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Title:
BACTERIAL STRAINS FOR INDUSTRIAL USE
Document Type and Number:
WIPO Patent Application WO/2020/234760
Kind Code:
A1
Abstract:
The present invention relates to a bacterial strain selected for production of enzymes with high dehairing capacity. It further relates to a supernatant obtained from said bacterial strain and a method that comprises the use of said supernatant for dehairing hides.

Inventors:
DEMATTÈ ELISA (IT)
SARTORI ELISA (IT)
JOUSSON OLIVIER (IT)
QUATTRONE ALESSANDRO (IT)
Application Number:
PCT/IB2020/054724
Publication Date:
November 26, 2020
Filing Date:
May 19, 2020
Export Citation:
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Assignee:
BIODERMOL AMBIENTE S R L (IT)
International Classes:
C14C1/06; C12R1/125
Domestic Patent References:
WO2008093353A12008-08-07
WO1997027841A11997-08-07
Foreign References:
Other References:
PRIYA PILLAI ET AL: "Hide depilation and feather disintegration studies with keratinolytic serine protease from a novel Bacillus subtilis isolate", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, SPRINGER, BERLIN, DE, vol. 78, no. 4, 9 February 2008 (2008-02-09), pages 643 - 650, XP019586332, ISSN: 1432-0614
PRIYA PILLAI ET AL: "Statistical optimization of production and tannery applications of a keratinolytic serine protease fromP13", PROCESS BIOCHEMISTRY, ELSEVIER LTD, GB, vol. 46, no. 5, 26 January 2011 (2011-01-26), pages 1110 - 1117, XP028372440, ISSN: 1359-5113, [retrieved on 20110202], DOI: 10.1016/J.PROCBIO.2011.01.030
Attorney, Agent or Firm:
RIGAMONTI, Dorotea et al. (IT)
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Claims:
CLAIMS

1. A bacterial strain for the production of enzymes with a high dehairing capacity, wherein said bacterial strain is selected in the group consisting of 1 Dm8, 1 Dm15, 3Am2, T2D3, the deposit number of each of said strains being DPS RE RSCIC 14, DPS RE RSCIC 13, DPS RE RSCIC 1 1 , DPS RE RSCIC 15, respectively.

2. The culture supernatant of at least one bacterial strain selected in the group consisting of 1 Dm8, 1 Dm15, 3Am2, T2D3, the deposit number of each of said strains being DPS RE RSCIC 14, DPS RE RSCIC 13, DPS RE RSCIC 1 1 , DPS RE RSCIC 15, respectively.

3. The supernatant according to claim 2, wherein said supernatant is a liquid culture medium.

4. The supernatant according to claim 2, wherein said culture medium is a minimal medium; it is preferably FMM (Feather Minimal Medium) or WMM (Wool Minimal Medium).

5. The supernatant according to one of claims 2 to 4, wherein the supernatant is treated, preferably it is dried and/or lyophilized.

6. A formulation which comprises at least one supernatant according to one of claims 2 to 5, and optionally additional supernatants from additional bacterial strains, optionally additional active substances.

7. A method for dehairing leather, wherein said method comprises

-Providing at least one supernatant according to one of claims 2 to 5, or a formulation according to claim 6;

-Exposing the leather to be treated to said at least one supernatant or to said formulation, at a temperature comprised between 18 and 40°C, at a pH comprised between 4.5 and 12.

8. The method according to claim 7, wherein said leather is exposed to said supernatant at a temperature comprised between 22 and 29°C, or between 24 and 27°C at a pH comprised between 6 and 9. 9. The method according to one of claims 7 or 8, which is for tanning leather and further comprises a soaking step and/or a liming step.

10. A method for cosmetic hair removal or peeling which comprises the topical administration of at least one supernatant according to one of claims 2 to 5 or of a formulation according to claim 6.

Description:
BACTERIAL STRAINS FOR INDUSTRIAL USE

FIELD OF THE INVENTION The present invention relates to selected bacterial strains and supernatants obtained therefrom for use in the process for tanning hides. The tanning process, although still strongly linked to craft processing, has for many years had industrial characteristics: in Italy alone, 12 thousand tonnes of finished hides were produced in 2016. Huge amounts of water, energy and chemical reagents are needed for carrying out the complete process, with a notable environmental impact due to the production of solid wastes, emissions to atmosphere and wastewater.

