WO2003080873A1

WO2003080873A1 - Cleaning animal skins

Info

Publication number: WO2003080873A1
Application number: PCT/GB2003/001113
Authority: WO
Inventors: Anthony Dale Covington; Christine Stella Evans
Original assignee: Klenzyme Ltd
Priority date: 2002-03-19
Filing date: 2003-03-18
Publication date: 2003-10-02
Also published as: EP1488012A1; GB0206464D0; AU2003214413A1

Abstract

An enzyme mixture, suitable for removal of dung from animal skins as a preparative step in production of leather, is prepared by cultivating white rot fungi under conditions that induce formation of a significant amount of laccase. The mixture preferably contains the three enzymes cellulase, xylanase and laccase (ligninase).

Description

CLEANING ANIMAL SKINS

This invention is concerned with cleaning animal skins by providing a composition for the removal of dung from animal hides and skins used for leather manufacture. This typically involves cattle, sheep and pig skins, but the problem of coated dung is especially prominent on skins from cattle. In particular, the present invention is concerned with the production of enzymes in a mixture, specifically designed to degrade the dung.

Dung on cattle creates problems for hygiene on the dairy farm and more particularly at the abattoir, where there is risk of contaminating the carcase with faecal organisms, notably including E. coli 0157. Typically this is not addressed by the respective industries, creating a residual problem that must be addressed by the global leather industry, particularly in respect of beef cattle which form the biggest source of hides for the leather industry.

Dung must be removed from the hides in the early stages of processing, as part of the cleansing operations, leading to tanning and the production of high quality leather. Removal of dung is difficult; the composite material created by hair and dry dung is resistant to solubilisation, even in the presence of surfactants. It is accepted in the industry that even the enzymes offered as soaking auxiliaries do not have any useful effect in this regard: those enzymes include proteases, lipases and amylases, but no claims are made by the supply houses for any positive effect on dung. In studies of the effects of enzymes on the solubilisation of dry dung, it was confirmed that those types of soaking enzymes are ineffective (Enzymatic removal of dung from hides. N. Auer, A. D. Covington, C. S. Evans, M. Natt, M. Tozan; J. Soc. Leather Technol. Chem., 83(4), 215, 1999.). Therefore, tanners are obliged to risk bacterial damage in prolonged soaking, or to remove the dung with hair, incurring additional chemical cost and limiting the options for disposing of the contaminated hair.

In GB 2,325,241 it is demonstrated that dung is removed efficiently and effectively by targeting the main components of the dung with specifically acting enzymes: the lignocellulosic material, from degraded plant cell walls, can be solubilised with a mixture of ccllulase, and xylanase, optionally ligninase.

Subsequent studies by the present inventors have showed that the lignocellulosic material is solubilised as the complex, rather than by selective damage of one or two of the constituents. This indicates that it is necessary to degrade the lignin which surrounds the fibres to expose the cellulose to attack, and at the same time to break the hemicellulose linkages between the cellulose chains, in order to dissolve the complex.

Accordingly, the present inventors' solution to the problem of dung in the tannery is to apply a treatment of the three enzymes, at an optimum activity ratio. However, although cellulase and xylanase are available in commercial quantities, there is at present no commercial source of ligninase. Therefore, there is a need to produce the ligninase by large scale fermentation of a suitable microorganism. In this context the present inventors have sought to create the conditions which would force a microorganism to express the mixture of enzymes required for dung removal.

This invention is based on the finding that white rot fungi can be induced to produce a mixture of enzymes suitable for removal of dung from animal skins as a preparative step in production of leather.

Accordingly, the present invention provides a method of preparing an enzyme mixture for cleaning animal skins which comprises cultivating a fungus selected from the class of White Rot Fungi in a liquid growth medium and harvesting the enzymes produced by the fungus from the liquid growth medium.

The principle aim of the present invention is to produce a mixture of the three enzymes cellulase, xylanase and laccase (ligninase).

