticed a good improvement in their facial skin condition after using the product and, also, they were satisfied with the overall quality of the HC serum.

4. Discussion

Recently, marine food production industries have expelled large amounts of by-products such as the skin of Asian seabass fish, which has become available as raw materials for the production of collagen, gelatin and hydrolysate with bioactivities [1]. This problem of excessive amounts of by-products from industries could be solved in a sustainable way if HC powder was utilized in the development of more products. The idea of utilizing by-products from industries and agriculture as sustainable materials is of great importance, especially for the production of green cosmetic products. In this study, HC powder was produced from Asian seabass skin, and its properties were evaluated as a cosmetic ingredient. The color of HC powder was grayish white, which depended on the amount of pigment cells in the epidermal layer of fish skin [15]. In salmon skin, astaxanthin is the main pigment related to its red and orange color; similarly, melanin could cause the HC to be dark in color, in some types of fish skin [15,16]. HC powder was stable in terms of pH and temperature; it had low molecular weight, good solubility and high antioxidant activity, which makes it suitable as a cosmetic ingredient in various formulations for nourishment and anti-aging of the skin. However, one of the critical problems of HC powder from fish was its fishy odor, which is not considered suitable in cosmetic products. Therefore, pretreatment and a two-step hydrolysis process, followed by spray drying were necessary to obtain an odorless antioxidative HC powder which could be used in cosmetic products. This process was effective in the reduction of the fishy odor because of proper pretreatment and spray-drying at high temperature, in which the lipid oxidation products generated during production could be removed [1]. Furthermore, the good solubility of HC powder in water made it a promising ingredient for product development of serum formulation, which needs a solution that is concentrated and clear in appearance. The increase in solubility of HC was due to the decrease in molecular weight as evidenced by the presence of smaller polypeptide fragments in hydrolysate [17]. Additionally, a suitable pH of the formulation also enabled complete solubilization of the HC powder.

HC obtained from Asian seabass skin is used as an ingredient in cosmetics because it has moisturizing and skin-regenerating properties, including the ability to nourish skin and heal wounds [4,5,6,7,18]. The serum containing HC also includes other ingredients such as vitamin B3, vitamin E and vitamin C which could make it even more of an effective cosmetic product. The HC serum was good in appearance and had high antioxidant activity. It was also stable and safe to use on facial skin, with a pH of 7.7, which would not irritate the skin. There was no presence of heavy metals and microbial contamination in terms of product safety. All of these results were sufficient to indicate that the HC serum was effective and safe before its use in volunteers.

The results of the facial skin evaluation showed that significant facial skin improvements such as reduced pores, wrinkles and UV acne, and increased skin moisture were observed in volunteers from at least 2 weeks of product usage. All the ingredients in the formulation worked in tandem to help improve the skin, especially HC which had the ability to bind water, and maintain proper water content in the skin during the day [7]. This was reflected by the highest score given to skin moisture by the volunteers after using the HC serum. Furthermore, the water balance maintained in the skin due to the improved skin moisture may also be able to reduce the production of acne from UV stimulation. Vitamin B3 or niacinamide can improve the skin’s protective barrier by stimulating the synthesis of ceramides within the skin. Ceramides in the skin help strengthen the skin barrier by forming strong bonds between the cells. It can also reduce fine lines, wrinkles and hyperpigmented spots in humans, which also corresponds to the results of the facial skin evaluation in this study [19,20]. Vitamin E is one of the ingredients which increases collagen production and protects the skin from various detrimental effects of solar radiation [21]. Vitamin C is also essential for collagen biosynthesis, and the topical use of vitamin C could increase collagen production in young as well as aged human skin, as shown in clinical studies [22]. However, vitamin C is not a stable compound which can make the product dark in color, because of oxidation. Therefore, in this study, a stable version of vitamin C known as ascorbyl glucoside was used to formulate a stable and effective cosmetic product.

5. Conclusions

This study utilized the by-products of the marine food industry, specifically HC powder derived from Asian seabass skin, to create a cosmeceutical product that is safe, stable and beneficial to users. The HC powder used as a raw material was produced by proper processes, and was ensured to have the required quality for use in cosmetic products. The facial skin evaluation of volunteers after using the HC serum presented positive results, which showed significant improvements to some of the facial skin parameters. Furthermore, the volunteers rated the HC serum with high scores, which indicated that the product was able to satisfy the users. In conclusion, the HC serum formulated in this study was proven to be effective as seen in the results, and it has great potential in becoming a commercial cosmeceutical product that can be used for anti-aging and skin lightening.