The production cycle consists essentially of a first step, called the preparation step, in which the hides are prepared for receiving the tanning agents added in the next steps of tanning and retanning. During the preparation step, the hide undergoes chemical treatments for various purposes: to rehydrate the raw hides (soaking), remove hair (dehairing) and swell the fibres of the hide (liming). In particular, the dehairing step envisages the use, besides the dehairing agents proper, typically of reducing agents such as sulphides/sulphydrates, which are highly toxic and polluting, and enormous amounts of water which, once used, must be suitably purified to lower the pollution values such as chemical oxygen demand (COD), biochemical oxygen demand (BOD), total dissolved solids (TDS), sulphides and sulphydrates (Layman’s report, Green LIFE project, 2017).

The enzyme-aided dehairing step is the environmentally friendly option that reduces the BOD values by almost 40% and the COD values by 50% in leather processing. Examples of enzymatic dehairing are described in US3,840,433, US4,636,222, W01994/06942, US5,834,299 and W02008/093353.

WO2010/043709 describes an enzymatic method for treating hides that comprises the use of carbohydrase and lipase.

US9,267,182 describes an enzymatic method for treating hides that comprises the use of glutamine endopeptidase.

The enzymes used are generally proteolytic enzymes that catalyse the breakdown of proteins. Examples of proteases that have been used are extracts of more or less raw proteases of bacterial or fungal origin containing various peptidases, as well as purer proteases such as elastase, subtilisins, trypsins, chymotrypsin, aspartic proteases, cysteine proteases and metalloproteases.

However, since skin and hides mainly consist of collagen, which is degradable by protease, there is a risk of damage to the skin or to hides when protease is used. Moreover, the proteases have not shown sufficient activity to be able to remove hair completely.

There is therefore considerable interest in alternative methods of dehairing that are effective and have low environmental impact.

Description of the invention

Description of the figures

Fig. 1 : Test of in-vitro enzymatic activity a) proteolytic activity; b) reductase activity; c) alpha-amylolytic activity; d) keratinolytic activity; e) proteolytic activity; f) collagenolytic activity.

Fig. 2: Heatmap of enzymatic activity measured in the 10 strains indicated. NB= Nutrient Broth, nutrient-rich medium. FMM= Feather Minimal Medium, minimal medium in which the only source of carbon and nitrogen is feathers. WMM = Wool Minimal Medium, minimal medium in which the only source of carbon and nitrogen is wool. Fig. 3: photographs showing the hides before and after the treatments in a pilot tumbler using the supernatants according to the present invention.

Fig. 4: large-scale treatment (A) example photograph of a hide before treatment; (B) photograph of the same hide after treatment.

The present invention relates firstly to a bacterial strain selected from the strains described in Table 1 .

Table 1

IZSLER=lstituto Zooprofilattico Sperimentale di Lombardia ed Emilia Romagna [Experimental Zooprophylactic Institute of Lombardia and Emilia

Romagna], via Bianchi 9-25124 Brescia, Italy

Key:

The bacterial strains according to the present invention, alone or mixed, proved to be particularly effective in the treatment for preparation of hides.

In a further embodiment, said bacterial strains, alone or mixed, find cosmetic use. As an example, in depilatory and/or peeling applications. One or more supernatants of said bacterial strains, individually or mixed, form a further aspect of the present invention. For the purposes of the present invention, the term "supernatant" means the culture medium in which at least one bacterial strain is grown. Said culture medium is preferably a liquid culture medium. Said supernatant is "as is", i.e. for example the culture medium in which at least one bacterial strain is grown, after suitable centrifugation, or treated, for example said culture medium in which at least one bacterial strain is grown and, after suitable centrifugation, is dried and/or lyophilized. In a preferred embodiment, said supernatant is from a culture that continued for at least 6 hours, preferably for at least 16 hours. In a preferred embodiment, said supernatant is formulated with excipients, stabilizers, for example is encapsulated.

