NZ708401B2 - Use of an enzymatic complex in the feeding of livestock animals - Google Patents

Abstract

The invention relates to the use of an enzymatic complex comprising a mixture of proteases, which is obtained by culturing a strain of Streptomyces fradiae, for supplementing the feeding of livestock animals, in which one of the proteases has an isoelectric point of about 7.0 and a specific activity of around 150,000 Anson units/mg of protein and another of the proteases has an isoelectric point of about 8.0 and a specific activity of around 38,000 Anson units/mg of protein, wherein greater than 80% of the activity of said enzymatic complex is due to these two types of proteases. The invention also relates to feed compositions for livestock animals comprising said enzymatic complex and also to the method for producing this complex. of around 150,000 Anson units/mg of protein and another of the proteases has an isoelectric point of about 8.0 and a specific activity of around 38,000 Anson units/mg of protein, wherein greater than 80% of the activity of said enzymatic complex is due to these two types of proteases. The invention also relates to feed compositions for livestock animals comprising said enzymatic complex and also to the method for producing this complex.

Description

USE OF AN ENZYMATIC COMPLEX IN THE FEEDING OF LIVESTOCK ANIMALS The technical field of the present ion is that of animal nutrition.

It is known that the Streptomyces fradiae strain is able to produce at least five types of protease known by Ia, Ib, II, III, IV and two peptidases (patent GB-1133579) In their patent application WO 87/07905, the applicants described a process to obtain a lytic x composed exclusively of type II, III and IV proteases with a predominance of type I I se. This complex is obtained from a Streptomyces fradiae WAKSMAN 3535 strain by fermentation, extraction of the complex by filtration, then ultrafiltration on a ne having a cut-off level corresponding to a molecular weight of between 2,000 and 12,000, and finally by atomization. The complex thus obtained in powder form has a titer of at least 2,000 Anson units/mg of protein.

In this application, the type II protease was characterized by a molecular weight approaching 18 kDa and an isoelectric point approaching 9.0.

This complex, used in animal foodstuffs, enables the animals to easily assimilate feed with high crude protein t thereby promoting their growth. Additionally, this complex, used for ing sows, promotes increased birth weights as well as a reduction in food consumption and an overall improvement in health. r, this process is not able to effectively eliminate further contaminating ducts.

In patent application WO 97/37681, improvements are made enabling the use of a product with a titer of at least 4,000 Anson units/mg of protein for therapeutic ations. Thus, the predominant proteinase has a specific activity of over 100,000 Anson units/mg of protein with a molecular weight of between 30 and 36 kDa and an isoelectric point approaching 7.0.

Subsequent analyses showed that the molecular weight of the protease was of 18-20 kDa.

The tterapeutic applications described essentially consist of a posi:ive action in the reduction of the secretion of mucus cnaracteristic of certain illnesses, in an improvement in intestinal absorption and blood flow or e"se in the figrt against infectious diseases. Additionally, the compositions are described as able to facilitate the regeneration of atrophied or damaged villi in the intestinal lining. "n view of the considerable therapeutic ties of the enzyma:ic complex e xtraCCed from Streptomyces fradiae cultures, it is important to use an enzymatic complex more completely cleared of biological impurities that might hinder its ac:ivity.

The aim of the t invention is thus to improve the nsly described. enzymatic x namely in order to enhance breeding conditions and animal health in factory farms.

The invention thus relates to the utilization of an enzymatic complex comprising a mixture of proteases obtained by culturing a Streptomyces fradiae strain to supplement farn animals feed, characterized in that among this ndxture o; proteases one of them has an isoelectric point of around 7.0 and another has an isoelectric point 0: around 8.3.

Said complex predominantly contains these two types of proteases, in o:her words a proportion of these two ses exceeding 80% of the activity of said mixture.