Suitable white rot fungi are found (but not exclusively) in the family Polyporaceae. Especially suitable are fungi of the species Coriolus, Pleurotus, and Ganoderma, in particular Coriolus versicolor (also known as Trametes versicolor), Pleurotus ostreatus and Ganoderma applanatum. Other suitable white rot fungi can easily be determined by routine testing for rate of growth, ability to produce all three enzymes, levels of enzyme activities etc..

Some white rot fungi decompose lignin by production of a peroxidase, which requires hydrogen peroxide as a cofactor, rather than laccase. A typical example is the species Phanerochaete, especially Phanerochaete chrysosporium. These white rot fungi are within the scope of the present invention, but the resultant enzyme mixtures are less preferable for the treatment of animal skins because of the need to provide a source of hydrogen peroxide for the peroxidase to act. The present inventors have found that white rot fungi that produce a mixture of cellulase, xylanase and laccase often do not produce laccase in sufficient quantities for optimum treatment of animal skins. However it has been discovered that this problem can be overcome by cultivating the fungus in the presence of a suitable inducer. Advantageously the inducer to promote production of enzymes to remove any particular faeces is the faeces itself, preferably in sterile form.

In tests carried out by the present inventors on the fungi Coriolus versicolor, Pleurotus ostreatus and Ganoderma applanatum, Coriolus versicolor and Pleurotus ostreatus were the fastest growing species, covering a 7 cm malt-agar Petri plate with hyphae from a central inoculum within six days, whereas Ganoderma applanatum, took twelve days.

C. versicolor and P. ostreatus produced similar amounts of cellulase and xylanase in the liquid media with cellulose or xylan as substrates over a ten day growth period, but differed in their production of laccase. P. ostreatus produced only low levels of laccase over ten days, with most laccase produced after growing for twenty days or more, when cellulase and xylanase activities had diminished considerably. Laccase activity was not increased significantly in the presence of a lignin mimic inducer in the first ten days of culture. In contrast, laccase production by C. versicolor doubled in the presence of an inducer compound, with the highest amount of laccase produced by any organism after eight days growth.

Surprisingly, it was found that the ratios of the three enzyme activities required to treat dung could be controlled by the nature of the growing medium, in particular, the difficulty of producing enough ligninase (laccase) could be overcome by adding a growth medium auxiliary as an inducer. Thus, the required enzyme mixture can be produced in a single fermentation step.

Unexpectedly, it was found that the inclusion of dung, most suitably dried, and preferably sterilised before use, significantly broadened the peak of laccase production. This is of great value in the context of commercial production, since it greatly assists in the ability to harvest a suitably proportioned enzyme mixture.

In the present invention, the fungi are cultivated in a liquid nutrient medium with a nitrogen source and a carbon source, and preferably an inducer in the form of sterile dung. After a suitable period of growth, fungal growth is removed and enzymes in the culture fluid are harvested. Suitably the fungi are added to the nutrient medium in pelletised form, to assist in subsequent removal by filtration, together with any dung residue. The filtrate containing the enzymes is preferably concentrated, for example using a membrane concentrator with a cut off at 10.000 Daltons. and then the concentrate is freeze dried, to obtain the desired enzymes as a lyophilised powder. The lyophilised powder may be stored or packaged for future use in leather preparation, to remove dung from animal skins as proposed in GB 2,325,241. Suitably, before use in the tannery, the enzyme powder is mixed with an inert bulking agent, so that technicians are able weigh out enzyme dosages in, for example 100 gm units rather than gram units. Alternatively the enzyme mixture, or bulked mixture, may be pre-packaged in unit doses. The bulking agent is selected so that it will not leave a residue on the treated skins, or affect treatment pH. Sodium chloride may be used.

If the fermentation to produce the enzyme mixture is carried out at or close to the tannery, then it may be suitable or appropriate to use the enzyme mixture as the liquid concentrate obtained after harvesting the enzymes by removing small molecules. Alternatively, the lyophilised powder may be reconstituted with water, to add the enzymes to the treatment bath as a liquid

Applying the teaching of this invention has two aspects: first the choice of microorganism(s) from the indicated white rot fungi, which determines the types of enzymes capable of being expressed and second the nature of the growing medium, which controls the amounts of the enzymes. The latter depends to a large degree on the choice of auxiliary/inducer, to produce the required enzyme mixture to treat a particular type of animal faeces: most suitably this will be the faeces itself.

