Field of the invention

The present invention relates to the field of reducing malodour which is due to bacterial conversion of molecules which are present in sweat. Indeed, the present invention discloses the usage of specific acids which steer the microbiome by inhibiting the growth of specific odour-producing bacteria and/or promoting the growth of bacteria known to be beneficial in relation to malodour. The acids of the present invention can be used in deodorants, washing powders, clothing finishing agents or any method to reduce malodour. The present invention thus relates to the usage of a topical composition as a prebiotic.

Background of the invention

Sterile sweat is odourless. The bacteria living in the underarm convert sweat molecules and depending on the kind of bacteria, that odour can be bad. The mode of action of current deodorants relies on the addition of perfume, to mask the malodour production, and the addition of antimicrobial compounds. Compounds that possess an antimicrobial and antifungal function and thus are commonly used in deodorants are triclosan, triclocarsan, quaternary ammonium compounds, metal salts, aliphatic alcohols and glycols and other fragrances (1)(2). Antiperspirants, on the other hand, are a subgroup of deodorants that act similarly but additionally, they prevent sweating by blocking the sweat glands. Antiperspirants have a third mode of action caused by aluminum salts, which is known to block the sweat glands by mechanical obstruction. The ingredients normally used in antiperspirants are aluminum chloride hexahydrate (ACH) (in low concentrations), aluminum chlorohydrate, aluminum sesquichlorohydrate, aluminum chlorohydrex and aluminum zirconium tetrachlorohydrate.

By using underarm antiperspirants and deodorants, a microbial shock is caused to the underarm microbiome which leads to an increase of the microbial diversity. A higher microbial diversity is associated with more underarm malodour. For certain individuals, the relative abundance of corynebacteria increased, which suggests that using an antiperspirant could lead toward a more malodorous microbiome (3)(4). It is impossible to remove all odour-producing bacteria by washing the underarm. Bacteria will always remain in the hair follicles and sweat glands. Deodorants of today are not designed to‘help’ the underarm microbiome.

Axillary malodour is generated due to bacterial biotransformation of underarm secretions. Typical human, unusual, methyl-branched, odd-numbered long-chain fatty acids (LCFA) are degraded via b-oxidation into short-chain, volatile fatty acids (VFAs) (5)(6). Additionally, the release of short- chain fatty acids, such as E-3-methyl-2-hexenoic acid (3M2H), 3-hydroxy-3-methyl-hexanoic acid (HMHA), 3 -hydroxy-3 -methylhexanoic acid (3M3H), and a wide range of other structurally unusual VFAs, secreted as L-glutamine conjugates in apocrine glands, are considered as major components of the axillary malodour (7)(8)(9). After secretion by apocrine sweat glands, bacteria remove the L-glutamine residue with N ^a -acyl-glutamine aminoacylase and consequently releasing the VFAs. Several thioalcohols, such as 3-methyl-3-sulfanyl-hexan-l-ol (3M3SH) and 2-methyl- 3-sulphanylbutan-l-ol (2M3SB), as well as their isomers were also reported as important contributors to axillary malodour ( 10)( 11 )( 12). Thioalcohols can produce a sulphuric, meaty, onion-like or even fruity smell (6). Thioalcohols are secreted by apocrine sweat glands as cysteine- or cysteine-glycine conjugates (9). Steroids secreted in the underarm through the apocrine glands secrete a series of steroids, through the ABCC11 gene (13). The bacterial breakdown products, although not fully characterized, are known to cause a specific malodour (14).

At present, there is still a need to find alternative methods to combat odour-producing bacteria. Methods upon today merely focus on inhibition of the malodour associated bacteria. For instance, US 6171582 uses agents that inactivate the malodour associated corynebacteria. Those agents include salicylic acid, benzoic acid, ferulic acid, amongst others (15). EP 1902754 describes a deodorant composition using a salt of zinc and salicylic acid, amongst others, that have a bactericidal effect, and thus inhibit the bacteria to obtain a deodorant activity. GB 2284762 describes the use of talc, starch, boric acid and salicylic acid to prevent the formation of microbes on feet that lead to bad odours. US7569530 discloses pyroglutamic acid, adipic acid, gluconic acid, gluconolactone acid, glutamic acid, glutaric acid, glycolic acid, tartaric acid, ascorbic acid, benzoic acid, salicylic acid, citric acid, malic acid, succinic acid, lactic acid as an antimicrobial composition. W02019011551 discloses anions of, for instance, lactates, citrates, and tartrates, to reduce the coryneform bacteria. JPH0622540 discloses malic acid, citric acid, tartaric acid and gluconic acid, amongst others, that have an antibacterial effect. The latter documents thus describe the inhibition or inactivation of bacteria to obtain a deodorant activity. Also the existing products on the market mainly focus on antibacterial ingredients to combat malodour.

JP2005270453 describes the use of gallic acid, tannic acid and caffeic acid to deodorize odors of sulfur and nitrogen compounds.

