A proof of concept: Clinical anti‐aging efficacy and safety of Lactiplantibacillus plantarum LB244R® applied topically in a double‐blinded placebo‐controlled study

With the increasing age of the westernized population, there is also increasing economic and aesthetic interest in reducing the signs of skin aging. Additionally, the physical aspect of aging can be displeasing and have detrimental effects psychologically in individuals. Probiotics have shown potential as anti‐aging agents, albeit proper studies are needed to confirm their potential.


| INTRODUC TI ON
Skin aging is a complex, albeit unavoidable, process.2][3] Wrinkles and pores become more prominent due to the weakening of extracellular matrix proteins, such as collagen and elastin fibers. 4Dullness and dry skin increase because of depleted moisture levels and lowered sebocyte activity, as well as reduced cell turnover and increased senescence. 5Spots and uneven skin tone get more prominent because of increased uneven melanin distribution. 2l these visual parameters can impact mental health as many people find the aging of the skin visually unappealing.In fact, the global cosmetics market reached 500 billion US dollar in 2017. 6ile many of the products currently on the market have some proven efficacy, such as pilot studies and cosmetic "before and after" studies, there is a need for properly designed, double-blinded placebo-controlled studies. 7e use of probiotics in combating aging, has shown great potential, but also has a huge gap when it comes to properly designed studies showing efficacy. 7,8Additionally, specific mode of action(s) has for some probiotic strains been indicated or hypothesized but uncovering the entire intricate synergy of interactions in vitro, ex vivo and in vivo is still lacking behind. 9This is further complicated when considering the human as a host with its added microbiota.A study by Landemaine et al. found that Staphylococcus epidermidis strains isolated from healthy skin produced high quantities of antioxidant indole metabolites (e.g., Indole-3-lactic acid) which induced the aryl hydrocarbon receptor (AHR) pathway when co-cultured with neonatal human epidermal keratinocytes.In contrast, strains isolated from nonhealthy atopic dermatitis skin produced low levels of indoles and did not induce the AHR pathway.This clearly shows the importance of local microorganisms and their metabolites. 10ctic acid bacteria (LAB) have previously shown great potential in inhibiting aging symptoms.This includes inhibition of melanogenesis, boosting collagen, reducing inflammation, and inhibition of matrix metalloproteinases (MMPs), all of which contribute to aging. 11,12Albeit the MoA is not fully elucidated, metabolites such as polyphenols and lipoteichoic acid have been implicated. 12,13The lack of properly designed studies, complex multifactorial MoAs, and formulation as well as stability issues are the major hurdles in the use of probiotics for anti-aging purposes.
In our previous pilot study, we found that a topical ointment containing live Lactiplantibacillus plantarum LB244R® (previously identified for its inhibitory activity towards S. aureus CC-type 1 14 ) could alleviate all measured aging signs in a time span of 56 days. 15e aim of this study was to build on our previous study and assess the anti-aging efficacy of the bacterial strain L. plantarum LB244R® for topical use as compared to its vehicle placebo, to specifically gain insight into the effect of the bacterium and use of topical LAB.This was done in a double-blinded, vehicle ointment placebo-controlled study with a total of 46 subjects enrolled.

| Investigational product
Two topical products were investigated: A probiotic, commercially available ointment (Healthy Aging Moisture Boost, BAK Skincare) and its placebo vehicle ointment without live bacteria.The active probiotic investigated in this study, namely the bacterium L. plantarum LB244R® was originally isolated from fermented cabbage. 14eeze-dried L. plantarum LB244R® (1 × 10 9 CFU/g) was formulated into an ointment containing Byturospermum parkii butter, Simmondsia chinensis seed oil, Brassica campestris seed oil, hydrogenated vegetable oil, Helianthus annuus hybrid oil, Prunus amygdalus dulcis oil, tocopherol, and Helianthus annuus seed oil.
Both topical ointments were kept cool, not exceeding 4°C until first use, whereafter they were stored at room temperatures not exceeding 25°C to ensure viability of the probiotics.

| Product compatibility, safety, and acceptability
Product acceptability and compatibility were assessed by a dermatologist under a standard daylight source at Day 0, Day 28 (+/− 2 days), and Day 56 (+/− 3 days), and by individual observation sheets.Any signs of reactivity such as erythema, oedema, vesicles, bulla, papules, scabs, dryness, coloration, and signs of discomfort, including heating, stinging, and itching were noted.