In a further embodiment, the present invention relates to a formulation that comprises a supernatant from one of the bacterial strains shown in Table 1 , or a mixture of two supernatants from two of the bacterial strains shown in Table 1 , or a mixture of at least two supernatants from at least two of the bacterial strains shown in Table 1 , suitably treated and/or suitably formulated, optionally with the addition of additional supernatants from additional bacterial strains and/or of additional active substances.

Advantageously, said treated supernatants or said formulation may be stored for later use, maintaining the activity of the enzymes contained therein.

The present invention further relates to a method for dehairing skin or hides, where said method comprises:

1 ) Providing at least one supernatant from at least one bacterial strain according to the present invention;

2) Exposing the skin or hide to be treated to said at least one supernatant, at a temperature between 18 and 40°C, at a pH between 4.5 and 12. In a preferred embodiment, said method is for preparatory treatment of hides intended for tanning. In a further application, said method is for cosmetic use.

Surprisingly, the supernatants isolated from the bacterial strains according to the present invention possess a depilatory activity such as to make it unnecessary to use treatment with reducing agents, which is typically used in this process. Said supernatants have in fact proved to be effective when used alone, with the advantage of avoiding treatment with reducing agents and the consequent environmental impact. Moreover, the absence of reducing agents allows dehairing to be less aggressive for the hides to be treated, so they are still in good condition at the end of the process. Moreover, the non-aggressiveness of said supernatants also makes them safe in applications of the cosmetic type.

Surprisingly, these supernatants maintain their effectiveness even when exposed to a reducing environment or to an oxidizing environment, as the following examples demonstrate.

In a preferred embodiment, said skin is exposed to said supernatant at a temperature between 22 and 29°C, or between 24 and 27°C at a pH between 6 and 9.

In a preferred embodiment, when applied in the tanning sector, said method also comprises a soaking step.

Said soaking is carried out by the methods known by a person skilled in the art, for example by exposing said skin to be treated with water and/or wetting agents and/or surfactants and/or alkalies such as, for example, sodium carbonate, sodium hydroxide, and/or bactericidal agents and/or enzyme mixtures.

In a further preferred embodiment, said method also comprises a liming step. Said liming preferably comprises exposing said skin at a pH of about 12 to a temperature between 23 and 32°C, preferably between 26 and 29°C, or between 27 and 28°C. In a preferred embodiment, said method comprises:

1 ) isolating the supernatant of the culture medium of at least one bacterial strain according to the present invention;

2) exposing the hides to be treated to a solution at pH between 5 and 9.5 and at a temperature between 22 and 30°C, preferably between 24 and 28°C;

3) adding said supernatant to said solution and, after at least 2 hours, or after at least 5 hours, or 10 hours, or 12 hours, or after 24 hours, adjusting the pH to about 12.

In a preferred embodiment, said pH in said step 2) is obtained with addition of carbonate and/or, in said step 3), said pH is obtained with addition of hydrated lime and/or caustic soda.

In a preferred embodiment, said supernatant is derived from the growth of at least one bacterial strain in a minimal medium, for example in FMM or WMM medium.

Preferably, in said step 2), called soaking, said hides are incubated in a solution that also comprises an emulsifier and/or an alkalizing agent.

Preferably, said emulsifier is an organic emulsifier, as an example it is Biodermol PRODEFAT (Biodermol, IT).

Preferably, in said step 2), called soaking, a controller of bacterial growth and/or phosphorus salts are also added. Examples are Biodermol BTM OS (Biodermol, IT) and/or Biodermol TP.

Advantageously, the bacterial strains according to the present invention and/or the supernatants thereof, make it possible to carry out the process of dehairing of hides efficiently, without damaging the hide itself.

The strains according to the present invention are advantageously able to grow on minimal medium, thus offering the advantage of reasonable production costs. The present inventors have also demonstrated that growth in said minimal medium means that an enzyme panel endowed with better depilatory activity becomes concentrated in said supernatant.

