Living, quiescent Lactobacillus plantarum Lp90 probiotic, delivered topically to full thickness tissues in vitro via a just‐add‐water cream delivery system, stimulates the expression of elastin protein

Delivering living probiotics to the skin can be challenging as most water‐containing cosmetic products require preservatives to maintain product stability. A recently introduced powdered technology [Stratabiosys™, Vantage Personal Care] allows for quiescent probiotic powders to be stored for extended periods of time. The powders can then be reconstituted to creams at the point of use by adding water and mixing and were examined in vitro on reconstructed human full thickness tissues to see if the probiotic had any influence of several important biomolecules expressed in the skin.


| INTRODUC TI ON
The development of probiotics intended to improve the health and function of the human gastrointestinal tract has been advancing for many years.To date, are offered many well-established living probiotics in various forms including oral tablets and food items like yogurts and drinks.Many companies sell commercially viable, quiescent probiotics in powdered form.These kinds of powders encapsulate the living probiotics and maintain them in a relatively stable, quiescent form that offers the probiotics for oral consumption where, upon entering the gut, the probiotics become activated and begin to function and even grow.To date, there have been emerging studies that suggest that oral consumption of probiotics may actually influence the health of the skin, although proving these kinds of links can be very challenging. 1,2ere is a desire within the topical cosmetic and therapeutic industries to also offer probiotics to the skin and scalp.4][5][6] However, unlike the oral administration of living, quiescent probiotics, delivering probiotics to the skin and scalp has some challenges.Most cosmetic and therapeutic skin care products contain water.And, because they do contain water, they are typically preserved to prevent the unwanted growth of microorganisms in the products.9][10][11][12][13][14] Some companies, like Lactbio A/S, that have developed probiotic formulations using strictly anhydrous oil-based formulations. 15These kinds of formulations are effective at maintaining the living, quiescent probiotics until they are placed onto the skin.Such formulations will feel oily and, of course, for the probiotics to become activated on the skin, they must find their way out of the oil matrix in which they are encapsulated and become activated by the limited moisture of the stratum corneum.Other companies, like Mother Dirt, have figured out ways to try and maintain the viability of the probiotics by formulating them in a salt stabilized aqueous solution. 16Another innovative company, Blis Technologies, has developed a kind of dual-chambered delivery system that provides a water-based cream in one container and the quiescent, living probiotics in an oil-based system in the other chamber. 17The two formulations are mixed in the pump nozzle which delivers the probiotics quickly to the skin where they are blended with rubbing with the water-based cream.
No doubt, other innovative mechanisms for delivering probiotics will continue to emerge as finished brand and consumer interest in ways to deliver such actives to the skin grows.The challenge will remain how to maintain the living probiotics while circumventing the need to use preservatives in water-based formulations.
Recently, Gruber et al, reported a novel way to store probiotics in preservative-free just-add-water powders that can maintain the probiotics in their desired quiescent state until the powders are hydrated at which point the powders convert to an elegant cream and the probiotics are activated by the addition of the water. 18One advantage of these kinds of interesting powders is they can be quite adaptable with the ingredients that are used to make the powders, offering opportunities for various formulation types, that is, milks, creams, lotions, pastes, etc.As noted, the powders are preservativefree meaning that until such time as water is added and the powder converts to a cream, the powders are carrying only the added probiotics in their living, quiescent state.
There are numerous commercially available probiotic powders in the market sold for nutritional purposes.Usually, these powders are dried using freeze-drying techniques to minimize the exposure of the probiotics to heat.However, the act of drying probiotics can be quite stressful to the microorganisms due to hydrostatic pressures that come about from the drying processes.To help maintain the living probiotics in their quiescent state, the manufacturers will typically add low molecular weight oligosaccharides, called cryoprotectants.These low molecular weight oligosaccharides help to maintain the integrity of the microbial cell walls and keep the microorganisms intact and living in the powdered form.It has been suggested that these cryoprotectants may serve also as potential prebiotics in topical applications. 19There are numerous references to the benefits of applying living Lactobacillus to the skin, particularly to troubled skin.1][22] The just prior to application to the tissues.In the case of a powder containing Lactobacillus plantarum Lp90, topical application significantly increased expression of elastin in the skin replicants after 48 h of exposure to the cream made with the probiotic.The elastin-stimulating effects are not coming from the oligosaccharide cryoprotectants used to maintain the probiotic powders in their quiescent, dried state.The results indicate that it is the living Lactobacillus plantarum probiotic that is stimulating the elastin expression in the skin tissues.
[25][26][27][28][29][30] In reviewing the scientific literature on the topical application of probiotic Lactobacillus it is important to note that there are differences between applying living probiotics to the skin and applying derivatives of Lactobacillus ferments such as supernatants, lysates and the like.One critical aspect of such treatments is the non-living probiotics cannot communicate with the skin cells in quite the same way that living probiotics can. 31 It is certainly possible that a nonliving component of a ferment might contain chemical elements that can communicate with the skin, but the response is not a two-way street as would happen when both the probiotic and the skin cells are alive.In addition, in the typical studies that have looked at the benefit of living probiotic Lactobacillus, reference to the influence of the cryoprotectants that are used during the freeze-drying processes is often overlooked.This may be a mistake.Many low molecular weight oligosaccharides are known to have biological activity particularly when applied to skin with a disrupted or damaged barrier.It is possible that these cryoprotectants are influencing the skin's biology in unknown, possibly beneficial ways. 32,33 the following study, living, quiescent probiotic Lactobacillus plantarum Lp90, formulated into a just-add-water powder was examined on Epiderm® Full Thickness tissues by converting the powders to cream immediately prior to placing the resulting cream onto the skin surface.[36][37] In the following work, the studies were run twice.In the first round of studies, the tissues were treated as described for 48 h (2 × 24-h treatment) and examined for four strategically important skin biomolecules including Type 1A collagen, elastin, filaggrin and hyaluronic acid.From the results of these initial studies, it was found that the creams appeared to have a dose-dependent influence on the expression of elastin.These initial studies were followed by a second set of studies with newly formulated powders to try and reproduce the initial findings on elastin and to examine if, with powders prepared with just the known cryoprotectants, that is, no probiotics, the findings on elastin expression were the result of the microorganisms or the added cryoprotectants.