According' to one characteristic of the invention, the protease whose isoelectric point is of around 7.0 has a specific activity 0: around 150,000 Anson units/mg of pro:ein. ing to one characteristic of the invention, the pro:ease whose isoelectric point is of around 8.0 has a specific activity of around 38,000 Ansor mg of protein.

According to another characteristic of the invention, the enzymatic complex is used as a feed supplement in , liquid or any other form suited to its deture with feed compositions to improve the general condition o_ factory farm animals.

According to another charaCteristic of the invention, the enzymatic complex in powder form is dry mixed with a feed composition in a rotating drum anti; homogenized. ing to aqother cqaraCteristic of tre invention, the enzymatic complex in liquid form is pulveri ed in a f“uidi ed bed onto a feed conpositioq, and is then grarulated.

According to another Ciaracteristic of the invention, the enzymatic complex is used in poultry feed.

According to r oiaracteriStic of tre invention, the enzymatic x is used in pig feed. ing to aiother cqaracteriStic of tre invention, the enzymatic complex is used in fish feed.

The invention also relates to a feed composition for farm animals comprising the enzymatic complex ing to the ion and possibly including excipients or vehicles for nutriments, or other additives. r1"he invention lastly relates to a manufacturing process for the enzymatic complex according to the invention, characterized in that a Streptomyces fradiae strain is cultured, in that the fermentation broth is filtered, in that the enzymatic complex is thereafter extraCted by ultrafiitration and ion exchange chromatography followed by an isoeiectric focusing and finally, the x thus obtained is ‘yophiii ed.

This invention provides a feed ment whose characteristics are strictly defined.

The invention firstly enables to adapt precisely the doses to be administered into the feed compositions so as to obtain thc r guirod thorapcutic effects.

The invention also has the advantage 0: improving the health of animals in the y farms. in addition, the invention also brings ar improvement to the general condition of the animals in the factory farms enhancing the profitability 0‘ such facilities.

The invention further enables a1 improved animal growth combined with a reduction in food ption.

Other teristics, ulars and advantages 0: the invention will become more apparent from, the additional description given hereafter of di ,— ferent embodiments given by way 0: e and with respect to the figures illustrating inteStinal villi. it is known that the enzyme mixture commercialized under the trademark Panstimase® (PANcreatic ST"Mu"ating diastASE) is an enzymatic x ed by fermertation of Streptomyces fradiae. This complex is described as essentially containing three proteases, n of wiich specifically act on tial proteins gen, elaStin, and keratin), but also on certain toxins of proteic nature ed by choleraic vibrio or certain pathogenic Escherichia coli strains. in the domain of animal husbandry, it is important to have a product that is technically defined, perfeCtly conStant and whose ties are thus clearly defined.

So as to obtain the enzymatic complex according to the invention a Streptomyces fradiae strain is cultured and the resulting fermentation broth is filtered. Thereafter, the enzymatic complex is extracted and terized by ultra— filtration, ion exchange chromatography and isoelectnic focusing. Lastly, the resulting complex is lfi ed.

For the Streptomyces fradiae culture, a strain. of the type WAKSMAN 353 for example is used, able to be genetically improved. to namely obtain an increase in the proteolytic activity or the suppression of any secretion of antibiotics.

The culture takes place in a large scale fermenter ir a suitable growing medium. This growing medium may, for example, be based on soybean meal: 15 g/l; glucose: 30 g/l; bipotassic phosphate: 1 g/l; calcium carbonate: 10 g/l.

The reaction takes place at a pH 0: between 7.0 and 7.5, at a fermentation temperature 0: around 28 0C with an on of 0.3 s of steri e air for one volume of mediJm per minute.

Once is culture is completed, the mycelium is eliminated by filtration of the fermentation broth using a clarifying agent.

The extraction of the enzymatic complex required the implementation of several techniques including ultra— tion, ion exchange chromatography and isoelectric focusing.

Ultra—filtration The ul:ra—filtration of she fil:ra:e is performed using a membrane With a CUt‘Om’ “eve of 50 (Da.

