Patent Description:
The skin microbiome is rich and complex and every skin niche has its own specific microbiome (<NUM>). Most of the microbiota living on the skin are harmless. Some bacteria are associated with the production of malodours, while others are not associated to malodours or are even associated to good odours (<NUM>). In the case of osmidrosis, body odour, bromhidrosis, the skin microbiome is different and considered responsible for the malodour formation (<NUM>).

Treatments of axillary body odour include the use of deodorants and antiperspirants (<NUM>), botulinum toxin injections (<NUM>), axillary liposuction (<NUM>), laser- or microwave-based ablation of sweat glands (<NUM>,<NUM>), antidepressants and antipsychotics (<NUM>), and topical antibiotics (<NUM>). Most methods focus on the sweat precipitation and not on the bacterial impact. To date, there is no lasting solution focusing against the microbiome of underarm body odour.

Deodorants and antiperspirants are widely used to mask body odour. Both use perfumes, to mask the malodour production, and generally use antimicrobial compounds, to limit the bacterial growth. Compounds with an antimicrobial and/or antifungal function that are commonly used are triclosan, triclocarsan, quaternary ammonium compounds, metal salts, aliphatic alcohols and glycols and other fragrances (<NUM>)(<NUM>). Antiperspirants generally contain aluminium salts, which blocks the sweat pores by mechanical obstruction. The ingredients with such action are aluminium chloride hexahydrate (ACH), aluminium chlorohydrate, aluminium sesquichlorohydrate, aluminium chlorohydrex and aluminium zirconium tetrachlorohydrate. The deodorants and antiperspirants of today do not adjust the microbiome. Contrary, most deodorants and antiperspirants will infer a shock to the microbial community and will lead to an increase in microbial diversity (<NUM>). This is unwanted as a higher microbial diversity is associated with a stronger underarm malodour. The abundance of corynebacteria may increase, which can lead to more malodour formation (<NUM>)(<NUM>).

The current deodorant and antiperspirants aims to reduce the microbial load, and thus also the enzymatic load.

Deodorant formulations upon today indeed target and suppress biochemical reactions that transform odour precursors into malodorous volatiles. For example, <CIT> discloses amino acid β-lyase inhibitors in deodorants. The latter document discloses this amino acid β-lyase as a malodour-forming enzyme and identifies ways to inhibit it. In other words, this document describes the inhibition of enzymes to block biochemical conversions. Also <CIT> and <CIT> describe inhibitors of bacterial sulfatases and glucuronidases, which are thought to reduce the steroidal malodour. Also here, agents to block and inhibit the enzymes responsible for biochemical conversion are disclosed. <CIT>) further describes agents to inhibit steroid reductase and thus malodour. <CIT>) (<NUM>) describes the enzyme Nα-acyl-glutamine-aminoacylase which is responsible for the release of thioalcohol-based malodour and a general formula containing ingredients to inhibit said enzyme. This enzyme can also be present in Staphylococcus spp. , namely S. hominis (<NUM>), and is therefore responsible for the release of the typical oniony sulphurous thioalcohol based malodour in the axillae. This document thus describes agents to inhibit the afore mentioned enzyme, in order to avoid the biochemical conversion with the release of the malodorous thioalcohol as result. Similarly, <CIT> describes a high throughput screening method for agents affecting the enzymes involved in the fatty acid biosynthesis.

However, the use of inhibitors in deodorants and applied on skin fall short. The biochemical reaction already largely occurred in the sweat glands and the hair roots, where the deodorant ingredients cannot or can barely penetrate. The inhibitors of microbial enzymes will therefore only be partially effective.

Hence, there is still a need to find alternative products and methods which are useful to combat malodour.

Contrary to the inhibition of enzymes, the use of enzymes to foster or encourage the biochemical conversion of sweat precursors has not been disclosed before.

The present invention discloses the addition of extra enzymes to fully convert the biochemical compounds in -as an example- the underarm. This invention does thus not describe the inhibition of microbial enzymes. In contrast, the present invention aims to add extra active enzymes to selectively steer the biochemical reaction towards the release of non-odorous volatiles. By promoting one biochemical reaction, we selectively create breakdown of sweat molecules that are not odorous, and avoid the biochemical breakdown of sweat molecules by the armpit microbiome into malodorous molecules.

In one aspect, the present invention discloses the use of specific, fatty acid degrading enzymes to enable fatty acid breakdown and sweat molecules on skin and clothes to encourage the complete breakdown of fatty acids release from sebaceous and apocrine sweat glands.

The present invention also further describes the use of specific enzymes to fully catabolize squalene and biochemically break it down towards non-malodorous molecules or useful building blocks. The present invention further discloses a series of specific enzymes that use squalene and converts it into farnesyl, mevalonate and acetyl. In other words, the present invention discloses the usage of enzymes to convert naturally secreted molecules on skin to produce other useful compounds on skin, rather than degrading and producing malodorous volatiles.

