Method to form supernatant

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

[0001] METHOD TO FORM SUPERNATANT

[0002] TECHNICAL FIELD

[0003] The present invention relates to a method to form a supernatant from calcium silicate and its use.

[0004] BACKGROUND

[0005] Silicate ions are known to stimulate the formation of collagen. This effect is sought for when eating silica as food supplement to improve skin quality. Also, silicate ions are used to stimulate bone formation in bone void filler materials, also via the formation of collagen. There is a need for formulations that can deliver silicate ions locally also to hair, skin and nails and thereby achieve a high local high concentration of silicate to stimulate collagen formation in hair, skin or nails.

[0006] WO 2021 / 177882A1 discloses formulations for use on nails, skin or hair, comprising calcium silicates powders for local release of calcium and silicon ions.

[0007] There is still a need for method of producing formulations to be used in promoting collagen formation.

[0008] SUMMARY

[0009] It is an objective of the present invention to produce formulations that can be used in promoting collagen formation.

[0010] This and other objectives are met by embodiments disclosed herein.

[0011] The present invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0012] An aspect of the invention relates to a method of producing an ion-containing supernatant. The method comprises contacting water or an aqueous solution with a solid phase comprising calcium silicate at a mass ratio of water or the aqueous solution to the solid calcium silicate containing phase selected within an interval of from 1 :1 up to 1,000:1. The method also comprises removing any solid calcium silicate containing phase from the water or the aqueous solution to form a calcium silicate supernatant comprising calcium ions and silicate ions. The method further comprises adjusting, if a pH of the calcium silicate supernatant exceeds 8.5, the pH of the calcium silicate supernatant by addition of an acid to a pH selected within an interval of from 4.5 up to 8.5. Further aspects of the invention relate to a calcium silicate supernatant in the form of water or an aqueous solution comprising calcium ions and silicate ions and having a pH selected within an interval of from 4.5 up to 8.5, and a cosmetic formulation comprising such a calcium silicate supernatant.

[0013] Another aspect of the invention relates to a method of local delivery of silicate ions comprising applying a calcium silicate supernatant or a cosmetic formulation according to above to at least a portion of the hair, skin and / or nails of a subject, preferably a human subject.

[0014] Yet another aspect of the invention relates to a calcium silicate supernatant or a cosmetic formulation according to above for use in stimulating collagen formation in hair, skin and / or nails of a subject. The calcium silicate supernatant or the cosmetic formulation is then formulated for local application to at least a portion of the hair, skin and / or nails of a subject.

[0015] The present invention provides a method of producing a calcium silicate supernatant that can be used for local delivery of silicate ions to, for instance, the hair, skin and / or nails to thereby stimulate local collagen formation.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:

[0018] Fig. 1 is a graph showing ion concentration as a function of time during manufacture of a calcium silicate supernatant according to an embodiment;

[0019] Fig. 2 is a graph showing ion concentration as a function of time during manufacture of a calcium silicate supernatant according to another embodiment; and

[0020] Fig. 3 is a flow chart illustrating a method of producing an ion-containing supernatant according to an embodiment.

[0021] ABBREVATIONS AND DEFINITIONS

[0022] Monocalcium silicate - CaOSiO2, also expressed as CaO SiO? or CaSiOs and referred to as CS; Dicalcium silicate - (CaOJsSiC , also expressed as 2CaO SiO2 or Ca2SiO4 and referred to as C2S;

[0023] Tricalcium silicate - (CaO)aSiO2, also expressed as 3CaO SiO2 or CasSiOs and referred to as C3S;

[0024] Bioglass - means bioglass 45S5, also referred to as calcium sodium phosphosilicate, which is a bioactive glass composed of 45 wt% SiO2, 24.5 wt% CaO, 24.5 wt% Na2O, and 6.0 wt% P2O5;

[0025] % by weight (wt%) - the weight fraction of a compound in relation to the total weight of a mixture or composition including the component, expressed in percent;

[0026] Calcium ion - Ca2+;

[0027] Silicate ion - wherein 0 < x < 2 and 1 < n < 2, and includes orthosilicate SiO ~(x = 0, n = 1), metasilicate SiOl~{x = 1 , n = 1), and pyrosilicate Si2O^~(x = 0.5, n = 2); and

[0028] Hydroxide - OH-.

