Effect of oral intake of heat-killed Fructobacillus fructosus OS-1010 on human skin: a randomized, double-blind, placebo-controlled, parallel-group study

Abstract

Fructobacillus spp. comprise a large genus of fructophilic lactic acid bacteria whose growth characteristics differ from those of other common lactic acid bacteria. The genus has been extensively investigated microbiologically and phylogenetically. However, knowledge regarding Fructobacillus nutritional benefits remains unclear. In particular, Fructobacillus fructosus OS-1010 (OS-1010) was recently shown to act on intestinal cells to release extracellular vesicle exosomes that act on distant target cells. The released exosomes reportedly enhanced the expression of longevity-associated genes and intracellular mitochondrial activity in muscle cells. OS-1010 is expected to be a functional ingredient that improves the function of distant tissues such as muscles and skin upon oral intake. This study examined the effects of the oral intake of heat-killed OS-1010 on human skin in a randomized, double-blind, placebo-controlled, parallel-group study of healthy participants. Significant improvement in skin elasticity was observed after eight weeks of oral OS-1010 intake. Furthermore, although no significant difference was found between the two groups in a wrinkle-related parameter, the OS-1010 group demonstrated improvements in the percentage wrinkle area and overall average wrinkle depth of crow’s feet, which were not observed in the placebo group. These results indicate that OS-1010 can contribute to the improvement in skin conditions.

INTRODUCTION

Lactic acid bacteria (LAB) are a well-known group of microorganisms that exist in diverse environments, such as soil, plant surfaces, and animal intestines; moreover, they are among the most common microorganisms in the human diet. Indeed, humans have orally ingested various types of LAB through natural and traditional fermented foods for the longest time. Numerous studies have revealed a wide range of functional properties of LAB, which are widely available as probiotics for medical and commercial use. LAB that have been used or found to be effective as probiotics belong to the genera Lactobacillus, Streptococcus, and Enterococcus and are highly diverse [1]. Probiotics are generally live LAB; however, it has recently been reported that heat-killed LAB also have health benefits [2], which has garnered their functionality much interest in the food industry.

One of the benefits of LAB is their skincare effects, including skin-whitening, moisturizing, and anti-wrinkling effects [3]. Recent studies have shown that some LAB have various health effects on the skin of mice and humans when ingested orally [4,5,6,7]. The skin is the outermost organ of the human body. It plays a vital role in the life activities of all animals by preventing foreign substances from entering the internal environment, thus acting as a barrier to protect the body from injury by adverse physical stimuli (e.g., sub/supra-optimal levels of temperature, humidity, and/or sunlight), and functioning as a sensory organ. Furthermore, the skin can be highly suggestive of the healthy/unwell appearance of a person and, as it is generally accepted that a healthy, youthful appearance can affect the psychological well-being and social behavior of an individual. Therefore, consumer demand for cosmetics and probiotics that are beneficial to the skin is on the rise.

Fructophilic lactic acid bacteria are a relatively recently recognized subgroup of LAB, consisting primarily of Fructobacillus spp., which have unique properties, prefer fructose, and are widely distributed in fructose-rich environments, such as flowers and fruits [8, 9]. Additionally, Fructobacillus spp. are present in fermented foods prepared from fructose-rich fruits. For example, they have been isolated from taberna, a traditional Mexican alcoholic beverage; tempoyak, a Malaysian condiment; wine; fresh honey; bee pollen; and bee bread [10,11,12,13,14]. Fructobacillus spp. possess an incomplete gene encoding alcohol/acetaldehyde dehydrogenase, which results in an imbalance in nicotinamide adenine dinucleotide (NAD⁺)/ NAD⁺ hydrogen (NADH) levels and specific growth characteristics [15]. Additionally, these species lack several sugar metabolism-related genes that are essential for the metabolism of sugars utilized by other common LAB, limiting the variety of available sugars. Thus, Fructobacillus spp. are unique LAB significantly differing in both phylogeny and growth characteristics from other well-studied LAB. Recently, a unique feature of Fructobacillus spp. was identified under anaerobic and fructose-rich conditions: they accumulated high concentrations of NAD⁺ and its precursor, nicotinamide mononucleotide [16]. Although antibacterial and anti-inflammatory effects, which other LAB are known for, have also been demonstrated in vitro for Fructobacillus spp. [17], reports on their functionality in vivo are scarce at best. Similarly, there are only a few reports regarding their effects on intestinal regulation, the immune system, and the skin.