After 3h of filtering, the fil':ra:e (71% of the l volume) has a proteolytic activity reveaj_ed by the gelatin *i'm assay. This assay consists ir deteriorating a film of silver gelatin arranged on a solid frame. ion exchange chromatography assay A cation exchange chromatography is used made with colu ns of Cellu fine C—500 carboxymethy: gel (Amicon) o; suitable dimensiors.

The chromatographic bu” .Eer is a citrate bu ,. "er 10 mM at pH 5.5 and the elution is performed by a NaCl gradient 0: 0 to 1.0 M. The elution rate s on the size of the columns, for example, it is of l0 to 15 ml/min for a column 0: 2.5 cm in diameter and 27 on in height.

Isoelectric focusing 2O ical ctric ’oc ‘ng is per-formed using ready— to—use gels made by Pharmacia for pHs from 3 to 9 on Phast— system. tion is per:formed using Coomassie blue. in the case o: preparative isoelectric ng in a solid medium, the enzyme solution is dialyzed against distilled water (95 ml) and mixed with 5 ml 0:: 3—9 ampholytes with 4.7 g o: Ultrodex gel and 0.5 g o: glycine. The gel is dried for 6 h at 40°C. Migration takes place during the night, or equiva lent period, at a cor stant power of 3 Vt Revelation is per:formed us ing Coomassie blue.

The different gel strf-ps are taken, rinsed with 3 ml 0: water, filtered, and then rinsed with 3 ml of G: buffer.

The fractions thus obtained are then analyzed to determine their proteolytic activity and protein content. The s are then dialyzed against the Tris—HG: buffer 0.3 M pH 8.0.

In the case o: preparative isoe‘ectric focusing in a liquid medium, the enzyme solution dia:_yzed against distilled water is mixed with the ampholytes (3.5 ml). Migration is performed at a constant power of 8 W during ~ h.

The fraction s amples are ed then dialyzed against the TriS*HC" buffer 0.3 M pH 8.0.

The experimental conditions indicated above are to be adapted according to the quantities of product required to be obtained and to its utilization in rial conditions.

A predominant protease is thereby obtained in the mix:ure that has an isoelectric point (:EP) 0: around 7.0 and in a lesser propor:ion a protease whose iiP is 0: around 8.0.

The predominant protease whose "?P is 0“ around 7.0 is obtained in a volume of 9 ml with a protein tration 0; 0.49 mg/ml. Speci a; precautions for purification may be required because 0; :he itation of this protease at its isoelectric point. The solution 0btained has a titer of around 75,000 Anson inits (A.U)/ml.

This protease is purified after homogenization at a scale of 4 mg with a specific activity of 150,000 A.U/mg of protein.

One Anson unit (AU) is defined here as being the quantity of enzyme which, incubated for 10 minutes at 25 °C and at pH 7.5 in the C presence o; deratured hemoglobin, releases from this substrate the equivalent of l. microgranl of tyrosine, determined by spectro—photometric absorption at 280 nm on the filtrate that cannot be precipitated with trichloracetic acid. rThe protease, whose ifip is O“ around 8.0, is recovered join:ly with the previous one but in a greater volume, 0: 35 ml for example, and has a protei n concentration of 1.48 3O mg/mi. This solution ras a titer of around 56,000 A.U/ml.

This protease i s ed afte” homogenization at a scale o: 50 mg with a ic activity 0:5 around 38,000 A.U/mg of protein.

The previously described. enzymatic complex essentially contains pro:eases and other proteins. The purifications performed and ':he ophoresis technique used. prove an additional purification and the elimination of inating als that have no proteolytic activity.

The different proteases ed may be better characterized by determining tha: activities on proteins such as collagen, <eratin, n or hemoglobin.

Unfortunately, these dete”minat ons are complicated because of their lack of ivity and specificity. se m 4st ore be made to several activity assays, constituting detection methods, and principally the Anson method.

