EP4689038A1

EP4689038A1 - Use of metalloprotease

Info

Publication number: EP4689038A1
Application number: EP24716406.4A
Authority: EP
Inventors: Haidee Rachel BOOTH; Neil James Parry; Mark Lawrence THOMPSON; David Christopher Thorley
Original assignee: Unilever Global IP Ltd; Unilever IP Holdings BV
Current assignee: Unilever Global IP Ltd; Unilever IP Holdings BV
Priority date: 2023-04-06
Filing date: 2024-04-08
Publication date: 2026-02-11
Also published as: WO2024209106A1; CN121175401A

Abstract

The invention relates to the use of a metalloprotease to improve antiredeposition of blood onto a substrate by application of a home care composition comprising metalloprotease; the invention further relates to a method of improving antiredeposition of blood onto a textile.

Description

USE OF METALLOPROTEASE

Field of the Invention

The invention relates to the field of detergent compositions. of the Invention

Blood is a problematic stain to be removed from fabrics from consumers. Protease can be added to assist blood stain removal, however their use can result in a perceived loss of whiteness from the fabrics. This effect arising from a yellowing of the fabrics.

We have surprisingly found that the use of a metalloprotease in blood stain removal results in a much whiter fabric.

Summary of the Invention

The invention relates in a first aspect to the use of a metalloprotease to improve antiredeposition of blood onto a substrate by application of a home care composition comprising metalloprotease.

Preferably in the use, the home care composition is a home care detergent composition, more preferably a laundry detergent composition.

Preferably the composition is in the form of a liquid, solid, powder, pastille, bead or paste.

Preferably the substrate is a textile, more preferably a fabric, even more preferably a fabric comprising cotton and/or polyester.

In a second aspect, the invention relates to a method of improving antiredeposition of blood onto a textile, the method comprising the steps of:

(i) treating a textile with from 0.5 to 10 g/L, preferably from 1 to 8 g/L of an aqueous solution of a laundry detergent comprising a metalloprotease; and,

(ii) allowing said laundry detergent composition to remain in contact with said textile for from 5 minutes to 1 day, preferably from 10 minutes to 2 hours, then rinsing and drying the textile. Preferably in the method, the textile is a fabric, more preferably a fabric comprising cotton and/or polyester.

Preferably in the method, the metalloprotease is present in the laundry detergent at a level of from 0.0005 to 2 wt.%, preferably from 0.002 to 1 wt.%, more preferably from 0.002 to 0.5 wt.%, most preferably from 0.002 to 0.25 wt.%.

Detailed Description of the Invention

The metalloproteases provide a cleaning benefit to laundered fabrics. The cleaning benefit manifests itself as improved whiteness. The presence of protease enzymes within a laundry formulation can help the removal of the blood stains from fabrics, but when significant amounts of blood are removed redeposition of the blood onto fabrics can occur leading to a yellowing of the fabric. Applicants have found that metalloproteases provide a whiteness benefit, particularly in relation removal of blood stains. We have found that metalloproteases are considerably better than non-metalloproteases at preventing redeposition of the removed blood stains present in the wash liquor onto the fabric, thus maintaining the whiteness of the fabric.

A metalloprotease enzyme can be classified as a protease enzyme whose catalytic mechanism involves a metal.

Metalloprotease enzymes (E.C 3.4.24) hydrolyse bonds within peptides and proteins, in the laundry context this leads to enhanced removal of protein or peptide containing stains. Metalloproteases (metallopeptidases or metalloproteinases) is a class of hydrolases which cleave peptide bonds by the action of a water molecule which is activated by complexing to at least one bivalent metal ions belonging to the group of zinc, manganese, cobalt, nickel or copper ions, preferably zinc.

According to the classification of proteases based on protein structure and homology implemented in the MEROPS database (http://merops.sanger.ac.uk/cgi- bin/family_index?type=P), metalloproteases are found in 14 different families.

Preferably the protease is selected from the M4, M7 or M35 family, more preferably an M4 metalloprotease, most preferably a neutral metalloprotease. The metallo-proteases that may be used in this invention includes any of those which may be used in a homecare application. These metallo-proteases are, for example, derived from bacterium selected from the group consisting of bacillus amyloliquefaciens, bacillus subtilis, bacillus stearothermophilus, and bacillus thermoproteolyticus, and fungi selected from the group consisting of Aspergillus oryzae and Aspergillus niger.