Intestinal epithelial cells treated with the Fructobacillus fructosus OS-1010 strain (OS-1010) were recently reported to secrete exosomes, also known as extracellular vesicles, which activated mitochondria in muscle cells [18]. Mitochondria are dynamic organelles in eukaryotic cells. They are surrounded by two membranes and are essential for energy production and cellular metabolism. Recent studies have shown that mitochondria play key roles in skin homeostasis, cancer, inflammatory diseases, and rare genetic disorders [19, 20]. Here, we hypothesized that the oral intake of OS-1010 would have beneficial effects on skin health. This is the first report on the effects of Fructobacillus spp. on human health at the clinical level.

MATERIALS AND METHODS

Ethics

The study protocol complied with the principles of the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Participants of the Ministry of Health, Labour and Welfare, Japan. The protocol was approved by the Ethical Review Committee of Miura Clinic, Medical Corporation Kanonkai (Osaka, Japan), on September 15, 2022. The study was registered under the ID No. UMIN000049133 and was conducted between October and December 2022 at Miura Clinic, Medical Corporation Kanonkai. Skin measurements were performed at Maruishilabo Corporation (Osaka, Japan). This study was commissioned by Osaka Soda Co., Ltd. (Osaka, Japan) to Oneness Support Co., Ltd. (Osaka, Japan), which cooperated as a contract research organization and was responsible for recruiting the participants.

Participants

Sixty male and female participants that met the inclusion criteria were included in this study. The selection criteria were as follows: (1) healthy individuals aged between 30 and 59 years at the time of obtaining consent; (2) individuals who were concerned about dry and rough skin; and (3) individuals who had received an adequate explanation of the purpose and content of the study, had the capacity to provide consent, and volunteered to participate in the research based on their own free will. The exclusion criteria were as follows: (1) individuals suffering any heart, liver, or kidney disease (including complications from other disease); (2) individuals with a history of cardiovascular disease; (3) individuals with diabetes mellitus; (4) individuals who were undergoing medical treatment for a diagnosed medical condition; (5) individuals with allergies to food or medicine; (6) women who wished to become pregnant, were pregnant (including possible pregnancy), or were lactating; (7) individuals that were intense athletes and on an extreme diet; (8) individuals with severely irregular eating habits; (9) individuals who may be exposed to excessive ultraviolet irradiation during the test period; (10) individuals with skin problems, such as inflammation or eczema, in the measurement area (face or arms); (11) individuals who visited beauty salons for skin care; (12) individuals who did not consume health foods (including foods for specified health uses and foods with functional indications) or designated health foods during the study period; (13) individuals who drank an average of 60 g or more of pure alcohol per day; (14) individuals who smoked an average of 21 or more cigarettes a day; (15) individuals who were participating or would be participating in other clinical trials at the start of this study; and (16) individuals whom the principal investigator or sub-investigator determined to be unsuitable for this study.

Preparation of the heat-killed OS-1010-containing test tablets and placebo tablets

The test tablets comprised a mixture of heat-killed OS-1010 (Osaka Soda Co., Ltd., Osaka, Japan), dextrin, maltose, lactose, powdered cellulose, sucrose fatty acid esters, and fine silicon dioxide. Each test tablet contained 3 mg (approximately 1.2 × 10¹⁰ cells) of heat-killed OS-1010. The placebo tablet was prepared by replacing the OS-1010 in the test tablet with dextrin.

Clinical study design

This was a randomized, double-blind, placebo-controlled, parallel-group study. Before starting the intervention, potential participants who provided written informed consent underwent a screening test that included physical measurements (i.e., height, weight, body mass index, blood pressure, and pulse rate), a lifestyle questionnaire, a medical interview (to confirm their physical condition), various skin measurements, and a skin condition questionnaire. Skin measurements were performed after at least 20 min of acclimation in an environment with a constant temperature and humidity (temperature, 20°C ± 1°C; humidity, 50% ± 5%). The principal investigator conducted participant selection according to the selection and exclusion criteria detailed above. Moreover, the allocation manager, who was not involved in the study, created a randomized list and randomly assigned the participants to two groups, an OS-1010- and a placebo-treated group, using age, sex, skin elasticity (R7: biological elasticity), stratum corneum hydration as adjustment factors in the pre-intake measurement. The randomized list was sealed by the allocation manager and kept in a sealed container until it was opened. Blinding was maintained for the principal investigator, participants, and all other staff involved in the study.