Activity assays Anson assay This assay enables a proteolytic activity to be dosed, without icity. "t consists in dosing the hydrolysis of the denatured hemoglobin by spectrophotometric determination 0: the tyrosine contained in the oligopeptides released.

Azocasein assay This assay consists in determining by spectrophotometry at 440 nm the hydro“ ysis C 0’ the casein stained by an orange dye. This assay is not speci fic. After incubation with :he enzyme, the remaining azocasein is itated using :richloroacetic acid. irg the optical y reflects :he presence of the peptides released, and thus the enzymatic activity.

Elastolxtic activity assay Elastin Congo—Red assay This assay consists in spectrophotometrically dosirg at 495 im the stained peptides released by the hydrolysis of the elas:in stained by a red dye.

A possible problen1 of this dosage lies in :he insolubility of the substrate and thus its sensitivity depends on the sample accuracy, :he stirring of :he tion mediim and the adherence of the substrate :0 the walls of the incubation vessel.

The assays performed show a lack of linearity between the activity and quantity of enzyme. There is e”"ectively linearity between the activity and the incubation time, but the straight line representing it does not pass through the origin.

A on: Serence in absorbance can thus be measured between the incuba: ion times (2O and. 40 minutes). With 10 ul of complex d 10 times, a variation 0: 0.0~9 uDO/min is measured. The tic complex according to the invention thus does have a proteolytic activity wf-th respect to the elastin.

NBA assay (N—Bloc—L—Alaninate—para—nitrophenyl ester) Thi s assay consists in spectrophotometrically dosing at 347.5 rm the esterase activity present in the elastase by determiqing the itrophenol released (Vissier L. et al.

Biochim, s Acta 268 (1972) 207.260).

This dosage is not specific to an e iastolytic activity, but is ve ry fast (kinetics of 3 min) a nd has good sensi:ivity ndeed, wit? 50 u" 0: complex diluted 100 times, a varia:ion o f 0.235 uDO/min is measured. T 1e enzymatic complex thus does nave an. activity enabling tt e release of para— nitrophenol. enolxtic activity assay Azocoll assay This assay consists in spectrophotometrically dosing at 520 nm the peptides released by the ysis of the azocoll (ground collagen stained with a Bordeaux red dye) (Chavira, Analytical Biochemistry 136 (1984) 446—450).

This assay sucfiers from the same drawbacks as that of n Red (insoluble substrate), but has good sensitivity: with 50 pl of complex diluted 50 times, a D0 of 0.398 is Heasured after incubation during 10 Hdnutes. The enzymatic complex according to the invention thus does have a proteolytic activity with respect :0 the azocoll.

The proteolytic activity of the enzyma:ic complex according to the invention is thus established. The purifiication : 0' said complex has not altered its activity.

The pro:ei1 complex thus obtained and characterized can be ased in animal therapeutics, namely by way of an immunostimuiant agent and as (a regeneration agent for the Peyer’s Patches, in ular for older animals.

Additionally, the enzymatic complex enables the regeneration of intestinal vi‘li in older animals (rats, chickens, etc) and thus greatly improves the intestinal tion of essential nutrimerts.

The enzymatic complex according to the present invention is mainly intended for animal husbandry and more particularly for the improvement of cattle feed (bovine, porcine, ovine) and poultry, rabbit and tish teed as well as feed for any other monogastric anima l.

Incorporated at doses from 0.025 to l g/kg to feed compositions, this enzymatic complex enhances the metabo“ SID 0: these animals and contributes towards ensuring better general health. Pre ferably, the doses of the complex introduced into the feed. compositions are of 0.05 to 0.2 g/kg.

In the case of y farming, maintaining good general health is important as this avoids the propagation of illnesses caused by promiscuity or con:finement. The morbidity rate is thus ntially reduced and notably the mortality 2O rate by bacterial contamination o "arr1 animals.