WO 2020/052916 describes a saccharide isomerate for use as prebiotic ingredient for skin microbiome balancing. US20150202136 describes a list of ingredients claimed to increase the number of anaerobic and/or aerobic skin commensal microorganisms on skin. JP2017008095 discloses a galactooligosaccharide for promotion of growth of Staphylococcus epidermidis, Corynebacterium jeikeium, and Propionibacterium acnes. DEI 02005012476 discloses ingredients, such as carboxylic acids, dicarboxylic acid, metal salts, amongst others, that specifically target Staphylococcus hominis sp. US 20160089395 describes arginine bicarbonate and zinc carbonate to inhibit the growth of pathogenic Staphylococcus aureus and promote the growth of non-pathogenic Staphylococcus epidermidis.

There is still a need to provide compounds which are capable to selectively stimulate good-odour associated bacteria and/or to selectively inhibit malodorous bacteria.

Brief description of figures

Figure 1. Growth/inhibition of good-odour associated bacteria relative to malodour associated bacteria of phenotype microarray plate with an assortment of acids. Compounds on the left of the x-axis stimulate the growth of all good-odorous bacteria in this experiment combined, while compounds on the right stimulate the growth of malodorous bacteria in the experiment combined. Compounds such as gallic acid, tricarballylic acid, and lactic acid stimulate the growth of good- odorous bacteria minimum 0.5 times better than the growth of malodorous bacteria.

Summary of the invention

The present invention discloses 25 acids that can specifically enrich the good odour associated bacteria and/or specifically inhibit the malodour associated bacteria, both with the aim of reducing malodour in skin or textiles. In other words, the present invention discloses 25 acids which steer the microbiome to a better smelling microbiome in a very specific manner as the latter acids specifically promote the growth of good-odour associated bacteria such as Staphylococcus epidermidis, Acinetobacter Iwojfii, Propionibacterium acnes (Cutibacterium acnes) and Enhydrobacter aerosaccus bacteria and do not promote and/or inhibit the growth of mal-odour associate bacteria such as Corynebacterium tuberculostearicum, Corynebacterium amycolatum, Staphylococcus hominis, Micrococcus luteus and Enterobacter cloacae.

Hence, the present invention relates in first instance to the usage of a composition to selectively promote the growth, metabolism and/or colonization of good-odour associated bacteria and/or to selectively inhibit the growth, metabolism and/or colonization of malodour-associated bacteria, wherein said composition comprises at least one acid chosen from the list consisting of: gallic acid, fumaric acid, mucic acid, g-aminobutyric acid, a-keto-glutaric acid, quinic acid, D-saccharic acid, glyoxylic acid, D-glucosaminic acid, 4-hydroxybenzoic acid, D,L-a-amino-caprylic acid, N- acetyl-neuraminic acid, D,L-a-amino-N-butyric acid, orotic acid, e-amino-N-caproic acid, b- hydroxybutyric acid, tropic acid and hippuric acid.

The present invention further relates to the usage of a composition to selectively promote the growth, metabolism and/or colonization of good-odour associated bacteria, wherein said composition comprises at least one acid chosen from the list consisting of: tricarballylic acid, L- lactic acid, citric acid, D,L-malic acid, stearic acid, acetic acid and ferulic acid.

More specifically, the present invention relates to the usage as described above wherein said good- odour associated bacteria are at least Staphylococcus epidermidis, Acinetobacter spp, Propionibacterium acnes ( Cutibacterium acnes) or Enhydrobacter aerosaccus bacteria and wherein said malodour-associated bacteria are at least Corynebacterium tuberculostearicum, Corynebacterium amycolatum, Staphylococcus hominis, Micrococcus luteus or Enterobacter cloacae.

Furthermore, the present invention relates to the usage of a composition as described above, wherein said composition is provided as a topical formulation selected from the group selected from soap, spray, drop, aerosol, powder, roll-on, lotion, cream, stick, solution, sachet, colloidal suspension, film, patch, finishing agent and ointment.

The invention further relates to the usage of a composition as described above, wherein said composition has -upon application- a pH between 3.5 and 7. The invention further relates to the usage of a composition as described above to modify the underarm microbiome, skin microbiome, clothing microbiome or any other fabric microbiome. The invention further relates to the usage of a composition as described above to reduce the formation of short-chain fatty acids released from unusual, methyl-branched, odd-numbered long- chain fatty acids; reduce the formation of E-3-methyl-2-hexenoic acid, 3-hydroxy-3-methyl- hexanoic acid and 3-hydroxy-3-methylhexanoic acid; and, reduce the formation of thioalcohols such as 3-methyl-3-sulfanyl-hexan-l-ol and 2-methyl-3-sulphanylbutan-l-ol.

The present invention further relates to the usage of composition as described above as a prebiotic ingredient.