Examples

Example 1 : PRIMARY SCREENING

Primary screening was carried out by means of Skim Milk Agar Assay. The Nutrient Agar rich medium was supplemented with a solution at 5% (w/v) of condensed milk. The resultant medium is of opaque appearance. If the bacterial strain has proteolytic activity, a transparent halo will be created round the colonies of said strain owing to protease digestion of the casein contained in the Skim Milk. The digestion halo is measured in millimetres and the size of the halo is directly proportional to the amount of protease released by the bacterial strain. Table 2 presents the results observed.

Table 2

Screening revealed 26 strains that produce protease activity.

Some strains have growth of the migratory type, a factor that alters the test results. The strains affected by the migration process are 3Am9, 3Bm2 and 3Bm3.

Example 2: SECONDARY SCREENING

With the objective of selecting a strain that is not only endowed with good protease activity but would also be suitable for use in large-scale production processes, a further test was conducted that evaluated the capacity of the microorganisms to grow on minimal media. In the subsequent scale-up step, minimal media in fact make it possible for the production costs of the bioactive supernatant to be used as depilatory agent to be kept very low.

In particular, the minimal medium used for this screening step contains:

• 0.5 g/L NaCI

• 0.4 g/L K2HPO4 • 0.3 g/L KH2PO4

• 10 g/L keratin substrate

The keratinous substrates (the only source of carbon and nitrogen in the medium) used were chicken feathers, shorn sheep wool and cow hair derived from the process of recovery of hair in the tannery. Screening was carried out by inoculating the bacterial strains with the minimal culture media and incubating at 27°C and 30°C overnight, with stirring at 160 rpm.

In this step, the number of strains deemed valid for continuing the characterization fell from 26 to 10, as only 10 strains proved to be capable of growing on the minimal media tested. In particular, the 10 strains selected are: 1 Dm3, 1 Dm4, 1 Dm8, 1 Dm14, 1 Dm15, 3Am2, 3Am4, T1 R2, T2D2, T2D3.

Example 3: GENOMIC CHARACTERIZATION

The 10 strains that passed the primary and secondary screening were prepared for a step of genomic characterization, in which complete sequencing of the genome of said strains was carried out using an lllumina MiSeq platform.

The protocol for preparation of the DNA libraries of the samples for sequencing consists of 4 steps:

1 ) Tagmentation, or the step of fragmentation of the DNA filaments into fragments of about 300 base pairs;

2) Amplification, or a classical PCR reaction for increasing the number of fragments;

3) Purification of the product obtained in the amplification step by removing non-specific reaction products;

4) Quantification of the library obtained and quality control thereof. Application of sequencing technology made it possible to identify the selected strains taxonomically. The results are given in Table 3 hereunder.

Table 3

AN I score = average nucleotide identity

Example 4: CHARACTERIZATION OF IN VITRO AND IN VIVO ACTIVITY

For the purpose of characterizing the enzymatic activity generated by the culture supernatants of the 10 selected bacterial strains, a series of experiments was conducted for comparing the in-vitro enzymatic activity and the depilatory activity exerted on real samples of hide.

The enzymatic activities measured were:

a) proteolytic activity;

b) keratinolytic activity; c) reductase activity;

d) alpha-amylolytic activity;

e) collagenolytic activity.

Each in-vitro experiment was carried out in triplicate

a) PROTEOLYTIC ACTIVITY

The proteolytic activity, i.e. the capacity of an enzyme to break the peptide bond, was measured in three different tests.

a1 ) Skim Milk Agar Plate assay

5 pi of supernatant, centrifuged (10 000 g, 15 minutes, 4°C) and filtered, was deposited on a plate of Nutrient Agar solid medium containing a 3% (w/v) solution of skim milk. The plates were incubated for 24 hours at 30°C and then the millimetres (mm) of halo present were measured.

a2) Assay with Azocasein (Sigma Aldrich)

400 mI of azocasein (20 mg/ml solution) was incubated with 400 mI of centrifuged (10 000 g, 15 minutes, 4°C) and filtered supernatant. Incubation took place overnight at 30°C, with stirring (160 rpm). Then 800 mI of 5% trichloroacetic acid (TCA) was added and the samples were centrifuged (10 000 g, 3 minutes, 4°C). Readings of the value of absorbance (400 nm) were taken. For the control samples (blanks), incubation overnight was only carried out with the azocasein solution and the supernatant was only added after blocking the reaction with TCA.