The feed composit ions based OH this enzyme mixture contain one or several excipients or vehicles for nut:iments, such as for example, grain flour (wheat, maize, rye, rice, millet, and sorghum), n, ca rbohydrates se, mannose), loading elements (casein, bran, cellulose, cellulose derivatives) and/or mineral elements (chalk, clay, bentonite, ) and all other elements used in animal ion.

These compositions are thoroughly dry mixed with the complex in a rOtating drum, or the enzymatic complex, in a liquid and particular ly s form, is sprayed as a fluidized bed on a feed composition and then granulated. Such operations may be performed. at variable atures from °C to 60°C.

The use 0: an tic complex that is purer and more active thereby enables the biological ty of the nutritive compositions according to the invention to be better dosed and a satis:factory homogenization o: the results to be obtained.

For this reason, feed compositions according to the invention have an enzymatic complex t that is clearly determined and nt.

Thus, here is a non—exhaustive list or examples 0‘ Seed compositions intended for farm animals that contain the enzymatic complex according to the invention mixed with excipien:s or appropriate vehicles of nutrition. y feed composition A composition is ed for a batch of 10 kg adding the enzymatic complex: Wheat flour 2.5 kg Lactose 4.5 kg Bran 3 kg Enzymatic complex according the invention 0.2 g/kg.

Such a composition is incorporated at a proportion of 0.5 kg per me':ric ton 0'‘ poultry Seed. :y feed composition A composition is prepared for a batch o? 20.1 kg to which the enzymatic complex is added and which is then incorporated into the poultcy ‘eed: Wheat flour 1.5 kg Oat s'our 4.5 kg Cellulose 14 kg Colloidal silica 0.100 kg Enzymatic complex according the invention 0.1 g/kg.

Pig feed composition A ition is prepared cor a batch of 10 kg to which the enzymatic complex is added and which is then incorporated into the pig feed: Wheat flour 4 Bran 2 Casein 3 9otato starch 1 Enzymatic complex ing the invention 0.2 g/kg Cattle feed composition A composition is prepared in the same way using: Rice starch 0.5 kg ?otato starch 2.5 kg tic complex according the invention 0.2 g/kg.

The premix thus formed is added little by little to a mixture 0: 6 kg of mu:ton protein powder and 14 kg of casein.

Tie homogenized preparation is intended to be incorporated into the cat:le :eed in a proportion of 0.5 kg per metric ton 0; feed. y _eed composition5 The composition is prepared for a batch o: 10 kg to which the enzymatic complex is added: Maize 5.65 kg Soybean meal 3.23 kg seed meal 0.3 kg Wheat germ flour 0.4 kg Chalk 0.13 kg Di—calcium phosphate 0.16 kg .aC; 0.03 kg Amino acid complex 0.1 kg Enzymatic complex according the invention 0.2 g/kg.

The enzymatic complex according to the ion may also be added to o””—Lhe—shelf poultry feed compositions so as to supplemert these preparations.

Fish feed composition The iollowing1) composition. is prepared. and incorporated into the Sish ‘eed to‘lowing the customary practice.

Fish meal 50.8% Animals fats and oils 28% Soybean meal 9.9% Wheat flour 10% Vitamin complex i 0\0 (A, D, E, C,m) Enzymatic complex 0.3 % according the invention Experimental part To determine the action of the complex according to the invention on intestinal villi, assays are performed on relatively old rats (more than seven months old).

These rats are given food based on vegetal proteins ively of tre type wheat flour, soybean, or maize. The enzymatic complex according’ to the invention as described previously is added to this feed in the proportion 0“ O.’ OI 0.2 g/kg.

The results are explained in figures 1 to 4. These figures are photonic microscopic views (enlargement x120) 0; :he villi of the duodenum and jejunum Figure 1 shows the duodenal villi 0: rats not having received the enzymatic complex according to the invention in their feed.

Figure 2 shows the al villi 0: rats having received :eed incorporating the enzymatic complex according' to the invention.