| Skin ultrasonography
Skin ultrasonography was measured as described previously. 15ssue density and dermis structures were assessed in the malar region, randomly assigned either left or right side, using a DermaScan C ultrasound system (Cortex technology, Denmark) with a modified 20 Mhz probe.Measurements were used to calculate the subepidermal low echogenic band (SLEB) thickness as well as skin density.

| Skin biomechanical evaluation
Skin biomechanical parameters were measured as previously, 15 using a Dual-cutometer MPA 580® (Courage & Khazaka Electronic GmbH, Germany).Measurements were performed using a 2 mm probe, where negative pressure is created in the device (450 mBar) and after 2 s the skin is released.Penetration depth is determined using a non-optical measuring system.The penetration depth is used as a measurement for skin firmness and the skin's ability to return to its original position after release as a measurement for skin elasticity.The resistance of the skin to the negative pressure can be used as a measure for firmness, and the ability of the skin to return to its original position as a measure for elasticity.Therefore, decreasing values mean decreased ability of suction device to deform skin and increased firmness of the skin.

| Skin hydration
Skin hydration assessments were measured using a Corneometer CM825 probe connected to a Cutometer dual MPA 580 (Courage & Khazaka, Germany).Measurements were performed in the malar area of the face, with measurements randomly assigned to either the left or right side.The hydration is assessed by measuring the capacitance of the skin expressed in arbitrary units (AU) to a factory standard.

| Skin pH
Skin pH was evaluated using a pH meter® PH905 (Courage & Khazaka Electronic GmbH, Germany).

| Skin structure visualization
Skin conditions and collagen structures were visualized and characterized using confocal microscopy with Vivascope® 1500 (Mavig, Germany).The epidermis and reticular dermis were imaged and inspected by in vivo confocal laser scanning microscopy (CLSM) or line-field confocal optical coherence tomography (LC-OCT) (Damae, France).Images were generated using an 830 nm or 800 nm laser hitting defined spots at specific skin depths.Images can be used to create a 3D object by scanning several optical planes and stacking them using confocal Vivascope software.

| Clinical evaluations
The efficacy of the ointments was assessed by a dermatologist at baseline (D0) and after application for 28 and 56 days.The following parameters were assessed: Crow's feet wrinkle clinical score was assessed using the Bazin scale as described previously from Grade 0 to 6 (with Grade 6 meaning very apparent and deep crow's feet and Grade 0 meaning no crow's feet), 15 Spot clinical score according to the Bazin scale from Grade 0 to 7 (with Grade 7 meaning a lot of age pigmentation and aging spots, and Grade 0 no age pigmentation or aging spots), smoothness of the skin score on a scale from 0 to 9 (with 9 meaning very smooth skin and 0 meaning not smooth skin), and complexion radiance score on a scale from 0 to 9 (9 meaning very radiant complexion and 0 meaning not radiant complexion).These observations were supported by selfassessment questionnaires.

| Statistical analysis
Student's T test or Wilcoxon's signed ranks test was performed for all the continuous data comparisons; between Day 0, 28, and 56 as well as both products.For subjective data, binomial tests were used.
A significance level of 95% was adopted for all tests.

| Study design
In this study, 46 postmenopausal female subjects were included with two dropouts in Vehicle group and none in Active group.Subjects had a mean age of 58.7 years in both groups, primarily phototype II and III and a skin reactivity dominated by sensitive skin (Table 1).

| Product compatibility, safety, and acceptability
Subjects in both groups showed no reactions or signs of discomfort attributed to the products throughout the study and after 56 days of consecutive application.

| Skin ultrasonography
SLEB thickness was reduced by both the vehicle (−2.2%, p < 0.05) and the active (−8.5%,p < 0.05) after 28 days of application.After 56 days of application, no change was observed for the vehicle while the active reduced the SLEB thickness with −13.1% (p < 0.05) (Table 2, Figure 1A).Skin density increased for the active at both time points with +8.7% (p < 0.05) and + 20.1% (p < 0.05), respectively.The vehicle increased dermal density with +2.2% and + 5.2%, although not significant (Table 2, Figure 1B).Comparing the vehicle and active, there was a statistically significant difference in skin density change after 28 and 56 days, in favor of the active ointment (Table 2).

