Cera- Glow, ferment lysates of Lacticaseibacillus rhamnosus IDCC 3201, improves skin barrier function in clinical study

Background: Ceramides are essential lipids in stratum corneum for skin permeability barrier function in that they retain the skin moisture and protect from the inva-sion of foreign pathogens. Previously, we demonstrated that ferment lysates of Lacticaseibacillus rhamnosus IDCC 3201 enhanced ceramide production in human epidermal keratinocytes. Furthermore, for comprehensive knowledge of this effect, in vitro experiments and multi- omics analysis were conducted to explore the underlying mechanisms. Aims: This study was designed to identify whether a cosmetic sample (i.e., Cera- Glow) containing the lysates improves the skin


| INTRODUC TI ON
Probiotics, meaning "for life" as defined by Lilly and Stillwell in 1965, were recently defined as "live microorganisms which when administered in adequate amounts confer a health benefit on the host" by the Food and Agriculture Organization of the United Nations/ World Health According to the Organization (FAO/WHO). 1,2 The genera Lactobacilli (i.e., Lactiplantibacillus, Limosilactibacillus, and Lacticaseibacillus) and Bifidobacteria are the major well-known probiotics. 3 Probiotics, as form of a food supplement, exhibit a variety of health benefits, including improving gut health, enhancing the immune system, controlling serum cholesterol, anti-enteropathgenic compound secretion, reducing the risk of colon cancer and symptoms of lactose tolerance, and controlling blood pressure. [4][5][6][7][8][9][10] Recently, increasing interest in probiotics has accelerated the use of ferment lysates of probiotics (e.g., Lactobacillus ferment lysate) as a new cosmetic ingredient to improve the skin barrier function and enhance the appearance of the skin through brightening and antiaging. 11 Particularly, the skin barrier, the stratum corneum (outermost layer) plays an important role in maintaining skin moisture and protecting the skin from external stresses. 12 Specifically, ceramides, the main polar lipids of stratum corneum polar lipids together with cholesterol and saturated fatty acids, create a water-impermeable skin barrier to prevent excessive water loss. 12 Thus, ceramide deficiency is often observed in the skin of patients with psoriasis vulgaris and atopic dermatitis. 13 Previously, we reported that ferment lysates of Lacticaseibacillus rhamnosus IDCC 3201 enhance ceramide production in human epidermal keratinocytes through enhanced sphingomyelinase activity. 14 In this study, we evaluated a Cera-Glow whether it would improve the skin barrier function in clinical study. To examine the skin barrier function of Cera-Glow, transepidermal water loss and skin hydration values were measured for 5 days in 24 clinical subjects and for 4 weeks in 21 clinical subjects, respectively.

| Preparation of Cera-Glow as a cream
Lacticaseibacillus rhamnosus IDCC 3201 (ATCC-BAA-2836) isolated from the feces of breast-fed infants was cultured in commercial broth anaerobically at 37°C in a 1 ton fermentor. The bacterial cells from the culture were collected using centrifugation at 9200 x g (1736R; LaboGene) and freeze-dried. The dried cells were suspended in deionized water and disrupted using a high-pressure homogenizer (MN400BF; Micronox) at 4°C. Then, the lysates were collected by centrifugation and filtration through membrane with pore size of 0.22 μm. Finally, the Cera-Glow as a cream was mixed with the lysates and formulated ingredients at 80°C for 10 min with a homogenizer (T.K Homo Mixer Mark II 2.5; PRIMIX), followed by cooling the mixture to 45°C for 10 min.

| Subjects
Twenty-four and 21 healthy women, who were aged between 20 and 60 years, were enrolled for the study regarding improvement of skin barrier damage and skin hydration, respectively ( Table 1). These subjects, with a healthy skin condition, had no acute or chronic physical illnesses, including skin diseases. The exclusion criteria included subjects with allergic or sensitive skin, those taking drugs to treat skin diseases, and those who received dermatological treatment at the application site within 3 months before the start of the study.
Participants who were pregnant, breast-feeding, or did not meet the quality requirements for this experiment were also excluded. Before enrollment in the study, each participant signed a written informed consent form.