[0029] DETAILED DESCRIPTION

[0030] The present invention generally relates to the production of a calcium silicate supernatant, such a calcium silicate supernatant and the use thereof in cosmetic formulations for local delivery of silicate ions to stimulate local collagen production.

[0031] Calcium silicate is a chemical compound comprising calcium and silicate, commonly in the form of a powder. It exists in different forms, such as monocalcium silicate (CaOSiO2), dicalcium silicate ((CaO)2SiO2), and tricalcium silicate ((CaO)3SiO2). It can also be referred to, and is often traded as, CS, C2S, C3S, meta-silicate or Cai-Sil or Calsil.

[0032] Calcium silicate is used in different pharmaceutical and food applications as an anti-caking agent, as an antiacid, but it can also be used in other applications, for example, in agriculture as a source of silicon.

[0033] The following calcium silicate phases are suitable for use according to the invention, monocalcium silicate (CaOSiCh), dicalcium silicate ((CaO)2SiO2), and tricalcium silicate ((CaO)3SiO2). Preferably the monocalcium silicate phase. Such calcium silicate salts are soluble in water (or humid environment) and provide a source for forming a supernatant containing calcium ions (Ca2+), silicate ions and hydroxide.

[0034] The pH of such formed supernatant is, prior to any pH adjustment, above 9 at room temperature in water.

[0035] The present invention describes a method to form a supernatant from calcium silicate and the use of the supernatant as collagen growth promotor agent.

[0036] The solubility of calcium silicates, which are a Ca-salts, increases with the amount of calcium in the calcium silicate. Without being bound by any theory, the present invention takes advantage of the solubility of the calcium silicate and release of different ions, such as hydroxide, calcium ions and silicate ions to promote collagen formation in humans and animals.

[0037] As another part of the invention described herein the calcium silicate can be replaced by a bioglass. Hence, herein ‘calcium silicate’ can be exchanged for ‘bioglass’ and, thus, in one aspect of the invention there is a there is a bioglass powder material. The supernatant from a bioglass powder can be used as collagen promotor.

[0038] The parameters for the supernatant formation:

[0039] • Ratio: water to powder by weight: 1 :1 to 1000:1 ;

[0040] • Temperature: 10 degrees to 90 degrees Celsius; and

[0041] • Time: 5 minutes to 2 hours, even shorter time can be used for e.g., C3S at 90 degrees Celsius.

[0042] The remaining powder can be sieved or preferably centrifuged from the supernatant. pH of the calcium silicate supernatant > 9 when demineralized water is used.

[0043] An apparatus with where the calcium silicate is in contact water-based liquid without the need of filtering or centrifuging is also included in the invention. The calcium silicate is in a “tea bag” of suitable material and porosity and dipped into water. Water can thereby also be in a continuous process.

[0044] An aspect of the invention relates to method of producing an ion-containing supernatant, see Fig. 3. The method comprises contacting, in step S1 , water or an aqueous solution with a solid phase comprising calcium silicate at a mass ratio of the water or the aqueous solution to the solid phase selected within an interval of from 1 :1 up to 1 ,000:1. The method also comprises removing, in step S2, any solid phase from the water or the aqueous solution to form a calcium silicate supernatant, also referred to as calcium silicate solution herein, comprising calcium ions and silicate ions. The method further comprising adjusting, in step S4 and if a pH of the calcium silicate supernatant exceeds 8.5, the pH of the calcium silicate supernatant by addition of an acid to a pH selected within an interval of from 4.5 up to 8.5.

[0045] Step S1 of the method thereby comprises contacting water or an aqueous solution with a solid phase comprising calcium silicate, also referred to as solid calcium silicate containing phase herein, at a selected mass ratio. The removal of any remaining solid phase from the water or the aqueous solution in step S2 results in a calcium silicate supernatant typically having a basic pH above 8.5, typically equally to or above 9, or indeed even higher. Accordingly, the method preferably also comprises adjusting the pH of the calcium silicate supernatant in step S4 to a pH that is suitable for local delivery of silicate ions to, for instance, the hair, skin and / or nails to thereby stimulate local collagen formation. Accordingly, the pH of the calcium silicate supernatant is adjusted to a pH selected within an interval of from 4.5 up to 8.5 in step S4.