| Skin biomechanical evaluation
Skin firmness was increased for both ointments at both timepoints.
After 28 days, the vehicle showed a −4.8% decrease, whereas the active showed a −4.6% decrease.On Day 56 the vehicle had resulted in a −10.8% decrease and the active resulted in an −11.1% decrease.
Both values at day 56 were statistically significant compared to baseline measurements but the difference between the performance of the two was not significant (Table 2).

| Skin barrier function
TEWL was reduced at all timepoints for both ointments with −0.7% for the vehicle and −3.2% for the active after 28 days, as well as −6.2% and −7.2%, respectively, after 56 days.Comparing the products, the changes, albeit better for the active at both timepoints, were not significant.

| Skin hydration
Skin hydration was increased for both the vehicle (+12.3%) and the active (+44.3%,p < 0.05) after 28 days and after 56 days (+21.9%,p < 0.05 and + 66.7%, p < 0.05, respectively).When comparing the active and the vehicle, the active produced a statistically significant difference in skin hydration at both 28 and 56 days.

| Skin pH
Skin pH was increased at all measured timepoints compared to baseline.After 28 days the vehicle showed a +17.0%increase in pH, whereas the active showed a +7.6% increase.After 56 days this increase was +11.3% and +11.1% respectively, with no significant difference between the two.

| Skin structure visualization
Skin collagen fibers were evaluated using confocal microscopy.The active ointment showed an increase (p < 0.05) in collagen fiber perimeter evaluation compared to baseline (D0) for both timepoints (+12.4% after 28 days and +15.9% 56 days).The vehicle showed an increase at Day 28 and Day 56, although not statistically significant compared to its baseline (+7.2% and +9.8% respectively).
The differences were not significant between ointment and active (Figure 2A).
Additionally, collagen fiber density was also evaluated, of which both ointments produced a significant increase compared to the baseline measurements.No significant difference was found between vehicle and active.For Day 28 the changes were +7.5% for the vehicle and +14.1% for the active, for Day 56 the changes were +11.0% and +12.7%, respectively (Figure 2B).TA B L E 1 Trial demographics.All subjects included were female.

| Clinical evaluations
Crow's feet wrinkles were reduced only for the active ointment with a reduction from 3.1 to 3.0, a percent change of −4.9% at Day 56 (Figure 3A), which is statistically significant compared to the baseline (D0) and to the vehicle ointment.For the spot score, no significant differences were seen after 28 days, but after 56 days, the active showed a significant reduction compared to its baseline.When comparing active and vehicle, no significant difference was seen (Figure 3B).
For the smoothness score and the complexion radiance score, both the vehicle and the active increased the scores after both time points.No significant differences between the active and vehicle were seen (Figure 3C,D).