The OS-1010- and placebo-treatment groups were administered the test and placebo tablet, respectively, one tablet once daily with water or lukewarm water for a period of eight weeks. Eight weeks after treatments started, the participants visited the clinic and underwent prescribed physical measurements and various skin measurements. Additionally, they were asked to respond to a skin condition questionnaire. Moreover, prior to conducting the study, the investigators instructed all participants not to violate the following restrictions and prohibitions: during the study period, the participants would not (1) make any significant lifestyle changes, (2) change the cosmetics they used, (3) visit a beauty salon for skin care, and (4) perspire or consume high-fat or oily food on the day before and day of skin measurements.

Measurements

The primary efficacy outcome measures included R7, the stratum corneum hydration, and transepidermal water loss (TEWL). In turn, the secondary outcome measures included skin elasticity (R0: flexibility), skin color values, crow’s feet, and a skin condition questionnaire (visual analog scale, VAS). Skin elasticity, stratum corneum hydration, TEWL, and skin color were measured five times, and the average values were used.

Skin elasticity was measured on the cheeks using a Cutometer MPA 580 (Courage + Khazaka, Cologne, Germany), and R0 and R7 were determined. The stratum corneum hydration was measured on the inner forearm using a Corneometer CM 825 (Courage + Khazaka). TEWL was measured on the inner forearm using a Tewameter TM HEX (Courage + Khazaka). The sebum levels were measured on the forehead using a Sebumeter SM 815 (Courage + Khazaka). Skin pH was measured on the cheeks using a Skin-pH-Meter PH 905 (Courage + Khazaka). Skin color values were measured on the inner forearm using a CM-26d spectrophotometer (Konica Minolta, Tokyo, Japan) to determine melanin and erythema indices. Meanwhile, crow’s feet were measured using the 3D skin measuring device PRIMOS CR (Canfield Scientific, Parsippany, NJ, USA). Moreover, the accompanying software was used to analyze wrinkle parameters, including percentage wrinkle area, overall average wrinkle depth, average maximum wrinkle depth, and maximum largest wrinkle depth. A VAS was used to evaluate skin moisture, elasticity, luster, wrinkles, and blemishes. For skin moisture, elasticity, and luster, the left end was set to “feel very much” and the right end was set to “do not feel at all”. For skin wrinkles and blemishes, the left end was set to “do not care at all”, and the right end was set to “care very much”. The respondents were asked to indicate where their current condition was located on a 100-mm line segment, and the length from the left end was measured and used for the evaluation. Measurements were obtained at week 0 of the screening test and at week 8 after the start of treatment.

Statistical analysis

The analysis population was based on the intention-to-treat (ITT) principle [21], and missing data were imputed using the baseline observation carried forward (BOCF) approach.

The statistical significance of differences between the OS-1010 and placebo groups for each baseline value were analyzed using the unpaired t-test and for each endpoint were analyzed using analysis of covariance (ANCOVA) with age and baseline skin parameters as covariates. A paired t-test was used for within-group comparisons at weeks 0 and 8. The significance level for all tests (two-tailed) was set at 0.05, and statistical analyses were performed using the IBM SPSS Statistics 28 software (IBM Corp., Armonk, NY, USA).

RESULTS

Participants and their characteristics

The study began with 60 participants without any dropouts after randomization. During the study period, one patient in the placebo group dropped out of the study because of the discontinuation criteria (failure to come to the hospital on the day of the investigation for reasons unrelated to the study), bringing the total number of participants completing the study to 59. Figure 1 illustrates a flow diagram of the study, including the selection of participants and allocation of tests for the analysis. Table 1 presents the characteristics of the participants.

Fig. 1.

Flow diagram for this study.

A total of 90 individuals were registered and assessed for eligibility; 60 participants participated in the study.

Table 1. Baseline characteristics of the participants for efficacy analysis.

Characteristic	OS-1010 (n=30)	Placebo (n=30)	p-value
Sex (male/female)	8/22	9/21	-
Age (years)	51.9 ± 6.1	52.0 ± 7.6	0.855
Height (cm)	161.8 ± 5.9	162.4 ± 8.3	0.767
Body weight (kg)	57.0 ± 10.3	55.8 ± 9.5	0.620
BMI (kg/m2)	21.7 ± 3.4	21.1 ± 2.8	0.442
Systolic blood pressure (mmHg)	121.3 ± 14.3	125.2 ± 13.4	0.216
Diastolic blood pressure (mmHg)	78.3 ± 11.8	79.9 ± 11.5	0.505
Pulse rate (bpm)	72.9 ± 8.3	75.5 ± 10.9	0.321
Skin pH	6.6 ± 0.3	6.4 ± 0.3	0.104
Skin sebum	23.8 ± 19.3	27.5 ± 21.2	0.487

Data are expressed as mean ± standard deviation (SD). p-values were determined by unpaired t-tests. BMI: body mass index.