Figure 3 shows the jejunal vil li of rats not having received the enzymatic complex ing to the invention in their feed.

Figure 4 shows the jejunal villi of rats having received feed. incorporating the tic complex according‘ to the invention.

This establishes that the addi tion of the :ic complex in the feed of older animals (rats, chicken, etc.) increases the size and number of tre villi in both :he duodenum and the jejunum. Increases C Oi up to 70% have been observed.

These results are erable to intestinal microvilii.

Similar results are obtained with chickens, pigs and fish.

An increase in the surface area enables faster tion o: nutriments or medicines.

This improved absorp:ion is observed in trouts receiving feed containing an antibiotic. In the assay, the feed contains 4g/kg o: oxyte:racycline and the results are read after 48 hours. An increase in the tissue concentration of the antibiotic is thus observed of a magnitude of 100% in the liver (5.75 instead of 2.5 microgram/g), 400% in the muscles (1.75 d of 0.32 ram/g) and 1000% in the kidneys (5.75 instead of 0.5 microgram/g).

Similar s are obtained with chicken and pigs.

This increase in density may lead to an increase in the local production of physiologically active compounds such as intestinal hormones.

Definitions of specific embodiments of the invention as claimed herein follow.

According to a first embodiment of the invention, there is provided use the of an enzymatic complex comprising a mixture of proteases obtained by culturing a Streptomyces fradiae strain to supplement farm animal feed, wherein one of the proteases has an ctric point of around 7.0 and a specific ty of around 150,000 Anson units/mg of protein and another se has an isoelectric point of around 8.0 and a specific activity of around 38,000 Anson units/mg of protein, wherein greater than 80% of the activity of said enzymatic complex is due to these two types of proteases.

According to a second embodiment of the invention, there is ed a feed composition for farm s comprising the enzymatic complex according to the first embodiment and possibly including ents or vehicles for nutriments.

According to a third ment of the invention, there is provided a manufacturing process for the enzymatic complex according to the first or second ment, wherein a Streptomyces fradiae strain is cultured, in that the fermentation broth is filtered, in that the enzymatic complex is thereafter extracted by ultrafiltration and ion exchange chromatography followed by an isoelectric focusing and finally, the complex thus obtained is lyophilized.

Claims (9)

1. Use of an enzymatic complex comprising a mixture of proteases obtained by culturing a Streptomyces fradiae strain to supplement farm animal feed, n one of the proteases 5 has an isoelectric point of around 7.0 and a specific activity of around 150,000 Anson units/mg of protein and another protease has an isoelectric point of around 8.0 and a specific activity of around 38,000 Anson units/mg of protein, wherein greater than 80% of the activity of said enzymatic 10 complex is due to these two types of proteases.

2. The use according to Claim 1 as a feed supplement in powder, liquid or any other form suited to its mixture with feed compositions to improve the general condition of factory farm animals. 15

3. The use ing to Claim 2, wherein the enzymatic complex in powder form is dry mixed with a feed composition in a rotating drum until nized.

4. The use according to Claim 2, wherein the enzymatic complex in liquid form is ized in a fluidized bed onto 20 a feed composition, and is then granulated.

5. The use according to any one of Claims 1 to 4 in poultry feed.

6. The use ing to any one of Claims 1 to 4 in pig feed. 25

7. The use according to any one of Claims 1 to 4 in fish feed.

8. A feed composition for farm s comprising the enzymatic complex according to any one of Claims 1 to 4 and possibly including excipients or vehicles for nutriments. 30

9. A manufacturing process for the enzymatic complex ing to any one of Claims 1-8, wherein a Streptomyces fradiae strain is cultured, in that the fermentation broth is filtered, in that the enzymatic complex is thereafter extracted by ultrafiltration and ion exchange chromatography 35 followed by an isoelectric focusing and finally, the complex thus ed is lyophilized. ation] KEB