| Materials
The

| Tissue preparation
Upon arrival, the tissues were stored at 4°C until used.For use, the tissues were removed from the agarose-shipping tray and placed into a 6-well plate containing 2 mL of assay medium and incubated at 37 ± 2°C and 5 ± 1% CO 2 overnight.

| Tissue treatments
After the initial overnight incubation, the tissue culture media was replaced with 4 mL of fresh media and the tissues were then treated with the test materials which were prepared in ultrapure water and applied topically to the tissues.After a 24-h incubation period, the tissues were rinsed with PBS, fresh test materials were applied, and the tissues were incubated for a second 24-h period.At the end of this second incubation the tissues were homogenized in RIPA buffer, centrifuged to pellet any insoluble material and the supernatant was retained for analysis.The protein concentration of the supernatant was determined using a BCA assay.In addition to the tissue lysate, the media was also collected after the second 24-h incubation.

| BCA assay
A series of proteins standards were prepared using BSA ranging from 0 to 1000 μg/mL.Ten microliter aliquots of the standards and 5 μL aliquots of the cell lysate samples were then transferred to the wells of a 96-well plate.Two hundred microliters of working BCA reagent were added to each well and the plate was incubated for 30 min at 37°C.At the end of the incubation period the well plate was read at 540 nm using a plate reader.

| Filaggrin assay
The ELISA assay used to examine the filaggrin expression was purchased from Abexxa (Sugar Land, TX, Catalogue # abx151555).
For the ELISA assays, 100 μL of standards or diluted lysates (10 μL lysate diluted with 90 μL of sample buffer) were added to the wells of an ELISA plate and the plate was incubated for 90 min at 37°C.At the end of the incubation period the ELISA plate was emptied and 100 μL of biotinylated detection antibody solution was added to each well.The plate was then incubated for 60 min at 37°C and then washed three times with wash buffer.After the final wash was removed, 100 μL of an avidin-enzyme solution was added to each well and the plate was incubated for 30 min at 37°C.After this final incubation the plate was washed five times with wash buffer.Once the last wash was removed, 100 μL of substrate solution was added to each well.When a sufficient level of color development had occurred, 50 μL of stop solution was added to each well and the plate was read at 460 nm.