As indicated above, the current deodorants and antiperspirants aims to reduce the microbial load, and thus also to reduce the enzymatic load. In contrast, the present invention discloses to add extra specific enzymes to deodorants and antiperspirants in order to help the biochemical breakdown of sweat secretions. Indeed with the addition of extra specific enzymes, the present invention discloses to dissolve the sweat secretions and lipids into small compounds which don't have a bad odour.

In fact, the present invention relates to the finding that enzymes from Staphylococcus epidermidis have the ability to fully convert lipids secreted on the skin whereas Corynebacteria and other malodour associated bacteria lack the ability the fully catabolize the lipids. This is in contrast with the prior art wherein it is indicated that Corynebacteria contain a rich set of enzymes and that Corynebacterium spp. predominantly feed on skin secretions containing a variety of different lipid compounds (<NUM>). It is further indicated that to compensate for its incapability in synthesizing fatty acids, Corynebacterium spp. harbour a comprehensive set of enzymes involved in lipid metabolism encoded by multiple paralogous genes (<NUM>,<NUM>). This feature enables the organism to metabolize a broader range of lipid compounds (<NUM>). In contrast, the present invention shows that Staphylococcus epidermidis has a broader set of lipolytic enzymes than Corynebacterium spp. In addition, the present invention discloses that Staphylococcus epidermidis also has a broader set of squalene degrading enzymes. Squalene can convert into steroids, which can biochemically be broken down and can cause malodour (<NUM>).

The present invention thus discloses the usage of purified enzymes, bacterial fragments, dead or viable lyophilized bacteria, and/or bacterial lysates to combat skin malodour. The enzymes will fully catabolize skin lipids and squalene so that no malodour is generated. The enzymes may originate from Staphylococcus spp. , and more in particular from Staphylococcus epidermidis.

Lyophilization refers to the process in which bacteria are cultured and preserved by freezing very quickly and subsequently subjecting to a vacuum to remove the ice. Lyophilization is one of the most effective methods for the long-term preservation of cells. Freeze drying, known as lyophilization, is used to prepare bacterial culture that can be revived upon contact with moisture (<NUM>).

Hence, the present invention relates to the usage of lipolytic enzymes obtained from a bacterial Staphylococcus species to reduce the amount of malodorous short-chain fatty acids released from unusual, methyl-branched, odd-numbered long-chain fatty acids present in sweat. The human fatty acids present on the stratum corneum are often called "unusual", as no other animal contains such mixture of methyl-branched and odd-numbered fatty acids on the skin.

Fatty acids of the human skin consists of saturated fatty acids, among which and not limited to palmitic acid (C16), myristic acid (C14), stearic acid (C18), pentadecanoic acid (C15), heptadecanoic acid (C17); unsaturated fatty acids, among which and not limited to cis-hexadec-<NUM>-enoic acid (<NUM>:<NUM>Ω6), cis-octadec-<NUM>-enoic acid (<NUM>:<NUM>Ω8), oleic acid (<NUM>:<NUM>Ω9), petroselenic acid (<NUM>:<NUM>Ω6), cis-heptadec-<NUM>-enoic acid (<NUM>:<NUM>Ω6), cis-tetradec-<NUM>-enoic acid (<NUM>:<NUM>Ω6), sebaleic acid (<NUM>:<NUM>Ω5,<NUM>), cis-heptadec-<NUM>-enoic acid (<NUM>:<NUM>Ω8), linoleic acid (<NUM>:<NUM>Ω9,<NUM>), cis-eicos-<NUM>-enoic acid (<NUM>:<NUM>Ω10), cis-eicos-<NUM>,<NUM>-dienoic acid (<NUM>:<NUM>Ω7,<NUM>); iso-branched fatty acids, among which and not limited to <NUM>-methyltetradecanoic acid, cis-<NUM>-methylpentadec-<NUM>-enoic acid, cis-<NUM>-methylheptadec-<NUM>-enoic acid; anti-iso-branched fatty acids, among which and not limited to <NUM>-methyltetradecanoic acid, cis-<NUM>-methylhexadec-<NUM>-enoic acid (<NUM>,<NUM>,<NUM>). Malodorous short-chain fatty acids or 'malodorous fatty acids' are defined as, but not limited to, <NUM>-methyl-<NUM>-hexenoic acid (3M2H), <NUM>-hydroxy-<NUM>-methylhexanoic acid (HMHA) (<NUM>-<NUM>), <NUM>-methyl-<NUM>-octenoic acid, <NUM>-methyl-<NUM>-nonenoic acid, <NUM>-hydroxy-<NUM>-methylhexanoic acid, <NUM>-hydroxy-<NUM>-methylheptanoic acid, <NUM>-hydroxy-<NUM>-heptanoic acid, <NUM>-hydroxyoctanoic acid, <NUM>-hydroxy-<NUM>-methyloctanoic acid, <NUM>-hydroxy-<NUM>-methyloctanoic acid, <NUM>-hydroxy-<NUM>-methylnonacoic acid, <NUM>-hydroxydecanoic acid (<NUM>), isovaleric acid (<NUM>), <NUM>-ethyloctanoic acid, <NUM>-octenoic acid (<NUM>), <NUM>-octen-<NUM>-ol, <NUM>,<NUM>-octadien-<NUM>-one (<NUM>). Thorough reviews on the topic of short-chain fatty acids and other malodorants derived from human sweat can be found in Takeuchi et al. (<NUM>) and Martin et al.