| DISCUSS ION
The interest in using probiotics for anti-aging purposes has undeniably grown and continues to do so.Nevertheless, there are still limitations; Specifically concerning properly designed studies that prove in vivo efficacy, very complex, multifactorial MoAs and issues concerning viability in formulation.This study aimed to address these gaps and investigate and confirm the specific anti-aging effect of the bacterium L. plantarum LB244R® when topically applied to the skin of healthy aged female subjects for 56 days.
In this study, no reactions or signs of discomfort during the 56 days of application were found or attributed to either the vehicle group or the active group, meaning both ointments presented very Addressing the specific MoA remains difficult due to the many different mechanisms currently implicated in current research.
Probiotics have previously been found to affect cellular senescence, inflammation, the host microbiome, mammalian cell signaling, and have antioxidant capabilities, all of which, most likely, work in tandem or synergistically in affecting aging phenotypes of the host. 7 further complicate things, all the above mentioned mechanisms are considered strain specific, and comparison between individual strains is therefore difficult.The following sections address several of the mechanisms we believe contribute to the MoA of LB244R® in alleviating aging parameters seen in this and our previous study.
One MoA of probiotics is the ability to alleviate cellular senescence and thereby aging. 8Fang et al. showed how a combination of L. fermentum SX-0718, L. casei SX-1107, Bifidobacterium longum SX-1326 and B. animalis SX-0582 improved senescence associated phenotype in senescence accelerated mouse prone 8 (SAMP8) models. 8SLEB density is known to correlate with photo-aging, senescence, and inflammatory processes in the skin. 16In this study, SLEB improved in the active group compared to the vehicle group, which indicates that LB244R® reduces cellular senescence and inflammation (Figure 4).
The gradual increase of damage, senescence and inflammation that occurs cellularly with age is by now well-established, termed inflammaging. 17In inflammaging, reactive oxygen species (ROS) are known to play a key role, as their accumulation results in cell damage.LAB are known to produce several strong antioxidant molecules, including polyphenols and other small molecules such as taurine.Polyphenols are important in oral intake of probiotics concerning their anti-aging MoA. 18LB244R® produces polyphenols including indole-3-acetic acid and indole-3-lactic acid, 19 which could play a key role in the anti-aging efficacy observed in this study.Indole-3-acetic and indole-3-lactic acid have both shown to significantly reduce oxidative stress and inflammation. 13,20Albeit most previous studies have worked with these indoles in relation to the gut environment, Landemaine found that microbial derived indoles affected the epidermal cohesion and structure through the AHR pathway in healthy skin compared to atopic dermatitis skin, clearly demonstrating polyphenols also play a role in a topical skin setting. 10her small molecules produced by LB244R® that may affect aging include taurine and creatine, both of which can alleviate aging. 21e microbiome plays a role in aging as well. 3Centenarians have higher levels of Lactobacillus and additionally, Lactobacillus present on skin reduces with age. 2 This study supports the notion of putting LAB back on aging skin to alleviate the skin aging phenotype.
The changes seen by the active compared to the vehicle in this study could be partly due to re-application by LB244R® and thereby simulation of a younger skin microbiota.One study investigating postbiotic Epidermidibacterium keratini (EPI-7) further supports this notion, with similar findings to that of our study, seeing improvement of dermal density, skin barrier function and elasticity. 22m et al. found that their postbiotic could increase abundance of commensal microbes, and it is not unlikely that our probiotic ointment has a similar effect on the host microbiota.LB244R® was selected for its ability to inhibit the atopic dermatitis associated with S. aureus CC-type 1 strain, 14 and Staphylococcus has been shown to positively correlate with cellular senescence and inflammation markers. 2,14To sum up, LB244R® potentially modulates the microbiota present on skin, as well as simulates a microbiota associated with younger skin, which may contribute to the alleviation of aging signs and symptoms.
Another implicated MoA is mammalian cell signaling, specifically concerning MMPs.With age, the amount of MMP activity increases, resulting in damage to the extracellular matrix and its primary structural proteins, such as collagen and elastin. 23It has previously been shown that lipoteichoic acid, a surface-associated amphiphile cell wall component on Gram-positive bacteria, has an anti-aging effect as it inhibits melanogenesis and photo-aging by regulating the metalloproteinase-1. 11,12We hypothesize that inhibition of MMPs and thereby alleviation of collagen and ECM degradation are also some of the anti-aging MoAs of LB244R®, which is supported by the skin structure visualization data, albeit not significant relative to the vehicle group (Figure 5).
It is only currently possible to speculate on the MoA of LB244R®, but with further study, such as in vitro characterization of MoA and further in vivo clinical testing, the cumulative effect, as well as each of the individual mechanisms mentioned above, will be better understood.
This study is, to the best of our knowledge, the first placebocontrolled trial on anti-aging efficacy of topically applied LAB.
Overall, there was statistical improvement in 12 out of 13 parameters compared to the baseline (D0) for the active group and 5 out of the 12 parameters were statistically significant relative to the placebo vehicle group.These 5 parameters comprised SLEB, dermal density, elasticity, hydration, and crow's feet, all resulting in an antiaging effect on skin when topically applying LB244R®.
The significant attribution to improved parameters by the ointment itself was expected due to its composition.It has ingredients such as Byturospermum parkii butter (Shea butter), which has very well-established anti-aging effects such as the ability to help cell tissue regeneration, reduce aging signs, prevention of photo-aging, anti-inflammatory effects, and boosting collagen production. 24doubtedly, the formulation of the vehicle plays a great role in not obtaining significance between the active and the vehicle in many of the measured parameters.The fact that there are improvements still in so many parameters speak volumes for the effect of the bacterium L. plantarum LB244R®.
The main limitations of this study include the small sample size (n = 46) and the short time span of 56 days of the study.When considering traditional anti-aging topicals, such as retinoids, a time span of less than 4 months would be considered a short-term study. 4,25creasing study time could potentially show more effects of the bacterium which were not significant in this study.Perhaps increasing the timespan of a study similar to this one would mean statistical concerning skin structure assessments and collagen fibers.Although it was not significant, the active did show a larger improvement than For our future work, further investigating the specific MoA of LB244R®, for example by assessing its antioxidant capacity in vitro, assessing its effect on the skin microbiota and assessing its specific effect on the host immune system as well as signaling, is of vital importance for understanding the multifaceted MoA.