Detailed description of the invention

The present invention relates to the following compounds/acids that selectively steer the microbiome towards a better smelling microbiome: gallic acid, tricarballylic acid, L-lactic acid, fumaric acid, mucic acid, citric acid, D,L-malic acid, stearic acid, g-aminobutyric acid, a-keto- glutaric acid, acetic acid, quinic acid, D-saccharic acid, glyoxylic acid, D-glucosaminic acid, 4- hydroxybenzoic acid, D,L-a-amino-caprylic acid, N-acetyl-neuraminic acid, D,L-a-amino-N- butyric acid, orotic acid, ferulic acid, e-amino-N-caproic acid, b-hydroxybutyric acid, tropic acid, and hippuric acid.

The present invention thus relates in first instance to the usage of specific acids which promote the growth of good-odour-associated bacteria such as Staphylococcus epidermidis, Acinetobacter spp, Propionibacterium acnes (Cutibacterium acnes) and Enhydrobacter aerosaccus bacteria and do not promote (or inhibit) the growth of malodour-associated bacteria such as Corynebacterium tuberculostearicum, Corynebacterium amycolatum, Staphylococcus hominis, Micrococcus luteus and Enterobacter cloacae bacteria, wherein said composition comprises at least one acid chosen from the list consisting of: gallic acid, tricarballylic acid, L-lactic acid, fumaric acid, mucic acid, citric acid, D,L-malic acid, stearic acid, g-aminobutyric acid, a-keto-glutaric acid, acetic acid, quinic acid, D-saccharic acid, glyoxylic acid, D-glucosaminic acid, 4-hydroxybenzoic acid, D,L- a-amino-caprylic acid, N-acetyl-neuraminic acid, D,L-a-amino-N-butyric acid, orotic acid, ferulic acid, e-amino-N-caproic acid, b-hydroxybutyric acid, tropic acid, and hippuric acid. All of the above-listed compounds/acids are well-known and can be easily obtained through normal commercial canals. Compounds are relatively cheap, easily accessible, odourless or have a pleasant odour (or no malodour at the least), light or no colour and non-toxic to mammals/humans in the concentrations used in said experiments.

The following table shows the structure of each of the above-indicated acids:

Bacterial growth is measured with Optical Density (OD) measurements at wavelength 620nm with every sample analyzed in triplicate. Promoting bacterial growth relates to a higher bacterial biomass density in suspension after 24h, as measured with the spectrophotometer. Bacterial inhibition relates to a similar or lower bacterial density after 24h in suspension, as measured with the spectrophotometer. Promoting bacterial metabolism relates to a higher bacterial activity, and thus active gene expression of the bacteria of interest. Inhibiting bacterial metabolism relates to a lower bacterial activity, and thus less gene expression of the bacteria of interest. Bacterial metabolism can be measured by metatranscriptomics analysis, RNA-sequencing or any ELISA kit measuring specific enzymes of the metabolism.

Furthermore, the present invention relates to the usage of the acids as indicated above in a deodorant or skin cosmetics. A deodorant is any product applied in the underarm region to mask or reduce underarm odour. Deodorant compositions as described herein are administered, preferably topically, for the treatment of any one or more symptoms desirable of change, e.g., cutaneous (including axillary) malodour. A skin cosmetic is any product applied to the skin to improve its appearance. Dosage forms are solid or free- flowing. Dosage forms include, but are not limited to, soaps, sprays, drops, aerosols, powders, roll-ons, lotions, creams, sticks, solutions, sachets, colloidal suspensions, films, patches and ointments. Deodorants or skin cosmetics can be applied on skin -for example- the underarm or foot or other body parts, or, on clothes. Furthermore, the present invention relates to a fabric care product, textile finishing product, coating or washing powder. The compounds listed in this invention is applied or coated on clothing textiles, sport textiles, bed linen, intimate clothing, feet clothing (socks and shoes), carpets, or other textiles to reduce the malodour formation. The compounds listed in this invention is applied in the laundry machine or elsewhere in the household to reduce malodour formation. The compounds listed in this invention is used as washing powder to rinse the textiles. Dosage forms are solid or free-flowing. Dosage forms include, but are not limited to, powders, soaps, sprays, drops, aerosols, powders, roll-ons, lotions, creams, sticks, solutions, sachets, colloidal suspensions, films, patches and ointments. Hence, the present invention relates to the usage of a composition as described above, wherein said composition is provided as a topical formulation selected from the group selected from soap, spray, drop, aerosol, powder, roll-on, lotion, cream, stick, solution, sachet, colloidal suspension, film, patch, finishing agent and ointment. The invention further relates to the usage of a composition as described above to modify the underarm microbiome, skin microbiome, clothing microbiome or any other fabric microbiome.

Deodorant or clothing product/composition as described herein may have a pH of at least 3.0, 4.0. 5.0, 6.0, 7.0, 8.0, 9.0, or 10.0 upon administration and/but have preferably a pH between 3.5 and

7.