The basis of the invention is the means of producing the desired enzymes in suitable ratios for cleaning skins in a single fermentation. In the preparation of leather, the ability to remove the dung in the first soak, the dirtiest step, enables the pollution to be confined to a small volume for effluent treatment. Moreover, efficient early cleaning of the hide can open up new processing options for the leather industry, offering better leather quality and reduced environmental impact. In particular the simple change to removing the flesh after soaking is a highly desirable alternative to fleshing part processed hide, when it is alkaline with hair removed and thereby difficult to handle. A further use of the enzyme mixtures obtainable by this invention is to clean the skins of live animals before slaughter, to improve hygiene in the abattoir. The invention is further illustrated by the following Examples and by reference to the accompanying drawings, in which:

Figures la, lb and lc show production of enzymes after adding 1% (w/v) inducer after 3, 6, 9 or 12 days into cultures of C. versicolor containing 2% (w/v) carboxymethyl cellulose (CMC) as carbon source.

Figures 2a, 2b and 2c shows a comparison of shaker speeds in enzyme production. Figures 3a, 3b and 3c show further results from fermentation of C. versicolor on the 2 litre scale.

Example 1.

Induced enzyme production by C. versicolor in shake flask cultures.

The liquid growth media in these trials were based on a mineral salts medium with ammonium nitrate as nitrogen source (see - E. Abrams; National Bureau of Standards Misc. Publications no. 188. U.S. Dept. of Commerce, Washington) and included carboxymethyl cellulose (CMC) as carbon source. The inventors' proposed inducer for laccase was cow dung collected from a field grazed by cattle.

Dried and sterilised cow dung, was added at different time intervals during growth of the fungus in the growth medium. The results are shown graphically in the appended Figures la, lb and lc which show the effects on the production of enzymes of adding 1% (w/v) inducer after 3, 6, 9 or 12 days into cultures of C. versicolor containing 2% (w/v) CMC as carbon source.

For laccase production, addition of the inducer after three days of fungal growth gave the highest yield of laccase in cultures at day 8 of growth. For cellulase and xylanase activities, addition of the inducer after three days also yielded the highest enzymes activities, but from day 6 onwards.

Table I. The effect of inducer on the production of laccase.

Example 2.

The role of carbon source in stimulated enzyme production from C. versicolor in shake flask cultures.

The effects of different carbon sources on enzyme production were investigated: using glucose, crystalline cellulose powder or CMC, each at 2% w/v in the medium. The results are shown in Table II.

Table II. The effect of carbon source and inducer on the activities of enzymes cultured from C. versicolor.

From these tests, CMC is the preferred carbon source for production of all three enzymes. Addition of 0.5-1.0 % inducer at day 3 of growth stimulated enzyme production by 80-100 % from day 6 for xylanase and laccase and from day 8 for cellulase. Addition of different concentrations of CMC was investigated (0.5 to 2 %) for the effect on enzyme production: all enzyme activities increased as the concentration of CMC was increased in the medium, up to 2 % CMC.

The optimum conditions for simultaneous production of cellulase, xylanase and laccase activities in shake flasks were 2% w/v CMC in the medium as carbon source. 0.5-1.0% w/v inducer added at day 3 of growth. The cultures reached the optimum enzyme activities at 8 days growth, with approximately equal cellulase and xylanase activities. For application to treating dirty cattle hides, GB 2,325,241 indicates the preferred ratio of cellulase to xylanase as 2:1 , together with sufficient laccase activity. If 1 % glucose was added to the CMC medium, the titre for laccase activity was increased substantially, three fold, with cellulase unchanged, but xylanase titre reduced by 30%, see Table IV. This resulted in a cellulase to xylanase ratio closer to 2:1 , but with substantially increased laccase activity, which meets the preferred mixture requirements more closely.

Example 3. The effect of agitation on shake flask cultures of C. versicolor.