| Type 1A collagen assay
The ELISA assay test for Type 1A Collagen was purchased from Takara Bio USA (Ann Arbor, MI, catalogue # MK101).A series of type I C-peptide standards was prepared ranging from 0 ng/mL to 640 ng/mL.Next, an ELISA microplate was prepared by removing any unneeded strips from the plate frame followed by the addition of 100 μL of peroxidase-labeled anti procollagen type I-C peptide antibody to each well used in the assay.Twenty (20) μL of either sample (collected tissue culture media) or standard was then added to appropriate wells and the microplate was covered and allowed to incubate for 3 ± 0.25 h at 37°C.At the end of the incubation, the wells were aspirated and washed three times with 400 μL of wash buffer.
When the last wash was removed, 100 μL of peroxidase substrate solution (hydrogen peroxide + tetramethylbenzidine as a chromagen) was added to each well and the plate was incubated for 15 ± 5 min at room temperature.After the incubation, 100 μL of stop solution (1 N sulfuric acid) was added to each well and the plate was read using a microplate reader at 450 nm.

| Elastin assay
The elastin ELISA assay test was purchased from Cloud Clone Corporation (Katy, TX, catalogue #SEB337Hu).A series of elastin standards were prepared ranging from 0 ng/mL to 300 ng/mL.Next, an ELISA microplate was prepared by removing any unneeded strips from the plate frame followed by the addition of 100 μL of each standard or sample.The microplate was then covered and allowed to incubate for 1 h at 37°C.Upon completion of the incubation, the wells were decanted and 100 μL of detection reagent A was added and the plate was incubated again for 1 h at 37°C.Following this incubation, the well plate was washed three times with wash solution (the plate was allowed to sit for 1-2 min at room temperature with each wash solution).After the final wash was decanted, 100 μL of detection reagent B was added to each well and the plate was incubated for 30 min at 37°C and then washed as described above.Upon removal of the final wash, 90 μL of substrate solution was added to each well and the plate was incubated for 15 ± 5 min at room temperature.After the incubation, 50 μL of stop solution (1 N sulfuric acid) was added to each well and the plate was read using a microplate reader at 450 nm.

| Hyaluronic acid assay
The hyaluronic acid ELISA assay was purchased from Echelon Bioscience (Salt Lake City, UT, catalogue # K-1200).A series of hyaluronic acid standards were prepared ranging from 50 ng/mL to 3200 ng/mL.Next, 100 μL of each standard and sample were transferred to a well in an incubation plate.After adding 50 μL of detection solution to each well (except the reagent blank wells), the plate was incubated for 1 ± 0.25 h at 37 ± 2°C.Upon completion of the incubation, 100 μL of each sample/standard from the incubation plate was transferred to a corresponding well in the ELISA plate.The ELISA plate was covered and incubated for 30 ± 5 min at 4°C and then washed three times with 300 μL of wash buffer.After the final wash, 100 μL of enzyme solution was added to each well and the plate was incubated at 37 ± 2°C for 30 ± 5 min.Following this incubation, the wells were washed again as described above and then 100 μL of enzyme substrate solution was added to each well and the plate was incubated for 30-45 min at room temperature.At the end of this final incubation, 50 μL of stop solution was added to each well and the absorbance of the plate was measured at 405 nm using a plate reader.

| MTT assay
After completion of the treatment period, the EFT tissues were placed into a 6 well plate with 2.5 mL of DMEM supplemented with 1 mg/mL MTT.The well plate was incubated for 3 h at 37 ± 2°C and 5 ± 1% CO 2 .Following the incubation, the tissues were rinsed with PBS and placed into a 6-well plate containing 4 mL of isopropyl alcohol per well and incubated for 2 h at room temperature.At the end of this incubation 200 μL of each sample of extracted MTT was transferred to a 96-well plate and read at 540 nm using isopropyl alcohol as the blank.

| Statistical analysis
Treatment means were compared using an ANOVA, with an n = 3 per treatment.Statistical significance was set at p < 0.05.A Tukey posthoc analysis was conducted after the ANOVA to determine which treatment means were significantly different from the untreated group.

| MTT assay results
The results of the cell viability assay on each of the products tested are shown in Figure 1.
The results show that none of the powders converted to creams presented a cytotoxic issue even at the highest concentrations studied.

| Type 1A collagen assay results
The results of the initial studies looking at the influence of the probiotic-containing powder against placebo powder and untreated cells are shown in Figure 2.