[0046] In an embodiment, contacting the water or the aqueous solution in step S1 comprises contacting, in step S1 , the water or the aqueous solution with the solid phase at a temperature selected within an interval of from 10°C up to 90°C.

[0047] In a particular embodiment, the temperature is selected within an interval of from 20°C up to 80°C. In another particular embodiment, the temperature is selected within an interval of from 20°C up to 70°C.

[0048] In an embodiment, the mass ratio of the water or the aqueous solution to the solid phase is selected within an interval of from 1 :1 up to 100:1.

[0049] In a particular embodiment, the mass ratio of the water or the aqueous solution to the solid phase is selected within an interval of from 5:1 up to 100:1.

[0050] In an embodiment, contacting the water or the aqueous solution in step S1 comprises contacting, in step S1 , the water or the aqueous solution with the solid phase for a duration selected within an interval of from 1 minutes up to 2 hours.

[0051] In a particular embodiment, the duration is selected within an interval of from 2 minutes up to 2 hours. In another particular embodiment, the duration is selected within an interval of from 5 minutes up to 2 hours. In a further particular embodiment, the duration is selected within an interval of from 0.5 hours up to 1.5 hours.

[0052] In an embodiment, the solid phase is selected from the group consisting of monocalcium silicate (CaOSiOs), dicalcium silicate ((CaOJsSiCh), tricalcium silicate ((CaO)3SiO2), calcium sodium phosphosilicate, and any combination thereof.

[0053] In a particular embodiment, the solid phase is selected from the group consisting of monocalcium silicate (CaOSiOs), dicalcium silicate ((CaO)2SiO2), tricalcium silicate ((CaOjsSiCh), and any combination thereof. In another particular embodiment, the solid calcium silicate containing phase is monocalcium silicate (CaOSiO2).

[0054] In an embodiment, the calcium silicate supernatant has, prior to pH adjustment in step S4, a pH above 8.5, most often above 9 and higher when using water or a non-buffered aqueous solution.

[0055] In a particular embodiment, the calcium silicate supernatant has, prior to pH adjustment in step S4, a pH selected within in interval of from 8.5 up to 12.5 wherein the pH of the calcium silicate supernatant is at least partly dependent on the calcium content of the solid calcium silicate containing phase. For instance, a calcium silicate supernatant produced using C3S as solid calcium silicate containing phase generally has, prior to pH adjustment in step S4, a higher pH as compared to a calcium silicate supernatant produced using CS as solid calcium silicate containing phase.

[0056] In an embodiment, the method comprises measuring the pH of the calcium silicate supernatant in step S3. In such a case, if the measurement as performed in step S3 indicates that the pH of the calcium silicate supernatant exceeds 8.5 then the method continues to step S4, where the pH of the calcium silicate supernatant is adjusted to a pH selected within an interval of from 4.5 up to 8.5.

[0057] However, if the pH measured in step S3 does not exceed 8.5 and is thereby already within an interval of from 4.5 up to 8.5, then no pH adjustment is needed, and the method ends.

[0058] In an embodiment, step S4 comprises adjusting, if the pH of the calcium silicate supernatant exceeds 8, the pH of the calcium silicate supernatant by addition of an acid to a pH selected within an interval of from 6 up to 8. The particular target pH of the calcium silicate supernatant can be selected based on the intended use or application of the calcium silicate supernatant. For instance, if a neutral pH is desired, then the pH of the calcium silicate supernatant, following any adjustment in step S4 if needed, is preferably within an interval of from 6 up to 8, preferably within an interval of from 6.5 up to 7.5, and more preferably within an interval of from 6.75 up to 7.25, such as about 7. Correspondingly, if a slightly acidic pH is preferred then the pH of the calcium silicate supernatant, following any adjustment in step S4 if needed, is preferably within an interval of from 4.5 up to 7, preferably within an interval of from 4.5 up to 6.5, and more preferably within an interval of from 4.5 up to 6.

[0059] The pH adjustment in step S4 can be performed by addition of an acid, such as hydrogen chloride (HCI).