The present invention further relates to the usage of a composition/acids as described above to modify the microbiome. Staphylococcus epidermidis is a major member of the skin microbiome which is correlated to a better underarm/skin odour and a lower bacterial diversity. Other bacteria such as, and not limited to, Acinetobacter spp, Propionibacterium acnes (Cutibacterium acnes) and Enhydrobacter aerosaccus, are associated with a good skin odour. These bacteria are here called“good odour associated bacteria”. Corynebacterium spp. (containing and not limited to C. tuberculostearicum, C. amycolatum ) are correlated with more skin malodour and/or a higher bacterial diversity. Other bacteria, such as, and not limited to, Staphylococcus hominis, Micrococcus luteus and Enterobacter cloacae, are associated to malodour and/or a higher bacterial diversity. These bacteria are here referred to as“malodour associated bacteria”. All of the isolates combined represent about 81% of the average in vivo underarm microbiome (16), which gives a good representation of the underarm microbiome. Modifying the microbiome relates to steering the microbiome to a higher abundance of good-odour associated bacteria and a lower abundance of malodour associated bacteria. The microbiome can be modified in the underarm region, on skin, on clothes, on other skin regions of the human body or in washing machines. Good odour associated bacteria are associated with a positive hedonic value, which relates to the pleasantness of the odour - on a scale from -8 (very unpleasant) to 0 (neutral) to +8 (very pleasant). Good odour associated bacteria are also involved in a lower intensity of the odour, which relates to the quantity of the odour - on a scale from 0 (no odour) to +10 (very strong/intolerable). Malodour associated bacteria are associated with a low hedonic value and a high odour intensity.

Hence, the present invention specifically relates to the usage of a composition as defined above wherein said microbiome comprises bacteria of at least one of the following the species: Staphylococcus epidermidis, Acinetobacter Iwoffii, Propionibacterium acnes (Cutibacterium acnes), Staphylococcus hominis, Corynebacterium tuberculostearicum, Corynebacterium amycolatum, Micrococcus luteus, Enhydrobacter aerosaccus, Enterobacter cloacae.

The present invention relates -in other words- to a method to reduce malodour comprising administering an effective amount of a composition as defined above to a mammal in need thereof. An effective amount is amount of compound that can have an impact on the microbiome.

Skin and textile malodour is generated due to bacterial biotransformation of sweat secretions. Typical human, unusual, methyl-branched, odd-numbered long-chain fatty acids (LCFA) are degraded via b-oxidation into short-chain, volatile fatty acids (5,6). Additionally, the release of short-chain fatty acids, such as E-3-methyl-2-hexenoic acid (3M2H), 3-hydroxy-3-methyl- hexanoic acid (HMHA), 3-hydroxy-3-methylhexanoic acid (3M3H), and a wide range of other structurally unusual VFAs, secreted as L-glutamine conjugates in apocrine glands, are considered as major components of the axillary malodour (7-9). After secretion by apocrine sweat glands, bacteria remove the L-glutamine residue with N ^a -acyl-glutamine aminoacylase and consequently releasing the VFAs. Several thioalcohols, such as 3-methyl-3-sulfanyl-hexan-l-ol (3M3SH) and 2-methyl-3-sulphanylbutan-l-ol (2M3SB), as well as their isomers were also reported as important contributors to axillary malodour (10-12).

Therefore, the present invention further relates to a method -or the usage of a composition as described above- to reduce the formation of short-chain fatty acids released from unusual, methyl- branched, odd-numbered long-chain fatty acids; reduce the formation of E-3-methyl-2-hexenoic acid, 3-hydroxy-3-methyl-hexanoic acid and 3-hydroxy-3-methylhexanoic acid; and, reduce the formation of thioalcohols such as 3 -methyl-3 -sulfanyl-hexan-l-ol and 2-methyl-3-sulphanylbutan- 1 -ol comprising administering an effective amount of a composition as defined above to a mammal in need thereof.

The present invention relates to prebiotic ingredients to steer the microbiome towards a less odorous microbiome.

The present invention also relates a to a method as defined above wherein said malodour is situated on the skin or hair of said mammal.

The present invention also relates a to a method as defined above wherein said malodour is situated in the underarm of said mammal.

The present invention also relates a to a method as defined above wherein said malodour is situated in the household, clothing textiles, washing machine, fabrics or shoes of said mammal.

The present invention further relates to a method as defined above wherein said mammal is a human.

The present invention further relates to the usage of composition as described above as a prebiotic ingredient. With the term‘prebiotic ingredient’ is meant that the carboxylic acid stimulates the growth, metabolic activity and/or colonization of the beneficial bacteria. As such, the present invention steers the microbiome towards a less odorous and better microbiome.

The present invention will now be illustrated with the following, non-limiting examples.

