IT201900006994A1 - Bacterial strains for industrial use - Google Patents

Description

DESCRIPTION

Attached to a patent application for INDUSTRIAL INVENTION having the title

"Bacterial strains for industrial use"

The present invention relates to selected bacterial strains and supernatants obtained from them for use in the leather tanning process.

STATE OF THE ART

The tanning process, while remaining strongly linked to craftsmanship, has for years taken on industrial characteristics: in Italy alone in the year 2016 12,000 tons of finished leathers were produced. To complete the entire process, large quantities of water, energy and chemical reagents are required, with a significant environmental impact due to the production of solid waste, emissions into the atmosphere and water waste.

The production cycle is essentially constituted by a first phase, called riviera phase, where the hides are prepared to receive the tanning agents added in the subsequent tanning and retanning phases. During the riviera phase, chemical treatments are applied to the skin with different purposes: rehydrating the raw skins (soaking), removing the hair (hair removal) and swelling the skin fibers (liming). In particular, the depilation phase involves the use, in addition to the depilating agents themselves, typically reducing agents such as sulphides / sulphhydrates, highly toxic and polluting, of large quantities of water that once used must be purposely purified to reduce pollution values. such as chemical oxygen demand (COD), biochemical oxygen demand (BOD), total dissolved solid (TDS), sulphides and sulphhydrates (Layman's report, Green LIFE project, 2017).

The enzyme-assisted hair removal phase is the ecological option that reduces BOD values by almost 40% and COD values in leather processing by 50%.

Examples of enzymatic hair removal are described in US3,840,433, US4,636,222, WO1994 / 06942, US5,834,299 and WO2008 / 093353. WO2010 / 043709 describes an enzymatic method of treating skins which includes the use of carbohydrates and lipases.

US9,267,182 describes an enzymatic method of treating skins which includes the use of glutamine endopeptidase.

The enzymes used are generally proteolytic enzymes that catalyze the breakdown of proteins. Examples of proteases that have been used are more or less crude protease extracts 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 the skin and skins are mainly made up of collagen, which is susceptible to degradation by the protease, there is a risk of damage to the skin or skins with the use of proteases. Furthermore, the proteases did not show such activity as to be able to completely remove hair.

For this, alternative hair removal methods that are effective and with low environmental impact are strongly advised.

DESCRIPTION OF THE INVENTION

DESCRIPTION OF THE FIGURES

Figure 1: In vitro enzymatic activity test a) proteolytic activity; b) reductase activity; c) alpha-amylolytic activity; d) keratinolytic activity; e) proteolytic activity f) collagenolytic activity.

Figure 2: Heatmap of the enzymatic activity measured in the 10 indicated strains. NB = Nutrient Broth, a soil rich in nutrients. FMM = Feather Minimal Medium, minimal soil in which the only source of carbon and nitrogen are feathers. WMM = Wool Minimal Medium, minimal soil in which the only source of carbon and nitrogen is wool.

Figure 3: representative photographs of the hides before and after the treatments in pilot bottal using the supernatants according to the present invention.

A first object of the present invention is a bacterial strain selected from the strains described in table 1.

Table 1

IZLER = Experimental Zooprophylactic Institute of Lombardy and Emilia Romagna, via Bianchi 9 - 25124 Brescia, Italy

The bacterial strains according to the present invention, alone or in mixture, have proved to be particularly effective in the riviera treatment of hides.

In a further embodiment, said bacterial strains, alone or in a mixture, find cosmetic use. For example, in depilatory and / or peeling applications.

A further aspect of the present invention is one or more supernatants of said bacterial strains, singly or in mixture.

For the purpose of the present invention, the term "supernatant" means the culture medium where at least one bacterial strain has grown. Said culture medium is preferably a liquid culture medium. Said supernatant is as it is, or for example the culture medium in which at least one bacterial strain has grown, after appropriate centrifugation, or treated, for example said culture medium in which at least one bacterial strain has grown, after appropriate centrifugation, is dried and / or freeze-dried. In a preferred form, said supernatant derives from a culture which has lasted for at least 6 hours, preferably for at least 16 hours. In a preferred form, said supernatant is formulated with excipients, stabilizers, for example it is encapsulated.

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

Advantageously, said treated supernatants or said formulation can be preservatives for subsequent uses, maintaining the activity of the enzymes contained therein.