The term "M4 Metalloprotease Family" or "M4 Metalloprotease" or "M4" as used herein means a polypeptide falling into the M4 metalloprotease family according to Rawlings et al., Biochem. J., 290, 205-218 (1993) and as further described in MEROPS - (Rawlings et al., MEROPS: the peptidase database, Nucl Acids Res, 34 Database issue, D270-272, 2006). The M4 metalloproteases are neutral metalloproteases containing mainly endopeptidases. All peptidases in the family bind a single, catalytic zinc ion. M4 metal loprotease family members include the common HEXXH motif, where the histidine residues serve as zinc ligands and glutamate is an active site residue. M4 metalloproteases have a pH optimum mainly at neutral pH. The M4 metal loprotease family includes, e.g., Neutrase™ (Novozymes) (classified as MEROPS subclass M04.014), Thermolysin, Bacillolysin, vibriolysin, pseudolysin, Msp peptidase, coccolysin, aureolysin, vimelysin, lambda toxin neutral peptidase B, PA peptidase (Aeromonas- type), griselysin, stearolysin, Mprlll (Alteromonas sp. strain 0-7), pap6 peptidase, neutral peptidase (Thermoactinomyces-type), ZmpA peptidase (Burkholderia sp.), zpx peptidase, PrtS peptidase (Photorhabdus luminescens), protealysin, ZmpB peptidase (Burkholderia sp.). The M4 metalloprotease family of polypeptides have been further characterized and presently includes, according to MEROPS, at least twenty- two subclasses for which a distinct MEROPS ID (i.e., an identifier of the formula M04.xxx) has been assigned, as well as non-peptidase homologues and unassigned peptidases.

The term "Thermolysin-Like Metalloprotease" as used herein means (a) an M4 metal loprotease of the MEROPS subclass M04.001 ; (b) an M4 metalloprotease of the MEROPS subclass M04.018; (c) an M4 metalloprotease of the MEROPS subclass M04.021; (d) an M4 metal loprotease having an active cleft motif: TG[TS] [QS] D N GGVH [Tl] ; (e) an M4 metalloprotease having an active cleft motif: DPDHSKRYTG[TS][QS]DNGGVH[TI]NSGI; and (f) an M4 metalloprotease having an active cleft motif: NT[TS][QS]DNGGVH[TI]NSGI. In these motifs, the accepted IIIPAC single letter amino acid abbreviation is employed. Also in the above motifs, the use of brackets indicates alternative amino acid choices at a particular position. The Thermolysin-Like Metalloproteases are described, e.g., in Barret et al. (1998) Handbook of proteolytic enzymes. Academic Press, pp. 350-369. The amino acid sequences of several TLPs have been determined, and the three-dimensional structures of several TLPs have been solved. Focus has been on increasing the thermostability of the TLP and a number of publications describe the thermostability of Bacillus TLPs, e.g., Veltman et al., (1998) Biochemistry 37(15):5312-9. The TLPs consist of an alpha-helical C-terminal domain and an N- terminal domain mainly consisting of beta-strands. The domains are connected by a central alpha-helix. This helix is located at the bottom of the active site cleft and contains several of the catalytically important residues such as four substrate binding pockets S2, Si , Si' and S2' have been identified (Hangauer et al. (1984) Biochemistry 23:5730-5741)

The term "M7 Metalloprotease Family" or "M7 Metalloprotease" or "M7" or "snapalysin family" as used herein means a polypeptide falling into the M7 metalloprotease family according to Rawlings et al., Biochem. J., 290, 205-218 (1993) and as further described in MEROPS - (Rawlings et al., MEROPS: the peptidase database, Nucl Acids Res, 34 Database issue, D270- 272, 2006). The protease family M7 contains a metalloendopeptidase, snapalysin. Snapalysin is active at neutral pH. The only known activity is cleavage of proteins of skimmed milk to form clear plaques around the growing bacterial colonies. The Zinc is bound by two histidines and an aspartate in an HEXXHXXGXXD sequence motif; the glutamate is a catalytic residue. The M7 proteases have clear signal peptides recognized by the SignalP prediction program. They also all have a propeptide that is cleaved off.