Examples

Materials and Methods

1.1 GROWTH MEDIA & SOLUTIONS

1.1.1 Axillary growth medium

To grow the axillary bacteria on an agar plate, axillary culture blood agar (ACX) was used. To compose 500mL of this medium, one needs:

• 19.75g blood agar base no. 2 (Oxoid LTD, Basingstoke, England)

• 1.5g yeast extract (Oxoid LTD, Basingstoke, England)

• lg glucose (Carlroth GmbH, Karlsruhe, Germany)

• 2.5mL Tween 80 (Sigma Aldrich, St. Louis, USA) 25mL defibrinated horse blood (Oxoid Limited, Basingstoke, England)

When the first four components were added to a Schott bottle of 500mL, the solution is diluted with distilled water until a volume of ca. 475mL is reached. After autoclaving and cooling down at about 40°C, the defibrinated horse blood is added. Immediately after the blood is added, the medium is poured in plates in a sterile way and one have to wait until it is fixed. The plates were stored in the cold room (ca. 4°C).

1.1.2 Nutrient broth

Axillary bacteria were cultured in liquid medium using nutrient broth. To compose 500mL nutrient broth, 6.5g nutrient broth without agar (Oxoid LTD, Basingstoke, Hampshire, England) was added to a Schott bottle of 500mL. The bottle was diluted with distilled water until 500mL. Afterwards, the bottle was shaken and autoclaved. Finally, the bottle was distributed over lOmL tubes in a sterile manner. The tubes were stored in a cold room (ca. 4°C) to make sure they remain sterile.

1.1.3 M9 medium

M9 medium is a minimal-growth-medium for bacteria. Bacteria were diluted and distributed in 96-well plates using M9 medium, containing the minimal salts and micro-nutrients. This medium provides enough nutrients for the bacteria to survive, but not to grow. To create 1L of this medium, one needs at first a salt solution (1L) that consists of:

• 42g Na ₂ HP0 ₄ .7H ₂ 0

• 15g KH ₂ P0 ₄

• 2.5g NaCl

• 5g NH ₄ C1

All compounds (originating from Carl Roth GmbH, Karlsruhe, Germany) were added to a Schott bottle of 1L which is diluted with distilled water until one reaches 1L. Next, this solution was autoclaved. In addition to the salt solution, two separate solutions with 120g/L MgS0 ₄ and 1 lOg/L CaCl ₂ were prepared. The chemical compounds originated again from Carl Roth GmbH, Karlsruhe, Germany. These solutions were sterilized using a filter with a pore size of 0.22pm (Sartorius Stedim Biotech S.A., Brussels, Belgium). For 1L M9 medium, 200m E salt solution was combined with 2mL MgS0 ₄ solution and O.lmL CaCl ₂ solution. This solution was diluted with autoclaved distilled water until 1L is reached (17). 1.1.4 PBS-solution

To dilute the samples and make them suitable for measurements using the flow cytometry, a phosphate-buffered saline solution (PBS) was needed. One needed to put one tablet of 2g in 200m U of distilled water in order to receive a 0.01M phosphate buffer, 0.0027M potassium chloride and 0.137M sodium chloride with an overall pH of 7.4 at 25°C. After sterilization, the solution was ready for use.

1.1.5 Physiological water

Physiological water was used, containing 4.25g NaCl (Carl Roth GmbH, Karlsruhe, Germany) for 500mL of sterile distilled water. Before use, the solution was sterilized at 121°C for 30 min.

1.2 SELECTION OF BACTERIA

During the experiment, the following bacteria were used:

• Staphylococcus epidermidis

• Acinetobacter Iwoffii

• Propionibacterium acnes (Cutibacterium acnes)

• Enhydrobacter aerosaccus

• Corynebacterium tuberculostearicum

• Corynebacterium amycolatum

• Staphylococcus hominis

• Enterobacter cloacae

• Micrococcus luteus

All these bacteria were stored in a -80°C freezer with the cryoprotectant glycerol. The first four bacteria are known to be beneficial according to axillary malodour, the next seven are malodour- producing bacteria. The selected microorganisms can be obtained by any suitable manner known in the art. For example, the selected microorganism(s) may be isolated from a natural environment (e.g., the underarm skin of a person or the worn clothes or a person) or purchased from a suitable commercial source such as the American Type Culture Collection (ATCC) (10801 University Boulevard, Manassas, VA 20110 USA) or LM-UGent (BCCM/LMG Bacteria Collection, Karel Lodewijk Ledeganckstraat 35, 9000 Gent, Belgium). Corynebacterium tuberculostearicum TVK048 ( similar to ATCC 35692), Corynebacterium amycolatum TVK039 ( similar to ATCC 700207), and Micrococcus luteus TVK014 (similar to ATCC 4698) originate from human armpits, were rated by a human odour panel and assigned as malodorous. The odour panel was trained and selected and samples were rated as previously described (18). The odour panel rated the odours based on the hedonic value, which is the pleasantness of the odour - on a scale from -8 (very unpleasant) to 0 (neutral) to +8 (very pleasant), and the intensity of the odour, which is the quantity of the odour - on a scale from 0 (no odour) to +10 (very strong/intolerable). Acinetobacter Iwojfii spp. EDM025 (similar to ATCC 15309), Propionibacterium acnes (Cutibacterium acnes) EDM035 (similar to ATCC 6919) and Staphylococcus hominis EDM024 (similar to ATCC 25615) originate from worn clothes samples but can also originate from the above-mentioned culture collections. Staphylococcus epidermidis LMG 10273, Enhydrobacter aerosaccus LMG 21877 and Enterobacter cloacae LMG 2783 originate from human samples and were obtained from the culture collection of LM-UGent. Collectively, this selection of bacteria is a good representation of the underarm microbiome and the microbiome of the bacteria living on clothes in the underarm region (16). A person skilled in the art can verify the identity of the bacterial species by verifying the hypervariable region of the 16S rRNA gene. The 16SrRNA gene of the used species is shown below:

• Staphylococcus epidermidis (SEQ ID N° 1)

TCCTCTGACGTTAGCGGCGGACGGGTGAGTAACACGTGGATAACCTACCTAT

AAGACTGGGATAACTTCGGGAAACCGGAGCTAATACCGGATAATATATTGA

ACCGCATGGTTCAATAGTGAAAGACGGTTTTGCTGTCACTTATAGATGGATC

CGCGCCGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATGCG

TAGCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAG

ACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTG

ACGGAGCAACGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTT

ATTAGGGAAGAACAAATGTGTAAGTAACTATGCACGTCTTGACGGTACCTAA

TCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG

GCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTTTTA

AGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGG

AAAACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAAT

GCGCAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTA

ACTGACGCTGATGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTG GTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCT

TAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAG

GTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTG

GTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTCTGAC

CCCTCTAGAGATAGAGTTTTCCCCTTCGGGGGACAGAGTGACAGGTGGTGCA

TGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC

GCAACCCTTAAGCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGTTGACTGC

CGGTGACAAACCGGAAGAAAGGTGGGG

• Acinetobacter Iwoffii (SEQ ID N° 2)

TGGGGTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGA

TCCGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAG

TGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGT

GAAGAAGGCCTTTTGGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACCGA

GATTAATACTCTTGGATAGTGGACGTTACTCGCAGAATAAGCACCGGCTAAC

TCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTA

CTGGGCGTAAAGCGCGCGTAGGTGGCCAATTAAGTCAAATGTGAAATCCCCG

AGCTTAACTTGGGAATTGCATTCGATACTGGTTGGCTAGAGTATGGGAGAGG

ATGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATA

CCGATGGCGAAGGCAGCCATCTGGCCTAATACTGACACTGAGGTGCGAAAG

CATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGAT

GTCTACTAGCCGTTGGGGCCTTTGAGGCTTTAGTGGCGCAGCTAACGCGATA

AGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAATGAATTGA

CGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGA

AGAACCTTACCTGGTCTTGACATAGTAAGAACTTTCCAGAGATGGATTGGTG

CCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGT

GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTTGCCA

GCGGGTTAAGCCGGGAACTTTAAGGATACTGCCAGTGACAAACTGGAGGAA

GGCGGGGACGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGT

GCTACAATGGTCGGTACAAAG

• Propionibacterium acnes (Cutibacterium acnes) (SEQ ID N° 3) GGTAGCCGGCCTGAGAGGGTGACCGGCCACATTGGGACTGAGATACGGCCC AGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGGAAGCC TGATGCAGCAACGCCGCGTGCGGGATGACGGCCTTCGGGTTGTAAACCGCTT TCGCCTGTGACCAAGCGTGAGTGACGGTAATGGGTAAAGAAGCACCGGCTA ACTACGTGCCAGCAGCCGCGGTGATACGCAGGGTGCGAGCGTTGTCCGGATT TATTGGGCGTAAAGGGCTCGTAGGTGGTTGATCGCGTCGGAAGTGTAATCTT GGGGCTTAACCCTGAGCGTGCTTTCGATACGGGTTGACTTGAGGAAGGTAGG GGAGAATGGAATTCCTGGTGGAGCGGTGGAATGCGCAGATATCAGGAGGAA CACCAGTGGCGAAGGCGGTTCTCTGGGCCTTTCCTGACGCTGAGGAGCGAAA GCGTGGGGAGCGAACAGGCTTAGATACCCTGGTAGTCCACGCTGTAAACGGT GGGTACTAGGCGTGGGGTCCATTCCACCGGGTTCCGTGGCCGTAGCTAACGC TTCAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAA TTGACGGGGCCCCGCACAAGCGGCGGAGCATGCGGATTAATTAGATGCCAC GCCTAGAACCTTACCTGGGCTTGACACGGATCGGGAGTGCTCAGAGATGGGT GTGCGCTCTTTTGGGGTCGGTTCACAGGCGGTGCATGGCTGTCGTCAGCTCGT GTCGTGAGATGTTGGGATAAGTCCCGCAACGAGCGCAACCCTTCTTCCCTGC TGCCAGCACGTTATGGCGGGG