The agitation rate of the cultures (affecting availability of dissolved oxygen) was found to be critical in maximising enzyme production. At day 8 of growth, with the inducer added at day 3, cellulase, xylanase and laccase activities maintained higher levels when agitation was at 150 φm, compared with 200 φm. Optimum activities of all three enzymes occurred at day 8 under these conditions, as shown in Figures 2a, 2b and 2c comparing shaker speeds in enzyme production.

Example 4.

Simultaneous production of cellulase, xylanase and laccase by culturing C. versicolor in 2 litre bioreactors.

The medium optimised for shake flasks, using 2% CMC as carbon source and addition of inducer on day 3 of fungal growth, was used in the bioreactor, with agitation at 150 φm. Figures 3a, 3b and 3c show enzyme activities from typical fermentation, with maximum activities occurring from day 5 to day 10 of growth for cellulase and xylanase and at day 8 for laccase. Dissolved oxygen concentration was maintained between 20 and 100% throughout the fermentation,

Example 5.

Simultaneous production of cellulase, xylanase and laccase by culturing C. versicolor in 20 litre and 75 litre bioreactors.

The same conditions as described for a 2 litre bioreactor, given in Example 4, were used for growing the fungus in a 20 litre bioreactor. Dissolved oxygen concentration was maintained at 40 to 100%. It was observed that maximum cellulase, xylanase and laccase production was obtained between days 6 and 12 of growth.

Using a 75 litre bioreactor, similar enzyme titres were obtained, providing the dissolved oxygen concentration did not fall below 30%.

Example 6.

Stability of the enzyme mixture

Enzymes in the culture fluid, harvested on day 8 of growth, were concentrated through a membrane concentrator with a cut off at 10,000 Daltons, then the concentrate was freeze dried.

The powder was stored at room temperature, 4°C and -20°C and the activity was assayed over a three month period. Laccase activity disappeared after 3 months at room temperature and reduced by 50% at -20°C. Cellulase and xylanase activities had not decreased after three months at -20°C or 4°C, but a slight reduction was observed in xylanase activity after storage at room temperature.

Claims

1 A method of preparing an enzyme mixture for cleaning animal skins which comprises cultivating a fungus selected from the class of White Rot Fungi in a liquid growth medium in the presence of animal faeces as an inducer and harvesting the enzymes produced by the fungus from the liquid growth medium.

2. A method according to claim 1 in which the fungus is selected from the family Polyporaceae.

3. A method according to claim 1 or 2, in which the enzyme mixture includes cellulase, xylanase and laccase enzymes.

4. A method according to claim 3 in which the fungus is selected from the species Coriolus, Pleurotus and Ganoderma.

5. A method according to claim 3 in which the fungus is selected from Coriolus versicolor, Pleurotus ostreatus and Ganoderma applanatum.

6. A method according to claim 1, in which the enzyme mixture includes cellulase, xylanase and lignin peroxidase enzymes.

7. A method according to claim 6 in which the fungus is selected from the species Phanerochaete.

8. A method according to claim 6 in which the fungus is Phanerochaete chrysosporium.

9. A method according to any one of claims 1 to 8, in which the fungus is cultivated in the presence of cattle dung as an auxiliary growth medium.

10. A method according to any one of claims 1 to 9 in which, after a suitable growth period, residues are removed from the nutrient medium by filtration, and the enzyme mixture is harvested, and then freeze-dried.

1 1. An enzyme mixture which includes cellulase, xylanase and laccase enzymes, or cellulase, xylanase and lignin peroxidase enzymes in suitable proportions for cleaning animal skins, obtainable by cultivating a fungus selected from the class of White Rot Fungi in a liquid growth medium in the presence of cattle dung and harvesting the enzymes produced by the fungus.

12. An enzyme mixture according to claim 1 1 as a lyophilised powder.

13. An enzyme mixture according to claim 12 further comprising an inert bulking agent.

14. An enzyme mixture according to claim 11 as an aqueous solution.

15. Use of an enzyme mixture as claimed in any one of claims 11 to 14 as an agent for removing dung from animal skins before tanning.

16. Use of an enzyme mixture as claimed in any one of claims 11 to 14 as an agent for removing dung from the skin of live animals before slaughter.