The term "M35 Metalloprotease Family" or "M35 Metalloprotease" or "M35" or "deuterolysin family" as used herein means a polypeptide falling into the M35 metalloprotease family according to Proteolysis in Cell Function, pp13-21 , IOS Press, Amsterdam (1997), Rawlings et al., Biochem. J., 290, 205-218 (1993) and as further described in MEROPS - (Rawlings et al., MEROPS: the peptidase database, Nucl Acids Res, 34 Database issue, D270- 272, 2006). Family M35 members contain two zinc binding histidines and a catalytic glutamate in an HEXXH motif. There is a third zinc ligand, an Asp, found in a GTXDXXYG motif C-terminal to the His zinc ligands (see the Alignment). For this reason, the peptidases in this family are sometimes termed "aspzincins", although peptidases in which the third ligand of zinc is Asp also occur in families M6, M7 and M64.

Home Care Composition

The home care composition is suitable for uses in home care. Preferably the composition is a home care detergent composition, more preferably a laundry detergent composition. Preferably the home care detergent composition, more preferably a laundry detergent composition comprises one or more surfactants selected from anionic, nonionic and amphoteric surfactants.

Preferably the home care detergent composition, preferably a laundry detergent composition, is in the form of a liquid, solid, powder, pastille, bead or paste, more preferably a liquid, solid or powder, more preferably a liquid.

The invention will be further described with the following non-limiting examples.

The improved whiteness of the fabric substrate treated with metalloprotease is demonstrated by the following examples.

Example 1

The whiteness of a fabric as determined by the measured reflectance values was assessed for a control detergent, a control detergent + protease, and the control + metalloprotease.

Proteases used:-

Carnival Evity (a non-metalloprotease sourced from Novozymes)

Neutrase BA (a metal loprotease sourced from Sigma Aldrich)

Samples of 5 cm double applied defibrinated sheep’s blood on both cotton and polyester were sourced from Warwick Equest. The stains were washed for 30 minutes at 40°C in 25 FH water using a tergotometer. The liquor to cloth ratio was 20:1. This was achieved by adding 25g of stain fabrics swatches and 25 g of unstained fabric. The reflectance (DE*) values of both the stains and blank ballast fabrics were measured before and after washing using an X-Rite Color i7 spectrophotometer with the UV component excluded and a 17 mm aperture.

Stain removal index (SRI) was calculated as 100-DE * and percentage stain removal calculated as:

((SRI after washing - SRI before washing)/ SRI before washing) *100

The products used in the evaluation consisted of a surfactant blend dose at the following levels, amounts given as g/L in the wash.

Table 1 - Compositions tested

The fabrics were washed in the surfactant solution with and without a (non-metalloprotease) protease (Carnival Evity) or a metalloprotease (Neutrase BA) at a level of 0.0133 g/L (as received).

Polyester Fabrics The following results were obtained and shown in table 2.

Table 2 - Percentage stain removal of blood from polyester with statistics From the statistical treatment of the results shown in table 2, the formulations not connected by the same letter are significantly different.

The results from table 2 shows that the metalloprotease provides much improved stain removal as measured by reflectance. The metalloprotease (Neutrase BA) is superior at removal of fresh blood stains off the fabric (~ 95%) compared to the comparative protease (Carnival Evity) (-85%). Applicants believe that the stain removal for the non-metalloprotease is lower than for no enzyme due to the removed blood stain being redeposited onto the fabric. The metal loprotease not only cleans better but also has an improved effect on the reduced deposition of the removed blood stain onto the fabrics present in the wash. This is shown by the results in table 3. Table 3 - Whiteness of ballast fabric (polyester) after washing

From the statistical treatment of the results shown in table 3, the formulations not connected by the same letter are significantly different.