[0060] In some cases, no pH adjustment is needed and step S4 could thereby be omitted. For instance, if the aqueous solution used in step S1 is an aqueous buffer or a buffered aqueous solution the pH of the calcium silicate supernatant following step S2, such as measured in the optional step S3, could be equal to or below pH 8.5. If the pH of the so-obtained calcium silicate supernatant is at or at least close to a desired or target pH for the calcium silicate supernatant then no pH adjustment is needed and step S4 could be omitted.

[0061] Illustrative, but non-limiting, examples of buffered aqueous solutions that could be used according to the embodiments include phosphate buffers, such as comprising dihydrogen phosphate (H2PO4 ) and hydrogen phosphate (HPO22), Tris buffers comprising tris(hydroxymethyl)aminomethane-HCI (Tris-HCI), and HEPES buffers comprising 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid.

[0062] In an embodiment, the calcium silicate supernatant has a calcium concentration of at least 10 mg / L.

[0063] In a particular embodiment, the calcium silicate supernatant has a calcium concentration selected within an interval of from 10 up to 50 mg / L. In another particular embodiment, the calcium silicate supernatant has a calcium concentration selected within an interval of from 10 up to 30 mg / L.

[0064] The calcium silicate supernatant comprises the calcium in the form of calcium ions.

[0065] In an embodiment, the calcium silicate supernatant has a silicon concentration of at least 5 mg / L. In a particular embodiment, the calcium silicate supernatant has a silicon concentration selected within an interval of from 5 up to 30 mg / L. In another particular embodiment, the calcium silicate supernatant has a silicon concentration selected within an interval of from 5 up to 20 mg / L.

[0066] The calcium silicate supernatant comprises the silicon in the form of silicate ions.

[0067] In an embodiment, contacting the water or the aqueous solution in step S1 comprises contacting the water with the solid phase in step S1 .

[0068] In a particular embodiment, the water is deionized water. In another particular embodiment, the water is pure water or ultrapure water, preferably ultrapure water. Ultrapure water is referred to as type I water and is defined by the American Society for Testing and Materials (ASTM) as having a resistivity of >18 MQ-cm, a conductivity of <0.056 piS / cm and <50 ppb of Total Organic Carbons (TOC). An example of such ultrapure water is Milli-Q® water. Pure water is also referred to as type II water and is defined by ASTM as having a resistivity of >1 MQ-cm, a conductivity of <1 S / cm and <50 ppb of TOCs. In a further particular embodiment, the water is tap water.

[0069] In an embodiment, removing any solid phase in step S2 comprises centrifuging any solid phase and the water or the aqueous solution to form the calcium silicate supernatant comprising calcium ions and silicate ions.

[0070] In another embodiment, removing any solid phase in step S2 comprises filtering any solid phase and the water or the aqueous solution to form the calcium silicate supernatant comprising calcium ions and silicate ions as a filtrate.

[0071] Contacting the solid phase with the water or the aqueous solution in step S1 leads to release of calcium and silicon ions from the solid phase and into the liquid phase, i.e., into the water or the aqueous solution. Accordingly, the solid phase is at least partly dissolved in step S1. Generally, calcium silicate has a solubility in water of about 0.01 wt% at 20°C. Thus, if a comparatively small amount of the solid phase is contacted with the water or the aqueous solution in step S1 and allowing sufficient incubation for the solid phase to almost completely dissolve then not removal of the solid phase is needed and step S2 could be omitted. However, in a preferred embodiment, the method comprises removing, in step S2, the solid phase from the water or the aqueous solution to form the calcium silicate supernatant comprising calcium ions and silicate ions.

[0072] In an embodiment, contacting the water of the aqueous solution in step S1 comprises contacting the water or the aqueous solution with a semipermeable pouch or bag comprising the solid phase. In such an embodiment, removing the solid phase in step S2 comprises removing the semipermeable pouch or bag from the water or the aqueous solution to form the calcium silicate supernatant comprising calcium ions and silicate ions. In this embodiment, the semipermeable pouch or bag has a porosity allowing water or the aqueous solution to penetrate into the semipermeable pouch or bag to contact the solid phase contained therein and allow calcium and silicate ions dissolved in the water or the aqueous solution to pass through the semipermeable pouch or bag. However, the pores of the semipermeable pouch or bag restrict the solid calcium silicate containing phase from passing through the semipermeable pouch or bag.