a3) Assay with casein and Folin Ciocalteau reagent (Sigma Aldrich)

This assay used 2 ml of centrifuged (10 000 g, 15 minutes, 4°C) and filtered supernatant, incubated for 10 minutes at 37°C, with stirring at 160 rpm with 4 ml of casein solution (5% w/v). After the incubation time, 1 ml of 5% TCA is added to the reaction and incubation is continued for 30 minutes at 37°C, with stirring at 160 rpm. Next, it is centrifuged for 5 minutes at 10 000 g at 4°C. 2 ml of the supernatant thus obtained is added to 5 ml of NaCC>3 and to 1 ml of F-C reagent, as in the Folin Ciocalteau protocol (1929) J. Biol. Chem. 73, 627. Incubation of 30 minutes at 37°C, with stirring at 160 rpm, is followed by reading of the absorbance at 660 nm. The blanks are treated like the normal samples but the supernatant is added to the reaction only after the TCA. The absorbance reading was compared to a standard curve obtained with known concentrations of tyrosine.

b) KERATINOLYTIC ACTIVITY

The keratinolytic activity was evaluated using a keratin substrate conjugated with a dye, Keratin Azure (KA, Sigma Aldrich). In detail, 1 ml of centrifuged (10 000 g, 15 minutes at 4°C) and filtered supernatant is aliquoted into an Eppendorf tube containing 5 mg of KA. After incubation overnight at 30°C, with stirring at 160 rpm and centrifugation (10 000 g, 3 minutes, room temperature), the absorbance at 595 nm was recorded. The supernatant "as such" was used as a blank to avoid the intrinsic colour of each supernatant altering the spectrophotometric reading.

c) REDUCTASE ACTIVITY

The reductase activity was evaluated using a reagent conjugated with a dye, DTNB (Sigma Aldrich). In detail, 1 ml of centrifuged (10 000 g, 15 minutes at 4°C) and filtered supernatant is aliquoted into an Eppendorf tube containing 10 mg of feathers and 1 ml of buffer at pH 8. After incubation overnight at 30°C at 160 rpm and centrifugation (10 000 g, 3 minutes, room temperature), 1 ml of DTNB (4 mg/ml) is added. The absorbance at 412 nm was recorded. The absorbance reading was compared to a standard curve obtained with known concentrations of cysteine.

d) ALPHA-AMYLOLYTIC ACTIVITY 1 ml of centrifuged (10 000 g, 15 minutes at 4°C) and filtered supernatant is aliquoted into an Eppendorf tube containing 1 ml of solution of maize starch. After incubation for 3 minutes at 20°C at 160 rpm, 1 ml of Color Solution (Sigma Aldrich) is added and it is boiled for 15 minutes. After boiling, it is cooled in ice and 9 ml of H 2 0 is added.

The absorbance at 540 nm was recorded. The absorbance reading was compared to a standard curve obtained with known concentrations of maltose.

e) COLLAGENOLYTIC ACTIVITY

The collagenolytic activity was evaluated using a substrate of collagen conjugated with a dye, Azocol (Sigma Aldrich). In detail, 1 ml of centrifuged (10 000 g, 15 minutes at 4°C) and filtered supernatant is aliquoted into an Eppendorf tube containing 1.5 mg of Azocol. After incubation overnight at 30°C at 160 rpm and centrifugation (10 000 g, 3 minutes, room temperature), the absorbance at 516 nm was recorded. The supernatant "as such" was used as a blank to avoid the intrinsic colour of each supernatant altering the spectrophotometric reading

f) DEHAIRING ACTIVITY

To verify the dehairing activity of the bacterial supernatants, ball mill tests were carried out (in rotating jars with a volume of about 13 dm 3 ), an industrial prototype that reproduces on a small scale the preliminary steps in the tanning of hides.

Each test requires about 48 h and 100 g per jar of salted bovine hide, in pieces of about 10x10 cm. All the tests were carried out using pieces of bovine hide derived from the same animal and were carried out applying the protocol given in Table 4.