A further object of the present invention is a method for skin hair removal, where said method comprises:

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

2) Expose the skin 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 form, said method is for the beam treatment of hides intended for tanning. In a further application, this method is for cosmetic use.

Surprisingly, the supernatants isolated from bacterial strains according to the present invention possess such a depilatory activity as to not require treatment with reducing agents, typically used in this process. These supernatants have in fact proved effective when used alone, with the advantage of avoiding treatment with reducing agents and the consequent environmental impact. The absence of reducing agents also allows less aggressive hair removal for the skin to be treated, which are therefore well preserved at the end of the process. Furthermore, the non-aggressiveness of said supernatants makes them safe even in cosmetic applications.

The supernatants themselves surprisingly maintain their efficacy even when exposed to a reducing or oxidizing environment, as the following examples demonstrate.

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

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

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

In a preferred form, said method comprises:

1) Isolate 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 with a pH of between 5 and 9.5 and a temperature of between 22 and 30 ° C, preferably between 24 and 28 ° C;

3) Add 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, bring the pH to about 12.

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

In a preferred form, 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 which also comprises an emulsifying agent and / or an alkalizing agent. Preferably, said emulsifier is an organic emulsifier, by way of example it is Biodermol PRODEFAT (Biodermol, IT).

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

Advantageously, the bacterial strains according to the present invention and / or their supernatants, allow the implementation of the hair removal process of the skins efficiently, without causing damage to the skin itself.

The strains according to the present invention are advantageously able to grow on minimal soil, thus offering the advantage of low production costs. The authors of the present invention have also shown that the growth in said minimum medium causes an enzymatic panel with a better depilatory activity to be concentrated in said supernatant.

EXAMPLES

Example 1: PRIMARY SCREENING

Primary screening was performed using the Skim Milk agar assay. Nutrient Agar rich medium was spiked with a 5% (w / v) solution of condensed milk. The resulting soil has an opaque appearance. If the bacterial strain exhibits proteolytic activity, a transparent halo will be created around the colonies of the strain itself due to the digestion of the casein contained in the Skim Milk by proteases. The digestion halo is measured in millimeters and the amplitude of the halo is directly proportional to the amount of protease released by the bacterial strain. Table 2 reports the observed results.

Table 2

The screening revealed 26 protease producing strains. Some strains exhibit migratory growth, which alters the test results. The strains affected by the migration process are 3Am9, 3Bm2 and 3Bm3.

Example 2: SECONDARY SCREENING

With the aim of selecting the strain that not only had good protease activity but was also suitable for use in large-scale production processes, a further test was conducted that assessed the ability of microorganisms to grow on minimal media. . In fact, the minimum soils allow, in the subsequent scale-up phase, to substantially contain the production costs of the bioactive supernatant to be used as a depilating agent.

In particular, the minimum medium used for this screening phase contains:

• 0.5 g / L NaCl

• 0.4 g / L K2HPO4

• 0.3 g / L KH2PO4

• 10 g / L keratin substrate

As keratin substrates (the only source of carbon and nitrogen in the soil), hen feathers, shorn sheep's wool and cow hair deriving from the hair recovery process in the tannery were used. Screening was conducted by inoculating bacterial strains with minimal culture media and incubating at 27 ° C and 30 ° C overnight with agitation at 160 rpm.

In this phase, the number of strains deemed valid to continue the characterization dropped from 26 to 10, as only 10 strains proved capable of growing on the minimum tested media. In particular, the 10 selected strains are: 1Dm3, 1Dm4, 1Dm8, 1Dm14, 1Dm15, 3Am2, 3Am4, T1R2, T2D2, T2D3.

Example 3: GENOMIC CHARACTERIZATION

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

The sample DNA library preparation protocol for sequencing consists of 4 steps:

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

2) Amplification, that is a classic PCR reaction to increase the number of fragments;

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

4) Quantification of the obtained library and quality control of the same. The use of sequencing technology made it possible to taxonomically identify the selected strains. The results are shown in Table 3 below.

Table 3

ANI score = average nucleotide identity

Example 4: CHARACTERIZATION OF IN VITRO AND IN VIVO ACTIVITIES In order to characterize the enzymatic activity carried out by the culture supernatants of the 10 selected bacterial strains, a program of experiments was carried out to compare the enzymatic activity in vitro and the depilatory activity exerted. on real leather samples.