| Study design
Overall experimental design of clinical trials was illustrated in Figure 1.
This study was conducted as a randomized, intra-group comparative, investigator-blinded clinical trial between April and May 2022. A total of 24 women with an average age of 45.46 ± 9.78 years participated in the study for improvement of skin barrier damage, whereas a total of 21 women with an average age of 50.33 ± 5.74 years participated in the study for skin hydration ( Figure 1).
The use of cosmetic products was prohibited for 12 h before the visit. On the day of the visit, the subjects took a shower without using soap and sanitizer, only used a skin cleanser provided by the

| Measurement of transepidermal water loss
Transepidermal water loss is used to assess the skin barrier and is directly associated with the moisture content of the stratum corneum. TEWL was evaluated using a Tewameter (TM Hex, Courage + Khazaka electronic) before the application of the patch, after patch removal (Day 0, baseline), and at 1, 2, and 5 days after Cera-Glow sample application. The TEWL value was expressed as an average of five repetitions, and its measurement unit was g/h/ m 2 . The TEWL improvement rate (%) refers to the recovery rate of the damaged area and was calculated using the following equation: The application sites were photographed under the same brightness condition using a digital SLR camera.

| Evaluation of skin hydration using Epsilon
The water content in the cheek skin was measured using the After applying the Cera-Glow sample, the participants responded to a self-assessment questionnaire. The questionnaire consisted questions related to four attributes: abnormal responses, satisfaction with product, efficacy of product, and its comparison with other products.

| Assessment of the degree of irritation in subjects
The subjects were monitored for abnormal skin reactions, and a subjective assessment of the degree of irritation was performed at each visit (data not shown). The irritation symptoms included erythema, itching, stinging, burning, edema, dead skin cells, and blisters. The clinical scoring of irritation ranged from 0 to 4 (0 = none, 1 = weak, 2 = moderate, 3 = strong, and 4 = very strong).

| Evaluation of adverse reactions by researchers and dermatologists
Adverse reactions of the subjects were evaluated by researchers and dermatologists at each visit. The types of adverse reactions, including Baseline TEWL − TEWL at 1, 2, and 5 days after sample application Baseline TEWL − TEWL before the application of patch × 100 PV after sample application − PV before sample application PV before sample application × 100 F I G U R E 1 Schematic diagram of the clinical design to evaluate the Cera-Glow for skin barrier function. [Correction added on 14th April 2023, after first online publication: Figure 1 caption has been updated in this version.] erythema, edema, stinging, itching, prickling, burning, tightness, and blisters, were evaluated following a scoring system: 0 = no reaction; 1 = weak reaction; 2 = moderate reaction; and 3 = strong reaction.

| Improvement of skin barrier function
To investigate the effect of Cera-Glow on skin barrier function, the forearm skin of the subject was exposed to 40 μl of 1% (w/v) sodium lauryl sulfate (SLS) for 1 day, and Cera-Glow was applied to the damaged skin for 5 days. A total of 24 females with average age  the results showed that the activity of the sample was maintained for 4 weeks, which were the period of the clinical trial. Meanwhile, F I G U R E 2 Assessment of transepidermal water loss before the application of SLS-patch, after the patch removal (day 0, baseline), and at 1, 2, and 5 days after Cera-Glow sample application (*p < 0.05, **p < 0.01, ***p < 0.001).

| Skin moisturizing effect
adverse events (e.g., erythema, edema, itching, stinging, burning, and tightness) were not observed in Cera-Glow treated participants during the clinical trials.

| Subjective assessment through questionnaire
To determine overall satisfaction with Cera-Glow, a questionnaire was provided to the subjects ( Figure 4A). The results indicated that 90.5% of volunteers were satisfied with improved skin hydration after Cera-Glow application ( Figure 4B), and 76.2% were satisfied with the effectiveness of Cera-Glow, compared to the product they used ( Figure 4C). Meanwhile, adverse events (e.g., erythema, edema, itching, stinging, burning, and tightness) caused by Cera-Glow were not detected during the clinical trials, and no specific adverse skin conditions were recorded in the volunteers' feedback.

| DISCUSS ION
Here, we showed that Cera-Glow, ferment lysate of Lacticaseibacillus rhamnosus IDCC 3201, improved skin barrier function through a significant reduction in TEWL and improved skin moisture content in F I G U R E 3 Evaluation of the permittivity of the cheek skin before, 2 weeks after, and 4 weeks after application of Cera-Glow using the Epsilon (***p < 0.001).