The present invention thus relates to the usage of lipolytic or squalene degrading enzymes obtained from a bacterial Staphylococcus species to reduce the amount of malodorous fatty acids, preferably short-chain fatty acids, in sweat.

The present invention relates to the usage of lipolytic or squalene degrading enzymes as described above wherein said Staphylococcus species is Staphylococcus epidermidis.

The present invention also relates to the usage of lipolytic or squalene degrading enzymes as described above wherein said enzymes are administered as purified enzymes or as part of a bacterial fragment, dead bacterium or bacterial lysate or a viable lyophilized bacterium.

The present invention relates to the usage of lipolytic enzymes as described above wherein said enzymes are part of the beta-oxidation pathway or the fatty acid biosynthesis pathway or the synthesis and degradation of ketone bodies pathway or terpenoid backbone biosynthesis pathway or steroid biosynthesis pathway of a bacterial Staphylococcus species.

The present invention further relates to the usage of lipolytic enzymes as described above wherein said lipolytic enzymes which are part of the beta-oxidation pathway (or fatty acid degradation pathway) are chosen from the list consisting of: FadE (acyl CoA dehydrogenase), FadB (enoyl CoA hydratase), FadJ (<NUM>-hydroxyacyl-CoA dehydrogenase), FadA (β-ketothiolase).

The present invention also relates to the usage of lipolytic enzymes as described above wherein said lipolytic enzymes which are part of the fatty acid biosynthesis pathway are chosen from the list consisting of: AccA (acetyl-CoA carboxylase), AccB (acetyl-CoA carboxylase), AccC (acetyl-CoA carboxylase), AccD (acetyl-CoA carboxylase), FabD (malonyl-CoA:ACP transacylase), FabH (β-ketoacyl-ACP synthases), FabG (NADPH-dependent β-ketoacyl-ACP reductase), FabZ (<NUM>-hydroxyacyl-ACP dehydratase), FabA (β-hydroxydecanoyl-ACP dehydrase), FabB (<NUM>-ketoacyl-ACP synthases I), FabF (β-ketoacyl-ACP synthase (chain elongation)), FabI (enoyl-ACP reductase), FabL (enoyl-ACP reductase) and FabK (enoyl-ACP reductase).

The present invention further relates to the usage of lipolytic enzymes as described above wherein said enzymes which are part of the synthesis and degradation of ketone bodies are chosen from the list consisting of: acetyl-CoA C-acetyltransferase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA lyase, <NUM>-oxoacid CoA-transferase, acetoacetate decarboxylase, <NUM>-hydroxybutyrate dehydrogenase.

The present invention also relates to the usage of squalene degrading enzymes as describe above, wherein said squalene degrading enzymes are chosen from the list consisting of: farnesyl-diphosphate farnesyltransferase, farnesyl diphosphate synthase, diphosphomevalonate decarboxylase, phosphomevalonate kinase, mevalonate kinase, hydroxymethylglutaryl-CoA reductase, hydroxymethylglutaryl-CoA synthase, acetyl-CoA C-acetyltransferase. Through usage of the enzymes indicated above, the breakdown products of the lipid and squalene metabolism can be further used into the metabolism of bacteria or bacterial cell mass. Indeed, the present invention relates to the usage of squalene catabolizing enzymes to degrade to building blocks such as farnesyl, mevalonate and acetyl. Through the use of said enzymes, squalene is completely broken down, and no longer converted to steroids and can no longer lead to malodorous compounds.

The present invention further relates to the usage of lipolytic enzymes or squalene degrading enzymes as described above wherein lipases, amylases, proteases and/or cellulases which are obtained from any microbial species are further added to said lipolytic or squalene degrading enzymes.

The present invention also relates to the usage of lipolytic or squalene degrading enzymes as described above wherein said sweat is present on skin and/or textiles.