Table 3 shows the beneficial effect of the metalloprotease enzyme on the reduced redeposition of removed blood stains back onto the polyester fabric. The white ballast polyester fabric swatches start the washing process as unstained white fabric swatches. The wash load also has the blood-stained fabrics. The blood-stained fabrics have some degree of the blood stain removed during the wash process, which blood stain is still present in the wash liquor and can transfer (redeposit) back onto the fabrics present in the wash liquor (both the originally stained fabrics and the white ballast fabrics). The white ballast fabric swatches remain whiter (less discolouration) after washing with metalloprotease compared to the wash with the comparative protease, where for the comparative protease the white ballast fabric swatches are discoloured and appear more yellow and less white. The only difference between these formulations is the use of metalloprotease versus the non-metalloprotease. This is an indication that the metalloprotease has an improved antiredeposition benefit of blood stains onto polyester fabric compared to the non-metalloprotease. The metalloprotease ensures ballast polyester swatches remain mostly white following blood stain removal, indicating improved antiredeposition of blood onto the fabric, while the non-metalloprotease causes decolouration (yellowing) of ballast swatches, indicating redeposition of blood onto fabric.

Cotton

The results for the cotton fabrics are shown in table 4. Table 4 - Percentage stain removal of blood from cotton with statistics

From the statistical treatment of the results shown in table 4, the formulations not connected by the same letter are significantly different.

The results from table 4 shows that the metalloprotease performs similarly to the nonmetalloprotease in terms of blood stain removal from the cotton fabric. However, in a similar fashion to the polyester results, the metalloprotease has an improved effect on the reduced redeposition of the removed blood stain onto the cotton fabrics present in the wash. This is shown by the results in table 5.

Table 5 - Whiteness of ballast fabric (cotton) after washing

From the statistical treatment of the results shown in table 5, the formulations not connected by the same letter are significantly different.

Table 5 shows the beneficial effect of the metalloprotease enzyme on the reduced redeposition of removed blood stains back onto cotton fabric. The white ballast cotton fabric swatches start the washing process as unstained white fabric swatches. The wash load also has the bloodstained fabrics. The blood-stained fabrics have some degree of the blood stain removed during the wash process, which blood stain is still present in the wash liquor and can transfer (redeposit) back onto the fabrics present in the wash liquor (both the originally stained fabrics and the white ballast fabrics). The white ballast cotton fabric swatches remain whiter (less discolouration) after washing with metalloprotease compared to the wash with the comparative protease, where for the comparative protease the white ballast fabric swatches are discoloured and appear more yellow and less white. The only difference between these formulations is the use of metalloprotease versus the non-metalloprotease. This is an indication that the metalloprotease has an improved antiredeposition benefit of blood stains onto cotton fabric compared to the non-metalloprotease. The metalloprotease ensures ballast swatches remain mostly white following blood stain removal, indicating improved antiredeposition of blood onto the cotton fabric, while the non-metalloprotease causes decolouration (yellowing) of ballast swatches, indicating redeposition of blood onto fabric.

In conclusion the use of the metalloprotease provides a whiteness benefit to both polyester and cotton fabric, particularly in view of blood stains and the improved antiredeposition of such blood stains.

Claims

1. Use of a metalloprotease to improve antiredeposition of blood onto a substrate by application of a home care composition comprising metalloprotease.

2. Use according to any preceding claim, wherein the home care composition is a home care detergent composition.

3. Use according to any preceding claim, wherein the home care detergent composition is a laundry detergent composition.

4. Use according to any preceding claim, wherein the composition is in the form of a liquid, solid, powder, pastille, bead or paste.

5. Use according to any preceding claim, wherein the substrate is a textile, preferably a fabric, more preferably a fabric comprising cotton and/or polyester.

6. Use according to any preceding claim, wherein the metalloprotease is present in the home care composition at a level of from 0.0005 to 2 wt.%, preferably from 0.002 to 1 wt.%, more preferably from 0.002 to 0.5 wt.%, most preferably from 0.002 to 0.25 wt.%.

7. A method of improving antiredeposition of blood onto a textile, the method comprising the steps of:

(ii) allowing said laundry detergent composition to remain in contact with said textile for from 5 minutes to 1 day, preferably from 10 minutes to 2 hours, then rinsing and drying the textile.

8. A method according to claim 7, wherein the textile is a fabric, more preferably a fabric comprising cotton and/or polyester.

9. A method according to claim 7 or 8, wherein the metalloprotease is present in the laundry detergent at a level of from 0.0005 to 2 wt.%, preferably from 0.002 to 1 wt.%, more preferably from 0.002 to 0.5 wt.%, most preferably from 0.002 to 0.25 wt.%.