F I G U R E 4
Overall assessment of Cera-Glow, ferment lysates of Lacticaseibacillus rhamnosus IDCC 3201, through questionnaire. (A) Overall satisfaction, (B) skin hydration improvement, and (C) effectiveness of Cera-Glow compared to the product they used.
the treatment group compared with that in the untreated group. This could be the result of Cera-Glow-induced enhanced ceramide production in the skin, as observed in human epidermal keratinocytes in vitro. 14 Consistent with the results in this study, direct ceramide application to the skin has improved skin barrier function in many clinical trials. 15 The stratum corneum is mainly composed of corneocytes and a lipid matrix that includes cholesterol, free fatty acids, and ceramides. Ceramides are main component, ranging from 30% to 40% (w/w) of lipid matrix. 16 The well-ordered alignment of the lipid matrix forms a closed system, resulting in the reduction of the TEWL. 17 Therefore, the hydrated stratum corneum enables corneocytes to maintain enzymatic reactions and allows lipid matrix to increase plasticity and flexibility. 17 Thus, adequate ceramide synthesis in the stratum corneum typically leads to skin moisturization and reduces skin problems such as ringworm, 18 rosacea, and eczema. 16 Clinical evidences have established the correlation between ceramide deficiency, temporal skin disorders, and skin diseases. For example, a significant decrease in the total ceramide content was observed in the skin lesion such as atopic dermatitis. 13,19 Cosmetic ingredients with skin-barrier reinforcement properties have been explored in recent decades. The basic mechanisms of their effects include lipid synthesis (e.g., ceramides) and penetration into the skin as a component of the stratum corneum. For instance, the leaf extract of Aloe vera significantly improved skin hydration by seeping into the skin, and 3-(trihydroxygermyl) propionic acid increased ceramide levels in a three-dimensional human epidermal model. 20,21 The major resources actively inducing skin barrier improvement are mainly categorized into (1) plant oils (e.g., coconut oil), which act as a protective barrier to the skin by exerting occlusive effect, (2) plant extracts (e.g., Pinus pianster), which contain bioactive compounds such as hyaluronic acid, and (3) animal-derived materials (e.g., collagen and milk). 22,23 Recently, as indicated in this study, probiotics have been recognized as novel cosmetic materials based on scientific evidences.
The human skin microbiome is composed of a random combination of various microorganisms and the skin environment. 24 Recent studies have suggested that beneficial skin microbiome play an important role in maintaining healthy skin. 11 Probiotic bacterium is considered a microbe that is beneficial for skin health, and the beneficial effects of probiotics are related to their anti-inflammatory property, anti-oxidative function, and metabolites (i.e., vitamins, amino acids, minerals, and organic acids) secreted by them. 25 In this study, we found that L. rhamnosus IDCC 3201 produces 22 major metabolites including amino acids and organic compounds by metabolic analysis ( Table 2). Among the metabolites identified, direct

TA B L E 2 Metabolites produced by Lacticaseibacillus rhamnosus IDCC 3201
No.

Metabolite Function Reference
1 Acetic acid Alleviating the inflammatory response, the wound, and atopic dermatitis [28,29] 2 Alanine Inhibition of apoptosis; stimulation of lymphocyte proliferation [30] 3 Aucubin Inhibition of inflammatory response, protection against oxidative stress, and improvement of transepidermal water loss and skin hydration [26,31] 4 Glutamic acid Skin and wound regeneration [32] 5 Glycine Collagen production [33] 6 Isoleucine Improvement of atopic dermatitis [34] 7 Lactic acid Modulation of the inflammatory immune response [35] 8 Leucine Regulation of immune responses [36] 9 Malic acid Improvement of anti-oxidant capacity, hematological parameters, and expression of immune-related genes [37] 10 Methionine Increase of immune function [38] 11 Palmitic acid Moisturizing, regenerating and restructuring properties, which promotes the integrity of the epidermal cell wall and regulates epidermal hydration [39] 12
However, several bioactive compounds such as aucubin, palmitic acid, stearic acid, and threonine are effective in decreasing TEWL and can increase skin hydration. 26 As these metabolites were also detected to show anti-inflammation and anti-oxidant effects, they were thought to indirectly help improve the skin barrier.

ACK N OWLED G M ENTS
We thank Ellead Skin & Bio Research for the clinical tests. This work was supported by Ildong Bioscience and ICBIO.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available on request from the authors.