Effects of OS-1010 on primary outcomes

The analysis regarding the primary outcome items is shown in Fig. 2. Figure 2A shows that the R7 value of the OS-1010 group was significantly higher than that of the placebo group, indicating improved skin elasticity (p=0.044). No significant differences were observed for stratum corneum hydration and TEWL (Fig. 2B, 2C).

Fig. 2.

Effects of OS-1010 intake on R7, stratum corneum hydration, and transepidermal water loss (TEWL).

Dot plot diagram of R7 (A), stratum corneum hydration (B), and TEWL (C) before and after the intervention in the OS-1010 and placebo groups. The horizontal bars show the average of each case. The average, standard deviation (SD), and p-value determined by the statistical analysis for each group at 0 weeks and 8 weeks are summarized in the table below the plot area.

Effects of OS-1010 on secondary outcomes

The analysis regarding the secondary outcome items is summarized in Table 2. No significant differences were observed between the OS-1010 group and the placebo group for all items (Table 2). Meanwhile, significant changes in the items R0, melanin index, and erythema index were observed in the OS-1010 group at week 8 compared with the baseline; however, similar changes were also observed in the placebo group. In contrast, the percentage wrinkle area and overall average wrinkle depth decreased significantly during treatment with OS-1010 group (percentage wrinkle area, p=0.004; overall average wrinkle depth, p=0.015), whereas no statistically significant changes were observed in the placebo group. The VAS questionnaire results indicated that both the OS-1010 and placebo groups showed significant improvement in all items, compared with the baseline measurements. However, no significant differences were observed between the OS-1010 and placebo groups (data not shown).

Table 2. Effects of OS-1010 intake on secondary outcomes of skin parameters.

Item	Group	0 weeks	8 weeks	p1-value	p2-value
R0 (mm)	OS-1010	0.302 ± 0.048	0.356 ± 0.070	<0.001	0.536
	Placebo	0.317 ± 0.050	0.350 ± 0.057	0.001
Melanin index	OS-1010	184.1 ± 55.4	165.7 ± 44.5	<0.001	0.098
	Placebo	187.5 ± 67.3	162.4 ± 48.1	<0.001
Erythema index	OS-1010	252.0 ± 70.6	231.5 ± 57.5	<0.001	0.667
	Placebo	260.9 ± 68.1	228.9 ± 59.7	<0.001
Percentage wrinkle area (%)	OS-1010	47.2 ± 17.2	41.8 ± 14.1	0.004	0.101
	Placebo	48.1 ± 16.0	46.3 ± 15.3	0.422
Overall average wrinkle depth (μm)	OS-1010	64.1 ± 10.4	61.2 ± 9.2	0.015	0.285
	Placebo	62.3 ± 10.6	61.9 ± 9.8	0.788
Average maximum wrinkle depth (μm)	OS-1010	69.6 ± 13.5	66.5 ± 10.9	0.111	0.526
	Placebo	66.3 ± 10.6	66.1 ± 9.3	0.906
Maximum largest wrinkle depth (μm)	OS-1010	191.5 ± 67.6	194.2 ± 73.2	0.754	0.566
	Placebo	185.8 ± 63.3	183.3 ± 57.3	0.780

Data are expressed as mean ± standard deviation (SD). p-values below 0.05 are printed in bold.

p¹-values indicate within-group comparisons by paired t-test.

p²-values indicate comparisons between the two groups at 8 weeks by analysis of covariance (ANCOVA).

Adverse events

Adverse events occurred in 5 participants in OS-1010 group and 2 participants in the placebo group during the study period (Table 3). The principal investigator concluded that all adverse events were unrelated to the intake of the test tablets.

Table 3. Adverse events observed in study participants (SAS).

Event	OS-1010 (n=30)	Placebo (n=29)
Adverse events
Headache	3	2
Diarrhea	2	0
Nausea	1	1
Runny nose	1	1
Sneezing	1	0
Fatigue	1	0
Sore throat	1	0
Skin rash (back of the hand)	1	0
Stiff shoulders	1	0
Backache	1	0
Serious adverse events
	0	0

All values present patient numbers.

The adverse effects associated with injection of SARS-CoV-2 vaccine were excluded.

SAS: safety analysis set.

DISCUSSION

Previous studies have reported a relationship between LAB intake and skin appearance, with LAB intake reportedly showing anti-photoaging, moisturizing, and anti-wrinkle effects, all of which are thought to be due to the probiotic effects of LAB [5, 7, 22, 23]. However, there are no previous reports on the effects of Fructobacillus spp. on the skin. In this randomized, double-blind, placebo-controlled, parallel-group study, we investigated whether the oral intake of OS-1010 for eight weeks improved the condition of healthy adults with dry or rough skin.