The results indicate that the treatments had no influence on
Type 1A collagen expression in the tissues verse untreated control tissues.While it does seem that the overall amount of collagen appears to be dropping in a dose-dependent manner, this effect never reaches statistical significance against either the untreated controls or against the placebo treatments in this assay even at the highest concentration of probiotic powder tested (3%).

| Filaggrin assay results
The results of the filaggrin assay are shown below in Figure 3.
The results demonstrate that the probiotic containing powder had no influence on filaggrin expression verses untreated control tissues.

| Hyaluronic acid assay results
The results of the hyaluronic acid assay are shown below in Figure 4.
The results of the hyaluronic acid assay show that the probiotic powders had no influence on expression of hyaluronic acid verse untreated control tissues or as measured against the placebo tissues that did not contain the probiotic.

| Elastin assay results
The results of the elastin assay are shown below in Figure 5.

Type 1A Collagen
The results of the elastin assay indicate that at 2% and 3% treatment levels the creams statistically stimulated the expression of elastin protein in the tissues verses the Untreated controls.The Placebo controls showed no influence on elastin expression verses untreated controls.In this first study examining elastin, the 3% Probiotic powder treatment was significantly greater than both the 3% placebo and the untreated control, while the 2% Probiotic powder treatment was significantly greater than the 2% placebo and the untreated control.

| Second study results on elastin expression
The results of the second assay run to examine only elastin expression are shown below in Figure 6.
The results from the second study which was run to examine the isolated expression of elastin in the tissues demonstrated again that the powder containing the Lactobacillus probiotic stimulated the expression of elastin in the tissues, although in this study, 2% of the powder did not reach statistical significance there still appears to be a potential dose response similar to the previous findings.The results also demonstrate that the elastin stimulatory effects are not the result of the cryoprotectants that are used to help stabilize the quiescent probiotic powders.
In this follow up study examining just elastin expression, only the 3% Probiotic powder treatment had a statistically significant effect, but it was significantly greater than the untreated control group, the 3% Placebo group and both 3% cyroprotectant groups.The placebo and the cryoprotectants treatments were not significantly different than the Untreated Control group at any concentration.

| DISCUSS ION
The topical delivery of living probiotics remains an area of keen interest in the cosmetic and therapeutic industries.However, creating F I G U R E 3 Results of filaggrin assay on creams made from each powder verses placebo powder and untreated cells.systems that will allow for stable storage of living probiotics presents significant challenges because of the need to preserve products that contain water.As most cosmetic ingredients are offered, typically, as water-containing emulsions, this is a problem.More importantly, the study of topically applied probiotics is still a nascent science that demands continuing studies as to how various probiotics influence skin biology.How probiotics interact with not only the skin's microbiota, but also with the skin itself is still an emerging area of research.
In the present study, a mechanism for topically delivering living quiescent probiotics has been developed in which the probiotics are stored in powders that can be quickly and easily converted to emulsions by adding water and mixing.Even without the added probiotics, these powders offer a way to deliver single dose, preservative free formulations where the consumers may add the water and mix the formulations at home. 38It is important that should consumers reconstitute the powders in this fashion by adding water, then the formulations must be offered in a way that minimizes the potential to create creams and lotions that are preservative-free that remain reconstituted for extended periods of time.With such unique products, challenges remain.When the powder purposely includes known probiotics, then the challenges are even greater as the powder already contains potential living microorganisms by design.
Studies examining the topical influence of Lactobacillus-based probiotics have been principally done to examine the microorganism's influence on troubled skin.Lactobacillus is only a minor player in the skin's commensal microbiome.So, when a larger quantity of the microorganisms is applied to the skin, this will create a dysbiosis in the skin's normal microflora.It was recently reported that during a sanitizing event in which skin was treated with 3% hydrogen peroxide and then examined over time for the return of commensal microbial populations using ATP biofluorescence, the skin's microbiota appears to return within about 3 h. 39These results have been supported by studies from other labs in which skin treated with antibacterial hand soaps also appear to return within about 3 h of the treatment. 40How  41 Interestingly, in a clinical study that investigated the benefits of orally delivered Lactobacillus plantarum HY7714 to examine skin antiaging benefits, one target appeared to be particularly improved by the oral probiotics, that is, skin elasticity. 42It is unreasonable to make any mechanistic correlations between benefits of orally delivered probiotics and topically delivered probiotics.None the less, these findings are compelling.
More importantly, methods to examine how the skin is influenced by topical application of probiotics has resided principally in the clinical realm.The use of in vitro tissues models to examine the influence of living probiotics offers a potential way to start answering these important questions.In the present study, it was found in two repeat experiments that topical application of creams made just prior to application on the tissues from probiotic-containing just-add-water powders demonstrated a beneficial influence on elastin expression.
The effect appears to be dose dependent.Importantly, the effects do not appear to be due to the presence of known cryoprotectants that are used during the drying of the probiotics.It will be important for scientists conducting work in this area to keep in mind that these oligosaccharides are present and could influence testing results.