[0073] In an embodiment, the solid phase comprises solid calcium silicate containing powder or particles. In a preferred embodiment, the solid phase is in the form of calcium silicate powder, calcium silicate particles, or a mixture thereof.

[0074] In an embodiment, the solid calcium silicate containing powder or particles has or have a grain size of below 700 pm.

[0075] In a particular embodiment, the solid calcium silicate containing powder or particles has or have a grain size equal to or below 500 pirn. In another particular embodiment, the solid calcium silicate containing phase comprises solid calcium silicate containing powder having a grain size equal to or below 300 pm.

[0076] Another aspect of the invention relates to a calcium silicate supernatant in the form of water or an aqueous solution comprising calcium ions and silicate ions and having a pH selected within an interval of from 4.5 up to 8.5, preferably selected within an interval of from 6 up to 8.

[0077] In an embodiment, the calcium silicate supernatant has a calcium concentration of at least 10 mg / L. In a particular embodiment, the calcium silicate supernatant has a calcium concentration selected within an interval of from 10 up to 50 mg / L. In another particular embodiment, the calcium silicate supernatant has a calcium concentration selected within an interval of from 10 up to 30 mg / L.

[0078] In an embodiment, the calcium silicate supernatant has a silicon concentration of at least 5 mg / L.

[0079] In a particular embodiment, the calcium silicate supernatant has a silicon concentration selected within an interval of from 5 up to 30 mg / L. In another particular embodiment, the calcium silicate supernatant has a silicate ion concentration selected within an interval of from 5 up to 20 mg / L.

[0080] In an embodiment, the calcium silicate supernatant is in the form of water, preferably tap water, pure water or ultrapure water, and more preferably ultrapure water, comprising calcium ions and silicate ions.

[0081] In an embodiment, the calcium silicate supernatant is in the form of an aqueous solution, preferably a buffered aqueous solution, comprising calcium ions and silicate ions. Examples of such buffered aqueous solutions include a phosphate buffer, a Tris buffer and a HEPES buffer.

[0082] In an embodiment, the calcium silicate supernatant is obtainable by the method according to the method described herein.

[0083] The present invention also relates to a cosmetic formulation comprising a calcium silicate supernatant according to the invention.

[0084] In an embodiment, the cosmetic formulation is a nail formulation. In a particular embodiment, the nail formulation is selected from the group consisting of a nail oil, a nail polish, a nail gel, a nail cream, a nail serum, and a nail hardener.

[0085] In an embodiment, the cosmetic formulation is a hair formulation. In a particular embodiment, the hair formulation is selected from the group consisting of a hair serum, a hair conditioner, a hair shampoo, a hair cream, a hair mousse, and a hair gel.

[0086] In an embodiment, the cosmetic formulation is a skin formulation. In a particular embodiment, the skin formulation is selected from the group consisting of a skin cream, a skin serum, a skin oil, a skin gel, and a skin lotion. The cosmetic formulation could be obtained by adding the calcium silicate supernatant to the nail, hair or skin formulation followed by mixing, i.e., addition of the calcium silicate supernatant to an already preformed nail, hair or skin formulation. Alternatively, the calcium silicate supernatant is used as one of the ingredients during production of the nail, hair or skin formulation. In such a case, the calcium silicate supernatant preferably replaces at least part of the liquid phase, such as water, of the nail, hair or skin formulation during the production of the nail, hair or skin formulation. In such a case, all or part of the liquid phase, such as water, could be replaced by the calcium silicate supernatant.

[0087] The invention further relates to a method of local delivery of silicate ions. The method comprises applying a calcium silicate supernatant according to the invention or a cosmetic formulation according to the invention to at least a portion of the hair, skin and / or nails of a subject.

[0088] The invention also relates to a calcium silicate supernatant according to the invention or a cosmetic formulation according to the invention for use in stimulating collagen formation in hair, skin and / or nails of a subject. In an embodiment, the calcium silicate supernatant according to the invention or the cosmetic formulation according to the invention is formulated for local application to at least a portion of the hair, skin and / or nails of a subject.

[0089] The use of the calcium silicate supernatant or the cosmetic formulation as described herein is for a cosmetic use of the calcium silicate supernatant or the cosmetic formulation.

[0090] The subject is an animal subject, preferably a mammalian subject and more preferably a human subject.