The present invention relates to the following enzymes that fully catabolize sweat secretions into non malodorous side-products. The enzymes are part of the fatty acid degradation and fatty acid biosynthesis pathways: FadE (acyl CoA dehydrogenase), FadB (enoyl CoA hydratase), FadJ (<NUM>-hydroxyacyl-CoA dehydrogenase), FadA (β-ketothiolase), AccA (acetyl-CoA carboxylase), AccB (acetyl-CoA carboxylase), AccC (acetyl-CoA carboxylase), AccD (acetyl-CoA carboxylase), FabD (malonyl-CoA:ACP transacylase), FabH (β-ketoacyl-ACP synthases - initial condensation with acetyl-CoA and branched acyl-CoAs), FabG (NADPH-dependent β-ketoacyl-ACP reductase), FabZ (<NUM>-hydroxyacyl-ACP dehydratase), FabA (β-hydroxydecanoyl-ACP dehydrase), FabB (<NUM>-ketoacyl-ACP synthases I), FabF (β-ketoacyl-ACP synthase (chain elongation)), FabI (enoyl-ACP reductase), FabL (enoyl-ACP reductase) and FabK (enoyl-ACP reductase).

More specifically, the present invention relates to the following enzymes which are part of the KEGG synthesis and degradation of ketone bodies (and use acetyl-CoA for further uptake into bacterial metabolism): acetyl-CoA C-acetyltransferase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA lyase, <NUM>-oxoacid CoA-transferase, acetoacetate decarboxylase, <NUM>-hydroxybutyrate dehydrogenase. Through usage of these enzymes, the breakdown products of the lipid and squalene metabolism can be further used into the metabolism of bacteria or bacterial cell mass.

The present invention further relates to the usage of lipolytic enzymes as described above wherein lipases, amylases, proteases and/or cellulases which are obtained from any microbial species are further added to said lipolytic enzymes.

More specifically, the present invention relates to the following enzymes which degrade squalene into smaller non-odorous compounds: farnesyl-diphosphate farnesyltransferase, farnesyl diphosphate synthase, diphosphomevalonate decarboxylase, phosphomevalonate kinase, mevalonate kinase, hydroxymethylglutaryl-CoA reductase, hydroxymethylglutaryl-CoA synthase, acetyl-CoA C-acetyltransferase. The usage of squalene catabolizing enzymes promotes the conversion into useful building blocks for microbial biomass, non-malodorous molecules or even good odorous molecules, such as farnesyl, mevalonate and acetyl. Through the use of said enzymes, squalene is completely broken down, and no longer converted to steroids and can no longer lead to malodorous compounds. These enzymes are naturally present in Staphylococcus spp. and can be obtained and purified from their cells.

The present invention thus relates in first instance to composition which degrade lipids and/or fatty acids secreted by the skin into smaller compounds that do not have a bad smell. The composition can be enzymes and/or bacterial lysates and/or inactive bacteria and/or lyophilized bacteria that have the enzymatic potential to catabolize the precursors that normally lead to underarm malodour into molecules which no longer smell bad. The enzymes and/or bacterial lysates and/or inactive bacteria (i.e. bacterial fragment and or dead bacteria) and/or living or lyophilized bacteria of the present invention can be obtained by any manner known in the art and/or as specified further in the examples.

Underarm malodour is characterized by a sour, musty, sharp, oniony and/or faecal-like smell. The enzymes described in this invention can convert the precursor and/or intermediate breakdown products into molecules which no longer have the above described odour.

Skin and textile malodour is generated due to bacterial biotransformation of sweat secretions. There are four major routes that lead to underarm/skin malodour.

The present invention relates to methods that mainly impacts the first and second route to malodour. The present invention does not relate to the inhibition of specific enzymes that cleave off L-glutamine residues or cysteine residues from malodour precursors.

The present invention reduces the formation of malodorous (short-chain) fatty acids (released from human methyl-branched, odd-numbered long-chain fatty acids). The long-chain fatty acids present in human sweat, released from sebaceous and apocrine sweat, is with this invention completely broken down into ATP and building blocks for bacterial de novo fatty acid synthesis. The pathways leading to malodour are converted towards pathways that no longer cause malodorous volatiles. The fatty acids are fully catabolized by the administered enzymes, when applied in an effective amount of a composition as defined above to a mammal in need thereof.

Triplicate bacterial samples were taken from the axillary skin of <NUM> healthy subjects. Sample demographic characteristics can be found in Table <NUM>. Subjects younger than <NUM> years were accompanied by a parent. All participants were Belgian, except two participants from The Netherlands and one from France. Samples were collected on three different days spread over a period of six weeks. The participants were invited to Ghent and were asked not to use deodorants or antiperspirants three days prior to sampling and not to wash their axillae on the day of sampling. No attempts were made to control the subjects' diet. All participants were asked to fill in their personal metadata and a questionnaire regarding the effect of the malodour on their daily lives. All participants attached a sterile cotton pad under their left axilla for <NUM> for odour assessment. All participants gave their written and informed consent to this research.