| CON CLUS IONS
The primary limitation on studying probiotic influences on reconstructed skin mimics is that such models offer only a modest similarity to human skin.Such models do present a fully developed, albeit weaker stratum corneum than normal human skin but are absent important axillary appendages such as sweat glands, hair follicles and sebaceous glands. 43However, because these axillary appendages are missing, it is unlikely that the living probiotics penetrate to any extent into the viable epidermis and dermis of the tissues.The tissues also come packaged in containers that have the tissues growing up against the sides of the container, minimizing the potential for larger materials, like bacteria, to circumvent the tissue's surface.
Therefore, it can be reasonably thought that the influence of the Lactobacillus plantarum Lp90 studied here is likely due to chemical communications occurring between the living microorganisms and the skin's living keratinocytes and fibroblasts.Further studies to examine not only additional skin targets, but also other quiescent probiotics delivered from the same just-add-water technologies are ongoing.More importantly, use of reconstructed tissue models negates one additional important aspect of human skin biology and that is the lack of a functioning microbiome.No doubt, the presence of additional commensal microorganisms is influencing how topical probiotics are affecting the skin and these kinds of affects are presently not testable with in vitro tissue assays as they would be when tested clinically on actual human skin.
Stratabiosys™ just-add-water powder containing 200 M CFU/ gram of Lactobacillus plantarum Lp90 and the corresponding placebo powder without probiotics were provided by Vantage Personal Care [Fairfield, NJ].The two cryoprotectant oligosaccharides were provided by Creative Enzymes [Shirley, NY].The Epiderm® Full Thickness Tissues were provided by Mattek [Ashland, MA] and were derived from neonatal cells.The tissue lots included lot #34298 for the initial study and lot #37916 for the follow up study.The creams made from the powders were prepared in 1.5 mL centrifuge tubes just prior to treatments.The tubes were vortexed for 3-5 s at room temperature (max speed) and then visually checked to see how well the material was blended in the tube.The process of vortexing and checking the samples was repeated until the material had a smooth, cream appearance with no visible clumps of material.A pipet tip was used to stir the material to help check for and break up clumps of material, if needed.

F I G U R E 1 2
Results of MTT cell viability assay on creams made from each powder verses untreated cells.Results of Type 1A collagen assay on creams made from each powder verses placebo powder and untreated cells.

4
Results of hyaluronic acid assay of probiotic powders verses placebo powder and untreated controls.

F I G U R E 5
Results of elastin assay verses placebo powder and untreated controls.Asterisks indicate statistical significance against untreated cells.Results of second assay examining expression of elastin with treatments including placebo powder, probiotic powder and powders containing 2% of either Fructo-oligosaccharide or 2% of Isomalto-oligosaccharide.Asterisks indicate statistical significance against untreated control cells.can return to normal after a probiotic delivered dysbiosis is unknown but the rate of return of the skin's microbiome from a dysbiosis event may be an important measure of the skin's overall health.Teng et al. have suggested that probiotics, both oral and topical, may play a role in improving photoaged skin, by rejuvenating the skin's extracellular matrix proteins.