[0091] All embodiments disclosed herein relate to all aspects of the present invention and all embodiments may be combined unless stated otherwise.

[0092] EXAMPLES

[0093] EXAMPLE 1

[0094] Monocalcium silicate powder (CaOSiOs) with a grain size of below 200 pirn was mixed with deionized water for an hour at 60°C (ratio powder to water of 1 :10 by weight). Grain size of the powder was sieved to be below 200 pirn. The mixture was centrifuged, the supernatant separated and sterile filtered. The pH of the supernatant was 9 and was neutralized (pH 7) by addition of HCI. EXAMPLE 2

[0095] Monocalcium silicate powder (CaOSit ) with a grain size of below 100 pm was mixed with deionized water (ratio 1 :50 by weight) for an hour at 60°C. The mixture was centrifuged, the supernatant separated.

[0096] EXAMPLE 3

[0097] Tricalcium silicate powder ((CaOJsSIOs) with a grain size of below 200 pm was mixed with deionized water (ratio powder to water of 1 :10) for an hour at room temperature (about 23.5°C). Grain size of the powder was below 200 pm, sieved. The mixture was centrifuged, the supernatant separated. The pH of the supernatant was above 12. The pH was reduced by addition of HCI.

[0098] EXAMPLE 4

[0099] A pH study was conducted on a calcium silicate supernatant produced by soaking tablets of calcium silicate with a grain size below 700 pm in milli-Q water. All measurements were made at 23.5°C and with an initial pH at 7.7.

[0100] As is shown in Table 1, the pH increased during the first 15 minutes before reaching an asymptote at about pH 10.7.

[0101] Table 1 - pH

[0102] EXAMPLE 5

[0103] Tablets of calcium silicate with a grain size below 200 pm was soaked in deionized water or Milli-Q® water. The concentrations of calcium and silicon were measured in the supernatant after soaking time using inductively coupled plasma (ICP) spectroscopy.

[0104] The results are presented in Fig. 1 for Milli-Q® water and in Fig. 2 for deionized water. As is seen in the figures, the concentration of Si increased rapidly during the 15 min of soaking and then reached a plateau concentration of about 25 mg / L for Milli-Q® water and 15 mg / L for deionized water. The corresponding plateau concentrations of Ca are slightly below 40 mg / L for Milli-Q® water and about 25 mg / L for deionized water.

[0105] EXAMPLE 6

[0106] A leave in hair serum was formulated with 5 ml vegetable glycerin, one drop of vitamin E, 80 ml of deionized water and 15 ml of the calcium silicate supernatant from Example 5 following pH adjustment to pH by addition of HCI.

[0107] EXAMPLE 7

[0108] Monocalcium silicate powder (CaOSiO2) with a grain size of below 200 pm was mixed with tap water for 5 minutes at 20°C (ratio monocalcium silicate powder to water of 1 : 10 by weight). Grain size of the powder was sieved to be below 200 pm. The mixture was centrifuged, the supernatant separated and sterile filtered. The pH of the solution was 8.

[0109] EXAMPLE 8

[0110] Isolates of Escherichia coli MG1655 and Staphylococcus aureus ATCC 29213 were used as common representatives for gram-negative and gram-positive bacteria, respectively. Initially, an overnight culture was established by plating a cryoculture (-80°C) on LB agar. Cultivation was performed at 37°C. Two morphologically similar colonies were used to inoculate 10 mL of fresh media. This culture was incubated overnight at 37°C and 200 rpm. The plates were stored at 4°C and every 7 days a few colonies were taken to streak out on a new LB-plate.

[0111] The disk diffusion test was performed according to the standard operating procedure (SOP) of EUCAST for the evaluation of the resistance of microorganisms to antibiotics, with modifications. The modifications include a disc size of 10 mm, as well as the use of ODeoo as an indicator for the inoculum.