• Enhydrobacter aerosaccus (SEQ ID N° 4)

CCGGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAAC

CCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTTTGGTTGTAAAGCAC

TTT A AGC AGGG AGG AG AGGCT A ATGGTT A AT ACCC ATT AG ATT AG ACGTT AC

CTGCAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGA

GGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGAGTGTAGGTGGCTC

ATTAAGTCACATGTGAAATCCCCGGGCTTAACCTGGGAACTGCATGTGATAC

TGGTGGTGCTAGAATATGTGAGAGGGAAGTAGAATTCCAGGTGTAGCGGTG

AAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCTTCCTGGCA

TAATATTGACACTGAGATTCGAAAGCGTGGGTAGCAAACAGGATTAGATACC

CTGGTAGTCCACGCCGTAAACGATGTCTACTAGCCGTTGGGGTCCTTGAGAC

TTTAGTGGCGCAGTTAACGCGATAAGTAGACCGCCTGGGGAGTACGGCCGCA

AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATG

TGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTGCTATGACTCATAG CTGAGAATGCTGCTGCAGAGATGAGAGAGTGCCTTCGGGAACTCACATACA

GGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCC

GCAACGAGCGCAACCCTTTTC

• Corynebacterium tuberculostearicum (SEQ ID N° 5)

CGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCCTGGGA AACTGGGTCTAATACCGGATAGGAGCCATTTTTAGTGTGATGGTTGGAAAGT TTTTTCGGTGTAGGATGAGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAATG GCCTACCAAGGCGGCGACGGGTAGCCGGCCTGAGAGGGTGGACGGCCACAT TGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT TGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGACGGC CTTCGGGTTGTAAACTCCTTTCGCTAGGGACGAAGCTTTTTGTGACGGTACCT AGATAAAAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGG GTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCTCGTAGGTGGTTTGT CGCGTCGTCTGTGAAATTCCGGGGCTTAACTCCGGGCGTGCAGGCGATACGG CCATAACTTGAGTACTGTAGGGGTAACTGGAATTCCTGGGGTAGCGCTGAAA TGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTTACTGGGCAGT TACTGACGCTGAGAAGCGAAAG

• Corynebacterium amycolatum (SEQ ID N° 6)

GTGGCGAACGGGTGAGTAACACGTGGGTGACCTGCCCTGCACTTCGGGATAA GCCTGGGAAACTGGGTCTAATACCGGATAGGACCGCACCGTGAGGGTGTGG TGGAAAGTTTTTTCGGTGTGGGATGGGCCCGCGGCCTATCAGCTTGTTGGTG GGGTAATGGCCTACCAAGGCGGCGACGGGTAGCCGGCCTGAGAGGGTGGAC GGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTG GGGAATATTGCACAATGGGCGGAAGCCTGATGCAGCGACGCCGCGTGGGGG ATGACGGCCTTCGGGTTGTAAACTCCTTTCACCATCGACGAAGGGTTTCTGA CGGTAGATGGAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAA TACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGAGCTCGTAGG TGGTTTGTCGCGTCGTCTGTGAAATTCCGGGGCTTAACTCCGGGCGTGCAGG CGATACGGGCATAACTTGAGTACTGTAGGGGAGACTGGAATTCCTGGTGTAG CGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTC TGGGCAGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTA GATACCCTGGTAGTCCATGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCT

TCCACGGGATCCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTA

CGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGC

GGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGAC

ATATACAGGATCGCGCCAGAGATGGTGTTTCCCTTGTGGCTTGTATACAGGT

GGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA

ACGAGCGCAACCCTTGTCTTATGTTGCCAGCACGTTGTGGTGGGGGACTCGT

AAAGAAACTGCCCGGGGTTAAC

• Staphylococcus hominis (SEQ ID N° 7)

GTTAGCGGCGGACGGGTGAGTAACACGTAGGTAACCTACCTATAAGACTGG

GATAACTTCGGGAAACCGGAGCTAATACCGGATAATATTTCGAACCGCATGG

TTCGATAGTGAAAGATGGCTTTGCTATCACTTATAGATGGACCTGCGCCGTA

TTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCAACGATACGTAGCCGACC

TGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAGACTCCTACG

GGAGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAA

CGCCGCGTGAGTGATGAAGGTCTTCGGATCGTAAAACTCTGTTATTAGGGAA

GAACAAACGTGTAAGTAACTGTGCACGTCTTGACGGTACCTAATCAGAAAGC

C ACGGCT A ACT ACGTGCC AGC AGCCGCGGT A AT ACGT AGGTGGC A AGCGTT A

TCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGGTTTTTTAAGTCTGATGT

GAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGAAAACTTGAG

TGCAGAAGAGGAAAGTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGAGAT

ATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACGCTGA

TGTGCGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCC

GTAAACGATGAGTGCTAAGTGTTAAGGGGGTTTCCGCCCCTTAGTGCTGCAG

CTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCA

AAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGA

AGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACCCTTCTAGAAG

ATAGAAGTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGT

CAGCTCGTGTCGTGAGATGTTTGGGTTAAGTCCCGCAACGAGCGCAACCCTT

AAGCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGTTGACTGCCGGT

• Enterobacter cloacae (SEQ ID N° 8) GAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGAT

AACTACTGGAAACGGTAGCTAATACCGCATAATGTCGCAAGACCAAAGAGG

GGGACCTTCGGGCCTCTTGCCATCAGATGTGCCCAGATGGGATTAGCTAGTA

GGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATG

ACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCA

GTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTA

TGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGT

GGTTAATAACCGCAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACT

CCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTA

CTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCG

GGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTGGAGTCTTGTAGAGG

GGGGT AG A ATT

• Micrococcus luteus (SEQ ID N° 9)

GGGTGAGTAACACGTGAGTAACCTGCCCTTAACTCTGGGATAAGCCTGGGAA

ACTGGGTCTAATACCGGATAGGAGCGCCCACCGCATGGTGGGTGTTGGAAA

GATTTATCGGTTTTGGATGGACTCGCGGCCTATCAGCTTGTTGGTGAGGTAAT

GGCTCACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACA

CTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATA

TTGCACAATGGGCGAAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGG

CCTTCGGGTTGTAAACCTCTTTCAGTAGGGAAGAAGCGAAAGTGACGGTACC

TGCAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAG

GGTGCGAGCGTTATCCGGAATTATTGGGCGTAAAGAGCTCGTAGGCGGTTTG

TCGCGTCTGTCGTGAAAGTCCGGGGCTTAACCCCGGATCTGCGGTGGGTACG

GGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGA

ATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTG

TAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAGATACCC

TGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGACCATTCCACGG

TTTCCGCGCCGCAGCTAACGCATTAAGTGCCCCGCCTGGGGAGTACGGCCGC

AAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCAT

GCGGATTAATTCGATGCAACGCGAAAGAACCTTACCAAGGCTTGACATGTTC

TCGATCGCCGT Bacteria were grown until plateau phase before conducting in vitro tests. A bacterium, stored in the -80°C freezer, was defrosted and spread with a Drigalski spatula on an ACX-agar plate. This plate was incubated for 24 hours at 37°C. Afterwards, a colony was picked up from the medium and brought to a new ACX-plate by using the streak plate method. After 24h of incubation, a pure strain was brought into a lOmL tube with sterile nutrient broth. After 24h, the bacterium was ready for use. Growth curves of each selected strain was performed to check for exponential, plateau and decay phase. This was done measuring the maximal optical density (OD _max ) using a spectrophotometer.

1.3 IN VITRO EXPERIMENTS 1.3.1 Biolog plates

Phenotype microarray multiwell plates were filled with a series of acids and used to screen for potentially interesting acids increasing the growth of Staphylococcus epidermidis and other good- odour associated bacteria, versus decreasing the growth of malodour-associated bacteria. The microarray plates were inoculated with a fresh culture of the most prevalent underarm bacteria and incubated for 24h. Before, during and after that period, the growth was measures with a spectrophotometer. Incubation and measurement was also conducted for 48h. Measurements per compound was done in triplicate and the average of each measurement was used in downstream analyses.

Results

Experiment 1: Screening of 48 acid compounds

Phenotype microarray multiwell plates were assembled with a series of acids and used to screen for interesting compounds. Incubation occurred at 37°C for 24h and 48h. Results of the growth/inhibition of the good odour-associated bacteria relative to malodour-associated bacteria are presented in Figure 1. In the first phase, selection of interesting compounds occurs based on the following parameters:

Sum of growth/inhibition of beneficial deducted with the sum of growth/inhibition of detrimental bacteria Ability to lead to extra growth of S. epidermidis

Ability to inhibit Corynebacterium spp.

Ability to create extra growth of beneficial bacteria as compared to the detrimental bacteria In the second phase, selection of interesting compounds occurs based on the following parameters (Table 1):

Price

Colour

Odour

- Toxicity for humans/mammals.

Interesting compounds are listed in column“Compound” if good-odorous bacteria are increased in abundance and malodour-associated bacteria are not (significantly) increased or decreased in growth. The toxicity for human use (on skin) is checked as well as its odour, colour and its price. The toxicity is indicated with if it is toxic, irritant, or both for humans or on skin; if not toxic or irritant a“+” is assigned. Prices were looked up with Sigma- Aldrich and/or Alibaba. For odour, a“=” is assigned if no odour is present, a is assigned if it has a bad smell, and a“+” is assigned if it has a good odour. Information that is not available is indicated with NA. From the 48 acids, about 25 acids were withheld as interesting compounds (Table 1).

Table 1. Interesting compounds after 24h of incubation at 37°C for phenotype microarray plates. The toxicity, price (2018), odour and colour of the compound is checked.

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