Skin elasticity analysis revealed that the OS-1010 intake group showed a significantly improved R7 value compared with the placebo group. The R7 value is the ratio of immediate elastic recovery to the total deformation and is one of the most practical parameters in evaluating skin elasticity. Indeed, the value of this ratio negatively correlates with age [24, 25] and is recommended for evaluation of aging effects [26]. Therefore, it is mainly the structural environment of the dermis that influences the R7 value, specifically the collagen and elastic fibers in the dermis. Indeed, it has been shown that aging skin has reduced thickness, disorganized elastin fibers, and a reduced number of collagen bundles [27,28,29], all of which are collectively considered one major cause of reduced skin elasticity. From this perspective, the improvement in biological elasticity is likely due to the improvement in the dermal environment caused by the increased production of elastin and collagen in the dermis. Although no significant intergroup differences were observed with respect to wrinkle-related parameters, a statistically significant reduction in the percentage area of wrinkles and overall average wrinkle depth of crow’s feet was noted in the OS-1010 group in comparison with the baseline values, whereas such reductions were not observed in the placebo group. Consistently, a negative correlation between wrinkle depth and dermal thickness beneath the wrinkles has been reported [30]. The loss of skin elasticity associated with elastic fiber deterioration disrupts the structure of the dermis, causing wrinkle formation. The improvement in percentage wrinkle area and overall average wrinkle depth observed in this study was likely due to an improvement in the environment of the dermal layer by the same mechanism that improved the skin biological elasticity.

The significant improvement of the skin observed upon OS-1010 intake appears to be entirely related to changes in the dermal environment. Given that elastin and collagen are the dominant components of the dermal layer, contributing to its structural properties, and that these components have been well established to be tightly related to skin elasticity and wrinkles [31], it was therefore anticipated that the observed improvements would be due to the structural and environmental improvement resulting from the enrichment of elastin and collagen in the dermal layer. In the dermis, fibroblasts are responsible for the production of extracellular matrices such as elastin and collagen and support the skin structure and function. In addition, they are highly dependent on mitochondrial energy production because they require energy to perform critical functions, such as elastin and collagen synthesis, growth factor and cytokine secretion, and cell signaling. Mitochondrial dysfunction in fibroblasts reportedly reduces the capacity for the synthesis of elastin and collagen, as well as elasticity, leading to wrinkle formation [32]. Mitochondrial function in skin cells is allegedly intimately involved in aging-induced changes in skin conditions [33]. Moreover, aging causes changes in mitochondrial morphology, metabolism, dynamics, DNA, and other factors that altogether lead to cellular aging and aging-related diseases. Therefore, maintaining and improving mitochondrial function is expected to help maintain healthy skin. Interestingly, it has recently been demonstrated that interaction of the F. fructosus OS-1010 strain with intestinal cells results in the secretion of specific exosomes from the intestinal cells, which enhances mitochondrial activity in muscle cells [18] and skin cells (Katakura Y, Idogaki H, et al., in preparation). The exosomes analyzed in these studies were found to act on muscle and skin cells; increase the expression of the Sirt1 gene, as well as the number and area of mitochondria; and activate mitochondria. Additionally, miRNAs contained in the released exosomes were found to affect pathways involved in longevity, including FoxO, mTOR, and autophagy, all of which are associated with skin inflammation and aging. The improvement in skin conditions upon OS-1010 treatment may be part of the exosome-mediated therapeutic effect of OS-1010, as demonstrated previously [18].

In conclusion, we conducted the first functional study on Fructobacillus spp. and demonstrated the beneficial effects of OS-1010 intake on the improvement of skin conditions. All improvements observed were closely related to the dermis. However, as our study did not include an analysis of actual dermal properties (e.g., elastin and collagen content, mitochondrial activity) or expected exosomes in blood or dermal layers, the described mechanism is not beyond the hypothetical realm, and further research is necessary to fully elucidate the mechanism. Note, however, that the clinical trial on human skin reported herein not only demonstrates the potential of OS-1010 as a functional ingredient for improving skin conditions, as previously reported, but additionally supports the proposal of a novel mechanism for skin condition improvement mediated by intestinal-derived exosomes.

CONFLICTS OF INTEREST

This study was financially supported by Osaka Soda Co., Ltd. All the authors are employees of Osaka Soda Co., Ltd.