Table 4

The products indicated in the table are commercially available products (Biodermol Ambiente srl). The percentages shown are percentages w/w relative to the skin. For example, water 300% indicates that, per 100 g of skin, the charge is 300 g of water. A protocol such as in Table 4, in which NaS/NaHS reducing agents have been added as dehairing agents, is used as positive control. The same protocol without addition of any depilatory agent, neither enzymatic nor chemical, is applied as the negative control. After 48 h of processing, dehairing is evaluated qualitatively. For quantitative evaluation of the degree of dehairing, the photographs of the skin before and after treatment were reworked as graphs, obtaining a percentage of dehairing.

The data obtained from the characterization in vitro are presented in the graphs in Fig. 1 and are summarized together with those obtained from the tests with a sample of real skin in the Fleatmap in Fig. 2. The results obtained are shown, after growing the bacterial strains indicated in NB= Nutrient Broth, nutrient-rich medium; FMM= Feather Minimal Medium, minimal medium in which feathers are the only source of carbon and nitrogen; WMM = Wool Minimal Medium, minimal medium in which the only source of carbon and nitrogen is wool. It can be seen that growth in minimal medium increases almost every enzymatic activity tested.

Advantageously, and as is clear in panels b), c), d), e) and f), the enzymatic activity obtained with the supernatants according to the present invention is not limited to an activity of the protease type (panel a) but includes a number of additional enzymatic activities.

The depilatory effect observed on the samples of real skin led to 4 strains being selected, which are:

-1 Dm8

-1 Dm15

-3Am2

-T2D3

TESTS ON PILOT TUMBLER

The 4 strains selected, grown in FMM medium (48h at 27°C, with stirring for 160 rpm), were tested in a pilot tumbler for determining dehairing activity. In particular, 4 litres of the clarified supernatant of each strain was tested on a sample of real skin (approx. 8 kg) according to the protocol given in Table 5 (traditional liming) and in Table 6 (oxidative liming).

Table 5

The products designated Biodermol are the property of Biodermol Ambiente srl:

- Biodermol BTM OS: biocide based on polyvinylpyrrolidone

- Biodermol PRODEFAT: bioemulsifier

- Biodermol PRODEFAT M: enzyme-based bioemulsifier - Biodermol WPM: mix of enzymes including hyaluronidase, amylase and protease

- Biodermol TP: sodium tripolyphosphate and Salts

- Biodermol LIPOL T1/T2S: degreasing agent based on lipolytic enzymes

Table 6

Fig. 3 gives an example of the results obtained in the dehairing tests with the bacterial supernatants at the pilot scale. At the end of all four tests, processing of the samples of skin was continued up to the 5 Wetblue stage in order to evaluate possible damage to the structure of the skin. No visible structural damage was found in any of the four tests carried out.

Example 5: LARGE-SCALE PROCESS

The process as in example 4 was optimized, defining a minimal 10 medium containing a minimum percentage of salts and pulverized feathers.

40 kg of hides were then treated by the method in Table 7, using 20 I of supernatant strain 1 Dm15 according to the present invention. Table 7

The treatment resulted in complete dehairing of the substrate, while avoiding permanent damage to the structure of the skin. Fig. 4 shows the skin before treatment (A) and after treatment (B).

The same strain was then grown by fermentation in a 5-litre 5 bioreactor. It was observed that the growth parameters remained unchanged relative to the growth in a flask, and were kept constant during the growth process. At the end of the fermentation step, the supernatant was tested on samples of skin, following the method as in Table 7 using the supernatants obtained with the following various fermentations:

- Test 1 : batch fermentation, with rich medium (NB);

- Test 2: batch fermentation, with minimal medium supplied with pulverized feathers 5 g/L (F3M medium)

- Test 3: batch fermentation, with modified F3M medium (pulverized feathers 10 g/L).

The tests of effectiveness on skin confirmed better performance of the strain with fermentation on minimal medium, and demonstrated the dehairing capacity of the bacterial supernatant obtained from an industrially scalable fermentation process.