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 performed in triplicate.

a) PROTEOLITHIC ACTIVITY

The proteolytic activity, or the ability of an enzyme to break the peptide bond, was measured through three different tests.

a1) Skim Milk Agar Plate Test

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

a2) Azocaseine test (Sigma Aldrich)

400 µl of azocasein (20 mg / ml solution) were incubated with 400 µl of centrifuged supernatant (10,000 g, 15 minutes, 4 ° C) and filtered. The incubation was carried out overnight at 30 ° C under agitation (160 rpm). Then 800 µl of 5% trichloroacetic acid (TCA) was added and the samples were centrifuged (10,000 g, 3 minutes, 4 ° C).

The absorbance value (400 nm) was read.

For the control samples (blank), the overnight incubation was carried out only with the azocasein solution and the supernatant was added only after the blocking of the reaction carried out with TCA.

a3) Test with casein and Folin Ciocalteau's reagent (Sigma Aldrich) For this test 2 ml of supernatant centrifuged (10,000 g, 15 minutes, 4 ° C) and filtered were used, incubated for 10 minutes at 37 ° C under stirring at 160 rpm with 4 ml of casein solution (5% w / v). after the incubation period, 1 ml of 5% TCA is added to the reaction and continued with an incubation of 30 minutes at 37 ° C with stirring at 160 rpm. Subsequently it is centrifuged for 5 minutes at 10,000 g at 4 ° C. 2 ml of the supernatant thus obtained are added to 5 ml of NaCO3 and to 1 ml of F-C reagent, as per protocol Folin Ciocalteau (1929) J. Biol. Chem.

73, 627. Following an incubation of 30 minutes at 37 ° C with agitation at 160 rpm, the absorbance is read at 660 nm. The blanks are treated like normal samples but the supernatant is added to the reaction only after the TCA. The absorbance reading was related to a standard curve obtained with known concentrations of tyrosine.

b) KERATINOLYTIC ACTIVITY

The keratinolytic activity was evaluated with the use of a keratin substrate conjugated to a dye, Keratin Azure (KA, Sigma Aldrich). In detail, 1 ml of centrifuged supernatant (10,000 g 15 minutes at 4 ° C) and filtered is aliquoted in an eppendorf containing 5 mg of KA. After an overnight incubation at 30 ° C with agitation at 160 rpm and a 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 prevent the color of each supernatant from altering the spectrophotometric reading.

c) REDUCED ACTIVITY

The reductase activity was evaluated with the use of a reagent conjugated to a dye, the DTNB (Sigma Aldrich). In detail, 1 ml of centrifuged supernatant (10,000 g 15 minutes at 4 ° C) and filtered is aliquoted in an eppendorf containing 10 mg of feathers and 1 ml of buffer at pH 8. After overnight incubation at 30 ° C 160 rpm and a 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 related to a standard curve obtained with known concentrations of cysteine.

d) ALPHA-AMYLOLYTIC ACTIVITY

1 ml of centrifuged supernatant (10,000 g, 15 minutes at 4 ° C) and filtered is aliquoted in eppendorf containing 1 ml of corn starch solution. After 3 minutes incubation at 20 ° C 160 rpm, 1 ml of Color Solution (Sigma Aldrich) is added and boiled for 15 minutes. Following boiling, it is cooled on ice and 9 ml of H2O are added. The absorbance at 540 nm was recorded. The absorbance reading was related to a standard curve obtained with known concentrations of maltose.

e) COLLAGENOLYTIC ACTIVITY

The collagenolytic activity was evaluated with the use of a collagen substrate conjugated to a dye, Azocol (Sigma Aldrich). In detail, 1 ml of centrifuged supernatant (10,000 g 15 minutes at 4 ° C) and filtered is aliquoted in an eppendorf containing 1.5 mg of Azocol. After an overnight incubation at 30 ° C 160 rpm and a 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 prevent the color of each supernatant from altering the spectrophotometric reading.

f) DEPILATION ACTIVITIES

To verify the depilation activity of bacterial supernatants, tests were conducted in wandering (in rotating jars with a volume of 13 dm <3> approximately), an industrial prototype that reproduces the preliminary stages of tanning on a small scale.

Each test requires about 48 hours and 100 g per jar of salted cowhide, in pieces of about 10x10 cm. All the tests were carried out using pieces of cowhide from the same garment and were carried out by applying the protocol shown in table 4.