A sterile cotton swab (Biolab, Belgium) was moistened in physiological water and swabbed for <NUM> in the axillary region to detach and absorb the microorganisms. The tip was suspended and broken in a sterilized reaction tube filled with <NUM> of sterile physiological water (<NUM>). The bacterial samples were immediately stored at <NUM> not longer than <NUM> prior to further analysis. The participants attached a sterile cotton pad with a dressing under their left axilla for <NUM> to absorb the odour. Afterwards, the cotton pad was brought into a closed numbered goblet until odour assessment by an odour panel.

The odour panel was trained and selected and samples were rated as previously described (<NUM>). Odour measurement was performed by means of a cotton pad worn in the axilla for <NUM>. The cotton pads were presented in a numbered odourless sealed glass goblet in a random manner to a panel of six selected and screened human assessors (four men, four women). Assessors were selected by means of sensitivity to dilutions of n-butanol and wastewater, and by means of the triangle test (<NUM>). In the triangle test, each member of the panel was presented three flasks, two of which were the same but the third contained a different odour. The flask was shaken, the stopper was removed, after which the vapours were evaluated. The panellists had to correctly identify the flask with the different odour. In the dilution test, each member of the panel was presented six flasks with increasing concentration of n-butanol and wastewater, starting with a flask without addition of n-butanol or wastewater. The panellists had to correctly place the flasks according concentration. The triangle and dilution test was repeated three times, with a minimum of two days in between each measurement. Assessors with minimum <NUM>% correct answers were selected for the panel. A representative panel was recruited from a pool of the <NUM> people. Training of the assessors was conducted through odour references (ammonia, cheese, axillary sample) and experience. The references were assessed in group to compromise hedonic value and intensity. The room in which the tests were conducted, was free from extraneous odour stimuli e.g. caused by smoking, eating, soaps, perfume, etc. The odour assessors were familiar with the olfactometric procedures and met the following conditions: (i) older than <NUM> years and willing to follow the instructions; (ii) no smoking, eating, drinking (except water) or using chewing gum or sweets <NUM> before olfactometric measurement; (iii) free from colds, allergies or other infections; (iv) no interference by perfumes, deodorants, body lotions, cosmetics or personal body odour; (v) no communication during odour assessment. About <NUM> odour measurements were performed per day, spread over a timeframe of <NUM>, to prevent fatigue.

The samples of the observational study were assessed by the following odour characteristic: intensity (scale <NUM> to <NUM>). The intensity indicates the quantity of the odour and varies between <NUM> (no odour) to <NUM> (very strong / intolerable). Each time, a control odour measurement, a clean cotton pad with random number, was served to the odour panel together with the other samples. Odour assessment was in the observational study additionally performed via direct odour assessment (first <NUM> participants), and a self-evaluation (all participants).

Total DNA extraction was performed as previously described (<NUM>). Briefly, the bacterial sample, dissolved in <NUM> of saline water, was centrifuged (<NUM>, <NUM>,<NUM> rpm) to obtain a pellet, while the supernatant was discarded. The pellet was washed and resuspended in <NUM>µL <NUM>% Chelex <NUM> resin (BioRad, Munich, Germany) and incubated at <NUM> for <NUM>. The removal of PCR inhibitors and metal ions was accomplished by means of Chelex-<NUM>. The sample was then firmly vortexed and boiled at <NUM> for <NUM>. Subsequently, the sample was mixed and cooled for <NUM> on ice. Next, a centrifugation step (<NUM>, <NUM>,<NUM> rpm) was performed. The supernatant containing the DNA was removed and stored at -<NUM> until further analysis.

The polymerase chain reaction (PCR) is a technique to amplify a piece of DNA, generating millions of copies of a particular DNA sequence. This method consists of repeated cycles of heating and cooling, which promotes the melting and separation of the double-stranded DNA. A target region of interest in the <NUM> rRNA bacterial gene is selected as a marker for thermal cycling, consisting of the reaction for DNA melting and enzymatic replication of the DNA. At present, the bacterial ribosomal RNA operon, encompassing the <NUM> rRNA gene, is the most frequently used molecular marker. The bacterial ribosomal RNA operon, encompassing the <NUM> rRNA gene, is the most frequently used molecular marker. The hypervariable regions of the gene (V regions) has a high discriminatory potential (<NUM>) and contains the signatures of phylogenetic groups and even species. It enables an accurate description of the microbial populations in a community (<NUM>). Therefore, molecular techniques based on <NUM> rDNA can be useful tools to gain insight on phylogenetic and functional relationships among the microbiota of any given environment. Two primers were used to amplify a target region of the <NUM> rRNA gene. As PCR progresses, the sequences generated are used as a template for replication and exponentially amplified with each cycle.