[0112] After overnight incubation at 37°C and 200 rpm, 150 pL of the bacterial solution was used to inoculate 10 mL of fresh Muller-Hinton broth (MHB). This solution was then further incubated for 2 hours at 37°C with 200 rpm to ensure exponential growth. 5 mL of the bacterial solution in MHB with approximately 1 x108CFU / mL, corresponding to an ODeoo = 0.2, was centrifuged at a relative centrifugal force (RCF) of 4000 for 5 minutes. The medium was discarded, and the pellet was resuspended in 5 mL of sterilized phosphate-buffered saline (PBS). 50 pL of this suspension was spread on a Muller-Hinton agar (MHA) plate with a sterile loop using the three-direction swab method to ensure homogeneous growth. Blank discs (10 mm) were loaded with 50 L of two calcium silicate supernatants, dried for 5-10 minutes and then firmly attached to the MHA with sterilized tweezers. As a negative control (NC), discs were loaded with 50 L PBS. Slight pressure was applied on the loaded discs to ensure even contact with the MHA plates.

[0113] Calcium silicate solution 1 : Calcium silicate supernatant produced by contacting water with monocalcium silicate (CaOSiCb) particles. The calcium silicate supernatant originally had a pH of 9, which was adjusted by addition of HCI to obtain a pH of 7.6.

[0114] Calcium silicate solution 2: Calcium silicate supernatant produced by contacting water with white Portland cement ((CaOhSiCh) particles. The calcium silicate supernatant had a pH of 12.4.

[0115] Coins of pressed calcium silicate (CaOSiCh) and white Portland cement ((CaOJsSiOs) were also evaluated.

[0116] No more than five discs per Petri dish were used. The Petri dishes were inverted for incubation at 37°C for 18±2 h. The zone of inhibition was measured with a ruler.

[0117] The MHA plates were investigated after incubation with either S. aureus or E. coli and after exposure to calcium silicate solutions 1 and 2, calcium silicate coins and white Portland cement coins.

[0118] Bacterial growth was overall homogenously distributed on the Petri dishes loaded with discs containing calcium silicate solution 1 or calcium silicate coins, meaning no inhibition zones. In clear contrast, discs with calcium silicate solution 2 and the white Portland cement coins had a clear growth inhibition zone for both of S. aureu and E. coli.

[0119] The antibacterial effects of calcium silicate supernatants are thereby pH dependent as shown above where a calcium silicate supernatant having a pH of 12.4 inhibited growth of S. aureu and E. coli, whereas a calcium silicate supernatant having an adjusted pH of 7.6 did not inhibit growth of S. aureu and E. coli.

[0120] The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims.

Claims

CLAIMS1 . A method of producing an ion-containing supernatant, the method comprising: contacting (S1) water or an aqueous solution with a solid phase comprising calcium silicate at a mass ratio of the water or the aqueous solution to the solid phase selected within an interval of from 1 :1 up to 1,000:1; removing (S2) any solid phase from the water or the aqueous solution to form a calcium silicate supernatant comprising calcium ions and silicate ions; and adjusting (S4), if a pH of the calcium silicate supernatant exceeds 8.5, the pH of the calcium silicate supernatant by addition of an acid to be equal to a pH selected within an interval of from 4.5 up to 8.5.

2. The method according to claim 1, wherein adjusting (S4) the pH comprises adjusting (S4), if the pH of the calcium silicate supernatant exceeds 8, the pH of the calcium silicate supernatant by addition of an acid to be equal to a pH selected within an interval of from 6 up to 8.

3. The method according to claim 1 or 2, wherein contacting (S1) water or the aqueous solution comprises contacting (S1) the water or the aqueous solution with the solid phase at a temperature selected within an interval of from 10°C up to 90°C, preferably selected within an interval of from 20°C up to 80°C, and more preferably selected within an interval of from 20°C up to 70°C.

4. The method according to any one of claims 1 to 3, wherein the mass ratio of the water or the aqueous solution to the solid phase is selected within an interval of from 1 :1 up to 100:1 , preferably selected within an interval of from 5:1 up to 100:1 .

5. The method according to any one of claims 1 to 4, wherein contacting (S1) the water or the aqueous solution comprises contacting (S1) the water or the aqueous solution with the solid phase for a duration selected within an interval of from 1 minutes up to 2 hours, preferably selected within an interval of from 2 minutes up to 2 hours, more preferably selected within an interval of from 5 minutes up to 2 hours and most preferably selected within an interval of from 0.5 up to 1.5 hours.