| CON CLUS ION
This study demonstrates anti-aging efficacy on skin of the bacterium L. plantarum LB244R® for topical use.When using the active ointment, 12 out of 13 measured parameters improved with statistical significance relative to their baseline measurements and 5 out of 13 parameters were improved with statistical significance when comparing with the vehicle placebo.L. plantarum LB244R® increased SLEB density, dermal density, skin elasticity, hydration, and reduced Crow's feet wrinkles and successfully showed a better anti-aging effect than that seen of the placebo vehicle ointment.
Albeit more studies, e.g., with larger sample sizes and increased timespan, are needed to further reaffirm this effect, this study further cements the use of LAB, and more specifically L. plantarum LB244R®, topically as a cosmeceutical ingredient to alleviate skin aging.
The study was run at a center in Portugal (PhD trials, Av.Maria Helena Vieira da Silva 24A, 1750-182 Lisboa, Portugal) beginning April 27, 2023, and ending June 30, 2023.The clinical trial was designed as a single-center, double-blinded, placebo-controlled study (study number 25850524.A-B).All subjects were randomly assigned either the vehicle (topical ointment without bacteria, Vehicle group) or the active probiotic (topical ointment with live L. plantarum LB244R, Active group).Subjects were recruited from an internal list in PhD Trials.Inclusion criteria were postmenopausal females aged 40-65 with normal, mixed/dry, or dry skin with visible signs of aging, such as fine lines and pigmented spots on a minimum Grade 2 on the Bazin scale. 15Subjects were instructed to apply their assigned topical ointment to the face and periocular area twice daily, morning and evening, on clean skin and massage until complete absorption for 56 consecutive days.The study was conducted in the spirit of and according to good clinical practice guidelines.Protocol and test conditions were submitted to the Ethical Commission of PhD Trials with submission number 48243.All parameters were assessed at Day 0, Day 26, and Day 56 for included subjects by trained technicians or dermatologists.All assessments were performed after a minimal acclimatization process of 15 min in a fully controlled and acclimatized room with temperatures T = 21 ± 2°C and a relative controlled humidity of 55% ± 10%.

F I G U R E 1 F I G U R E 2
Relative change in percentages of four measurements compared to D0; (A) Subepidermal low echogenic band (SLEB) thickness, (B) dermal density, (C) skin hydration, and (D) skin elasticity evolution throughout the study (D28 compared to D0 and D56 relative to D0) of the place vehicle and the active groups.Bars are mean changes of all subjects.Lined bars are Vehicle group and black bars are Active group.*Indicates significance with p < 0.05 between vehicle and active.Relative change in percentage in collagen quantification of skin.(A) Collagen fiber perimeter and (B) collagen fiber density throughout the study, bars are mean changes of all subjects.Lined bars are Vehicle group and black bars are Active group.*Indicates significance with p < 0.05.F I G U R E 3 Clinical scores evolution expressed as percentages in relative changes to Day 0. Bars are mean value changes of all subjects.Striped bars are Vehicle group and black bars are Active group.(A) Crow's feet wrinkle score, (B) Spot score, (C) Smoothness score, and (D) Complexion radiance.*Indicates significance with p < 0.05.

F I G U R E 4
Ultrasonography images of subject 11B at the three different timepoints (D0, D28, and D56), who was treated with the active (Active group).that of the vehicle in both collagen fiber perimeters and collagen fiber density.Although this is the first large step in proving that topical use of the right probiotic LAB can alleviate skin aging, there are still huge gaps in knowledge, specifically concerning the MoA of the bacteria.Inhibition of inflammation, positive modulation of the microbiota, effects on mammalian signaling and thereby indirect effects, as well as production of antioxidant compounds have all been shown to contribute to the effects of different specific strains.Furthermore, we also believe all of these to contribute to the repertoire of anti-aging effects seen by the bacterium L. plantarum LB244R®.