Genomic DNA axillary samples from <NUM> participants were amplified with conserved <NUM> rRNA gene primers generating <NUM>-bp amplicons. The <NUM> rRNA gene regions were amplified by PCR using the 27F and 338R primers, targeting the V1 region and libraries for Illumina sequencing were constructed as previously described (<NUM>). Briefly, the forward primer contains a <NUM> nucleotide (nt) barcode45 and a <NUM> nt CA linker (<NUM>). Both primers comprised sequences complementary to the Illumina specific adaptors to the <NUM>'-ends. Amplification was performed in a total volume of <NUM>µL with 5x PrimeSTAR buffer, containing <NUM> deoxynucleoside triphosphate, <NUM> of each primer, <NUM>µL template DNA and <NUM>µL PrimeSTAR HS DNA polymerase (<NUM>. PCR was performed with the following conditions: initial denaturation (<NUM> - <NUM>), followed by <NUM> cycles of denaturation, annealing and elongation (<NUM> - <NUM> sec; <NUM> - <NUM> sec and <NUM> - <NUM> sec). One µL of this reaction mixture served as template in a second PCR performed under the same conditions, but for <NUM> cycles using PCR primers designed to integrate the sequence of the specific Illumina multiplexing sequencing primers and index primers. PCR amplicons were verified by agarose gel electrophoresis, purified using Macherey-Nagel <NUM>-well plate purification kits (Macherey-Nagel, Germany) following the manufacturer's instructions and quantified with the Quant-iT PicoGreen dsDNA reagent and kit (Invitrogen, UK). Libraries were prepared by pooling equimolar ratios of amplicons (<NUM> ng of each sample), all tagged with a unique barcode. Each library was precipitated on ice for <NUM> after addition of <NUM>µl of <NUM> NaCl and <NUM> volumes of ice-cold <NUM>% ethanol to remove contaminants or PCR artefacts. The precipitated DNA was centrifuged at <NUM>,<NUM>×g for <NUM> at <NUM>. The supernatant was removed, the pellet air dried, resuspended in <NUM>µL of double-distilled water and separated on a <NUM>% agarose gel. PCR products of the correct size were extracted and recovered using the QIAquick gel extraction kit (Qiagen, Belgium). Negative controls (water as template) were performed and were free of any amplification products after PCR. Libraries were sent for paired-end sequencing on a MiSeq Genome Analyzer (Illumina Inc. , California, USA). Image analysis and base calling were accomplished using the Illumina Pipeline (version <NUM>).

Demultiplexed and quality-controlled sequences were clustered against the Greengenes (<NUM>) database using the closed reference OTU picking protocol (<NUM>) as implemented in QIIME1. <NUM> (<NUM>). These processing steps were performed using default parameters. The OTU table used for primary analysis was filtered. Finally, the table was rarefied to normalize for sample effort at <NUM> sequences per sample (<NUM>). Taxonomy was determined and alpha- and beta-diversity calculations were performed using QIIME version <NUM>.

Indications of functionality from phylogenetic information was obtained with PICRUSt (<NUM>). The open-source software allows reconstruction of the bacterial metagenomes of the obtained results based on the bacterial <NUM> rRNA gene sequence. The generated OTU table from the <NUM> sequencing data was used as an input. The copy number per OTU was normalized before the metagenome was predicted using Kyoto Encyclopedia of Genes and Genomes (KEGG) database (<NUM>). The metagenome prediction provided an annotated table of predicted gene family counts for each sample, where gene families were grouped by KEGG Orthology (KO) identifiers (<FIG>).

The bacterial cells are grown to late log or early stationary phase for about <NUM> in LB medium till an OD<NUM> of about <NUM>. The cells are harvested by centrifugation (<NUM> rpm/<NUM>), after which the supernatant is discarded. The cells are washed in sterile M9 medium. To create <NUM> of M9 medium, one needs at first a salt solution (<NUM>) that consists of: <NUM> Na<NUM>HPO<NUM>. <NUM><NUM>O, <NUM> KH<NUM>PO<NUM>, <NUM> NaCl, <NUM> NH<NUM>Cl. All compounds are added to a Schott bottle of <NUM> which is diluted with distilled water until one reaches <NUM>. This solution is autoclaved prior to use. Again, the cells are harvested by centrifugation; after which the supernatant is discarded. The bacterial biomass is suspended in an equal volume of lyophilization medium. The lyophilization medium contains suitable lyoprotectants and excipients, preferably sucrose and mannitol. The mixture is mixed thoroughly by vortexing. The cell suspension is distributed over a flat surface prior to freezing. The cell suspension is kick-frozen in -<NUM> in liquid nitrogen prior to the eventual freeze-drying. The frozen suspension is freeze dried and lyophilized (under vacuum pressure). A cell count is performed after the freeze-drying protocol (dilution to extinction protocol). The final concentration should be around <NUM><NUM> CFU/g.