6. The method according to any one of claims 1 to 5, wherein the solid phase is selected from the group consisting of monocalcium silicate (CaOSiOs), dicalcium silicate ((CaO)2SiC>2), tricalcium silicate ((CaOJaSiCh), calcium sodium phosphosilicate, and any combination thereof, preferably selected from the group consisting of monocalcium silicate (CaOSiCh), dicalcium silicate ((CaOjsSiO?), tricalciumsilicate ((CaOJaSiOs), and any combination thereof, and more preferably monocalcium silicate (CaOSiCh).

7. The method according to any one of claims 1 to 6, wherein adjusting (S4) the pH comprises adjusting (S4), if the pH of the calcium silicate supernatant exceeds 8.5, the pH of the calcium silicate supernatant by addition of HCI to a pH selected within an interval of from 4.5 up to 8.5, preferably from 6 up to 8.

8. The method according to any one of claims 1 to 7, wherein the calcium silicate supernatant has a calcium concentration of at least 10 mg / L, preferably selected within an interval of from 10 up to 50 mg / L, and more preferably selected within an interval of from 10 up to 30 mg / L.

9. The method according to any one of claims 1 to 8, wherein the calcium silicate supernatant has a silicon concentration of at least 5 mg / L, preferably selected within an interval of from 5 up to 30 mg / L, and more preferably selected within an interval of from 5 up to 20 mg / L.

10. The method according to any one of claims 1 to 9, wherein contacting (S1) the water or the aqueous solution comprises contacting (S1) water, preferably ultrapure water, with the solid calcium silicate containing phase.

11. The method according to any one of claims 1 to 10, wherein removing (S2) the solid phase comprises centrifuging the solid phase and the water or the aqueous solution to form the calcium silicate supernatant comprising calcium ions and silicate ions.

12. The method according to any one of claims 1 to 11, wherein the solid phase comprises solid calcium silicate containing powder or particles, optionally having a grain size of below 700 pm, preferably equal to or below 500 pm, and more preferably equal to or below 300 pm.

13. A calcium silicate supernatant in the form of water or an aqueous solution comprising calcium ions and silicate ions, wherein the calcium silicate supernatant has a pH selected within an interval of from 4.5 up to 8.5.

14. The calcium silicate supernatant according to claim 13, wherein the calcium silicate supernatant has a calcium concentration of at least 10 mg / L, preferably selected within an interval of from 10 up to 50 mg / L, and more preferably selected within an interval of from 10 up to 30 mg / L.

15. The calcium silicate supernatant according to claim 13 or14, wherein the calcium silicate supernatant has a silicon concentration of at least 5 mg / L, preferably selected within an interval of from 5 up to 30 mg / L, and preferably selected within an interval of from 5 up to 20 mg / L.

16. The calcium silicate supernatant according to any one of claims 13 to 15, wherein the calcium silicate supernatant is in the form of water, preferably ultrapure water, comprising calcium ions and silicate ions.

17. The calcium silicate supernatant according to any one of claims 13 to 16, wherein the calcium silicate supernatant has a pH selected within an interval of from 6 up to 8.

18. The calcium silicate supernatant according to any one of claims 13 to 17 obtainable by the method according to any one of claims 1 to 12.

19. A cosmetic formulation comprising a calcium silicate supernatant according to any one of claims 13 to 18.

20. The cosmetic formulation according to claim 19, wherein the cosmetic formulation is a nail formulation, preferably selected from the group consisting of a nail oil, a nail polish, a nail gel, a nail cream, a nail serum, and a nail hardener; or a hair formulation, preferably selected from the group consisting of a hair serum, a hair conditioner, a hair shampoo, a hair cream, a hair mousse, and a hair gel; or a skin formulation, preferably selected from the group consisting of a skin cream, a skin serum, a skin oil, a skin gel, and a skin lotion.

21. A method of local delivery of silicate ions comprising applying a calcium silicate supernatant according to any one of claims 13 to 18 or a cosmetic formulation according to claim 19 or 20 to at least a portion of the hair, skin and / or nails of a subject, preferably a human subject.

22. A calcium silicate supernatant according to any one of claims 13 to 18 or a cosmetic formulation according to claim 19 or 20 for use in stimulating collagen formation in hair, skin and / or nails of a subject, wherein the calcium silicate supernatant according to any one of claims 13 to 18 or the cosmetic formulation according to claim 19 or 20 is formulated for local application to at least a portion of the hair, skin and / or nails of a subject.