Table 4

The products indicated in the table are commercially available products (Biodermol Ambiente srl).

The% are expressed as percentages w / w with respect to the skin. For example, 300% water indicates that, per 100g of leather, 300g of water is loaded.

A protocol such as that of Table 4 is used as a positive control, where reducing agents NaS / NaHS have been added as depilating agents.

As a negative control, the same protocol is applied without the addition of any depilating agent, neither enzymatic nor chemical.

At the end of 48 hours of processing, the hair removal is assessed on a qualitative level. For a quantitative assessment of the degree of hair removal, a graphic reworking of the photographs taken of the skin before and after the treatment was carried out, obtaining a percentage of hair removal.

The data obtained from the in vitro characterization are shown in the graphs of figure 1 and are summarized together with those obtained from the tests with real skin sample in the Heatmap of figure 2. The results obtained having grown the bacterial strains indicated in NB = Nutrient are reported Broth, nutrient-rich soil. FMM = Feather Minimal Medium, minimal soil in which the only source of carbon and nitrogen are feathers. WMM = Wool Minimal Medium, minimal soil in which the only source of carbon and nitrogen is wool. It is evident that the growth in minimal medium increases almost each of the enzymatic activities tested.

Advantageously, and it is clearly visible in panels b), c) d) and) f), the enzymatic activity obtained with the supernatants according to the present invention is not limited to a protease type activity (panel a) but contemplates a series of further enzymatic activities.

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

-1Dm8

-1Dm15

-3Am2

-T2D3

TESTS ON PILOT BOTTLE

The 4 selected strains, grown in FMM medium (48h at 27 ° C with 160 rpm agitation), were tested in pilot boot for the determination of hair removal activity. In particular, 4 liters of the clarified supernatant of each strain were tested on real leather sample (about 8 kg) according to the protocol reported in table 5 (traditional liming) and in table 6 (oxidative liming).

Table 5

The products called Biodermol are owned by Biodermol Ambiente srl:

- Biodermol BTM OS: polyvinylpyrrolidone based biocide

- Biodermol PRODEFAT: bioemulgator

- Biodermol PRODEFAT M: enzymatic based bioemulgator

- Biodermol WPM: mix of enzymes including hyaluronidase, amylase and protease - Biodermol TP: sodium tripoli phosphate and salts

- Biodermol LIPOL T1 / T2S: degreaser based on lipolytic enzymes Table 6

Figure 3 is an example of the results obtained from the hair removal tests with the bacterial supernatants on a pilot scale. At the end of all four tests, the processing of the leather samples was carried out up to the Wetblue stage in order to assess any damage to the skin structure. No visible structural damage was found on any of the four tests performed.

Claims (10)

CLAIMS 1. A bacterial strain for the production of enzymes with a high depilatory capacity, where said bacterial strain is selected in the group consisting of 1Dm8, 1Dm15, 3Am2, T2D3, the deposit number of each of said strains being DPS RE RSCIC 14, DPS RE RSCIC 13, DPS RE RSCIC 11, DPS RE RSCIC 15, respectively. 2. The culture supernatant of at least one bacterial strain selected in the group consisting of 1Dm8, 1Dm15, 3Am2, T2D3, the deposit number of each of said strains being DPS RE RSCIC 14, DPS RE RSCIC 13, DPS RE RSCIC 11, DPS RE RSCIC 15, respectively. The supernatant according to claim 2, wherein said supernatant is a liquid culture medium. The supernatant according to claim 2, wherein said culture medium is a minimum medium, preferably is FMM (Feather Minimal Medium) or WMM (Wool Minimal Medium). The supernatant according to one of claims 2 to 4, where supernatant is treated, is preferably dried and / or lyophilized. A formulation comprising at least one supernatant according to one of claims 2 to 5, optionally further supernatants from further bacterial strains, optionally further active. 7. A method for skin hair removal, where said method includes: - Providing at least one supernatant according to one of claims 2 to 5, or a formulation according to claim 6; - Exposing the skin to be treated to said at least one supernatant or to said formulation, at a temperature between 18 and 40 ° C, at a pH between 4.5 and 12. The method according to claim 7, wherein 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. The method according to one of claims 7 or 8 which is for tanning hides and also comprises a soaking step and / or a liming step. A method for cosmetic hair removal or peeling comprising topical administration of at least one supernatant according to one of claims 2 to 5 or a formulation according to claim 6.