The bacterial culture is grown for <NUM> in LB medium till an OD<NUM> of about <NUM>; till the bacteria are in plateau phase. The cells are harvested by centrifugation (<NUM> rpm/<NUM>), after which the supernatant is discarded. The dense culture (pellet) is frozen at -<NUM>. About <NUM> of the pellet is resuspended in <NUM> lysis buffer. The lysis buffer contains <NUM> Tris pH <NUM>; <NUM>% glycerol; <NUM>% Triton X-<NUM>; 100ug/ml lysozyme; <NUM> PMSF and /or more anti-proteases; DNAse 3U and <NUM> MgCl on the final concentration. The pellet and lysis buffer are incubated on <NUM> for <NUM> minutes or <NUM> on ice. The resulting solution is sonicated 3x20" till sample is no longer viscous. Ultrasonication at frequencies around <NUM> was used to kill bacteria and to release their enzymatic contents. It was for many years has been standard technique in microbiology for the disruption of living cells (<NUM>). The solution is subsequently centrifuged at <NUM>,000rpm for <NUM> at <NUM>. The solution is transferred to new tubes and re-suspended in <NUM> lysis buffer. Then 60ul of the solution is centrifuged, after which 20µl 4X sample buffer with <NUM> DTT. The rest of the lysates is frozen in -<NUM>. A cell count is performed after the lysis protocol (dilution to extinction protocol). The final concentration should be <NUM> CFU/g.

Staphylococcal enzymes were produced as follows: <NUM>/ sonication to disrupt the bacterial cells (<NUM>) as described before, <NUM>/ centrifugation to separate the large particles from the enzymes, <NUM>/ bringing it in solution, <NUM>/ purification and precipitation of proteins (enzymes) using ammonium sulphate and crystallization by changing the pH (<NUM>).

The activity of the enzymes of the bacterial lysates and the purified enzymes was verified using a turbidity assay. We tested for the ability to degrade lipids using a turbidity assessment assay as adopted from Lawrence et al. (<NUM>), and further modifications (<NUM>). Human lipids, as obtained from liposuction and as described earlier (<NUM>), were emulsified in agar and poured into Petri dishes. The agar consistency was very turbid as a result of the lipids in the agar. The bacterial lysates and purified enzymes were brought onto the solid agar in droplets. Hydrolysis of the lipid emulsion was observed by formation of a zone of clearance around the droplet. Lipophilic enzyme activity was measured by the clearing zone on and around the droplet. Degradation of dense human lipids and conversion into different compounds led to a change in turbidity. A positive control with living bacteria and a negative control using no bacteria or enzymes was also assessed.

A spray was assembled using living lyophilized bacteria, inactive/dead bacterial lysates containing viable enzymes and the purified enzymes. Subjects were selected with above-average malodorous axillae (Table <NUM>), as determined by the odour panel and six subjects were recruited (section <NUM>. A one-month follow-up was scheduled, where the underarm odour was measured every week. A spray was prepared containing the live bacteria, the bacterial lysates/purified enzymes. This spray was used on a daily basis (once or twice application per day).

Subjects with malodorous axillae were recruited to conduct an experiment, where they applied a spray solution containing lyophilized S. epidermidis bacteria. The content of the spray was as follows:.

It was tested and verified on agar plate that the final concentration of bacteria with one normal spray is about <NUM><NUM> CFU/spray. The subject's underarm odour was assessed, as described above (<NUM>. <NUM>), before, during and after application of the spray. The subject applied the spray on a daily basis during four consecutive weeks, where the armpit was sampled every week during that period.

Ultrasonication at frequencies around <NUM> was used to kill bacteria and to release their enzymatic contents. It was for many years has been standard technique in microbiology for the disruption of living cells (<NUM>). A spray was created containing the non-viable S. epidermidis bacterial lysates, containing active enzymes. Other enzymes, such as lipases, amylases, proteases, cellulases, farnesyl-diphosphate, were purchased through normal commercial canals: Christian Hansen, Denmark; Novozymes, Denmark; DuPont, USA; Lallemand, France; or DSM, The Netherlands. The enzymes were either obtained as purified enzymes or as microbial lysate. The content of the spray was similar as before:.

The subject's underarm odour was assessed, as described above (<NUM>. <NUM>), before, during and after application of the spray. The subject applied the spray on a daily basis during four consecutive weeks, where the armpit was sampled every week during that period.

<FIG> shows the enzymatic load of each bacterial group, which originates from the armpit observation study. It can be noticed that in the class of lipid metabolism enzymes Staphylococcus epidermis has a white colour (higher enzymatic content), while Corynebacterium has a black colour (lower enzymatic content). <FIG> shows a more detailed where the KEGG pathways were collapsed to level <NUM>, for the fat metabolising enzymes only. Again, clear differences are seen between staphylococcal and corynebacterial enzymes for fat metabolism. In <FIG> finally, the lipid degradation and synthesis have been highlighted onto enzyme level. It can clearly be seen that the white lines (enzymes) are more abundant for Staphylococcus than for Corynebacterium. Staphylococcus is correlated with better underarm odours, while Corynebacterium is correlated with unpleasant body odours (fecal-like, sour, dirty, pungent, etc). In conclusion: a higher load of lipid degrading/catabolizing/synthetizing enzymes are needed to improve body odour.

Hence, the present invention discloses the following enzymes which are used in the battle against body odour:.

The function of each of the enzymes is given hereunder:.

A more thorough review on the bacterial enzymes as described above can be found with Fujita et al. (<NUM>) (<NUM>).

Three subjects with above-average malodorous axillae were selected, as determined by the odour panel. The subjects used the lyophilized bacterial spray on a daily basis during four consecutive weeks. In the week preceding the treatment, no bacteria were applied in the underarms. The subjects wore a cotton pad in the underarm to capture the odour, after which the sample was frozen till odour assessment by the trained odour panel. The subjects self-assessed the underarm odour and marked significant improvements during the use of the spray. The improved odour was also reported on clothing and elsewhere on the skin when the bacterial spray were applied. The trained and selected odour panel reported a significant decrease (as determined by the Mann-Whitney U-test; p<<NUM>) in underarm intensity when the bacteria were applied, as compared to when no bacteria were applied (<FIG>).

Claim 1:
Use of lipolytic enzymes or squalene degrading enzymes obtained from a bacterial Staphylococcus species to reduce the amount of malodorous fatty acids in sweat wherein said lipolytic enzymes, are chosen from the list consisting of: FadE (acyl CoA dehydrogenase), FadB (enoyl CoA hydratase), FadJ (<NUM>-hydroxyacyl-CoA dehydrogenase), FadA (β-ketothiolase), AccA (acetyl-CoA carboxylase), AccB (acetyl-CoA carboxylase), AccC (acetyl-CoA carboxylase), AccD (acetyl-CoA carboxylase), FabD (malonyl-CoA:ACP transacylase), FabH (β -ketoacyl-ACP synthases), FabG (NADPH-dependent β -ketoacyl-ACP reductase), FabZ (<NUM>-hydroxyacyl-ACP dehydratase), FabA (β-hydroxydecanoyl-ACP dehydrase), FabB (<NUM>-ketoacyl-ACP synthases I), FabF (β -ketoacyl-ACP synthase (chain elongation)), FabI (enoyl-ACP reductase), FabL (enoyl-ACP reductase), FabK (enoyl-ACP reductase), acetyl-CoA C-acetyltransferase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA lyase, <NUM>-oxoacid CoA-transferase, acetoacetate decarboxylase, <NUM>-hydroxybutyrate dehydrogenase, and, wherein said squalene degrading enzymes are chosen from the list consisting of: farnesyl-diphosphate farnesyltransferase, farnesyl diphosphate synthase, diphosphomevalonate decarboxylase, phosphomevalonate kinase, mevalonate kinase, hydroxymethylglutaryl-CoA reductase, hydroxymethylglutaryl-CoA synthase and acetyl-CoA C-acetyltransferase, and, wherein said malodorous fatty acids are chosen from the list consisting of <NUM>-methyl-<NUM>-hexenoic acid (3M2H), <NUM>-hydroxy-<NUM>-methylhexanoic acid (HMHA), <NUM>-methyl-<NUM>-octenoic acid, <NUM>-methyl-<NUM>-nonenoic acid, <NUM>-hydroxy-<NUM>-methylhexanoic acid, <NUM>-hydroxy-<NUM>-methylheptanoic acid, <NUM>-hydroxy-<NUM>-heptanoic acid, <NUM>-hydroxyoctanoic acid, <NUM>-hydroxy-<NUM>-methyloctanoic acid, <NUM>-hydroxy-<NUM>-methyloctanoic acid, <NUM>-hydroxy-<NUM>-methylnonacoic acid, <NUM>-hydroxydecanoic acid, isovaleric acid, <NUM>-ethyloctanoic acid, <NUM>-octenoic acid, <NUM>-octen-<NUM>-ol, and <NUM>,<NUM>-octadien-<NUM>-one.