CA2256256A1 - Composition with controlled release of biologically active substances for zootechnical use - Google Patents
Composition with controlled release of biologically active substances for zootechnical use Download PDFInfo
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- CA2256256A1 CA2256256A1 CA002256256A CA2256256A CA2256256A1 CA 2256256 A1 CA2256256 A1 CA 2256256A1 CA 002256256 A CA002256256 A CA 002256256A CA 2256256 A CA2256256 A CA 2256256A CA 2256256 A1 CA2256256 A1 CA 2256256A1
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- active substances
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
- A23K40/35—Making capsules specially adapted for ruminants
Abstract
Compositions for zootechnical use for the administration of biologically active substances with nutritional and/or pharmacological properties, with delayed release, are described. The compositions comprise a vehicle constituted by a mixture of fats and waxes, wherein the biologically active substances are incorporated. The method for the production of the compositions comprises the melting of the mixture of fats and waxes, the incorporation of the biologically active substances in said molten mixture, and the subsequent solidification and fragmentation obtaining particles with dimensions of 400-500 m. The compositions of the invention are used for the activation of feeds, for the purpose of obtaining medicated and/or nutritionally integrated feeds, for instance with essential amino acids or vitamins.
Description
17. DIi'' ' 9$ r,c;lOl 14: 12 BUi,Nfc)N SEA RIA41N1 sy39 x:;541 78:'721 FS~~.
.
COMI'OSl'1'lON WffHCON'CROI.LI~DREL,FAS~ Of BIOI,OCrICAl,I,Y ACTIVE?
SI1BSTANCES FOR ZOOTECHNLC.A(, USR.
B~CKC;ROj 1Nn OF 'fl-1F? INVENTION
The present invention relates to a composition for zootechnical use for the oral administration with controlled release of biologically active substances having pharmacological and Jor nutritional properties.
The present invention further relates to a method for the preparation of said Q composition, as well as the use of said composition as an additive to feeds, in order to obtain medicated andlor nutritionally integrated feeds.
Biologically active substances having nutritional properties ( i.c. food integrators) and/or pharmacological properties are commonly administered to animals orally, generally added to the feeds.
15 In such administra.ticrns the biologically active substances undergo a chemical-enzymatic debn~adation during the transit time through the animal organism prior to reaching the intestine. Such degradation is dune severe in the case ofruminanis, since ' transit time in the ruminant system is very high and due to the presence of the microbic.
flora.
The biologically active substances may also undergo a degradation during the feed enrichment phase and during the storage of the enriched feeds prior to their administration to the animals.
It is also important to mask the taste of the biologically active substances, particularly those with pharmacological properties, in order to make rnctre attractive the 25 medicated portions of feed to be administered to the animals.
F?xamples of substances commonly added to feeds are some vitamins. Vltamrns are prone to >7eing degraded prior to reaching the intestine of the animal.
Such degradation is also caused by interference with the microllora which is present in the organs of the animals (and especially in the ruminal system of ruminants) which are 30 traversed hef~re the intestine is reached.
For instance, in the case of vitamin C, the rate of degradation during permanence in the rumen can even exceed ~)0%. More or less severe degradation is also experienced l 17. Dli..' ' ~; !yIG! 14: 1. BIJi;NIcSN SPA. R_ItetlNl ii'j ,= 'i541 ';
:._~7G1 F':~.c-:. . .~ , with other vitamins, suctr as vitamin A, vitamin B6, vitamin 1312, vitamin D
and vitamin L. Particularly high is the degradation of the free form of vitamin >r (ILL-alpha-tocophrrrol)less high is the degradation of the esterified form (DI~ alpha-t~copherylacet~te).
Other examples of substances that are added to feeds and arc subject to degradation a.re ribof7a.vin, folic acid, niacin a.nd chOlrllC. The latter plays an essential role in large lactiferous animals during the first phase of lactation, where a considerable mobilization of reserve fats taken place t.o compe.nsate for energy shortages.
The fatty acids accumulated in the liver are therein transformed into triglycerides, which in turn give rise to pathological hepatic steazosis. Choline is able to obviate such accumulation favoring the phospholipidic metabolism, and thus the lipoproteins that carry out the function of triglyceride transport and removal. 'fhe proper administration ohcholinc improves the animal's hepatic functionality and productivity. However, unprotected choline is degraded into trimethyla.rnine, an inactive substance that gives unpleasant I S tlavor and odor to the milk. Such degradation can reach levels of about '70% of the quantity initially administered.
Other substances that are added to feeds and are subject to degradation prior to reaching the intestine of ruminants are amino acids. Enrichment with amino acids is important since milk production is particularly sensitive to fhe required duantity of plasmatic amino acids, essential for de nnvu synthesis of milk proteins at the mammary gland level. nll amino acids necessary for the metabolism of Illllk proterns Illust be present in the blood in the quantity necessary for the proper "assembly" of the proteinaceous -cascous component.
Maximum proteinaceous eft7ciency is often compromised by the lack of two essential amino acids: L-lysine and DL-methionine. The massive administration of protein sources is not always able to obviate this problem. A protein excess can set off forms of competition upon absorption and/or tissue and cell utilisation, for instance i competition between L,-triptophane and branched amino acids or between h-lysine and L-arginine. Moreover, the excess of nitrogenous substances, if not carlihratccf. entails an overload of ammonia defecation, aggravating environmental impact and hygienic conditions inside the stock farm, particularly if enclosed.
Furthermore. a shortage of amino acids, especially L7L-methionine, limits lhc t~. vtc ' ~_, rc;~o> »: i su<::r;tor~ ,<.~Fw emmrm _~e~~ ~~~~~i ?c;~;li r~;~:;.
growth, productivity and fertility of the animals.
Examples of pharmacologically active substances added to feeds and administered to animals are some antibiotics, both synthetic and fermentativc; in many eases, the antibiotics so added, especially macrolidic or quinolinic ones, in addition to being S degraded, also modify the taste of the feeds making thorn unattractive.
'fhe need to improve the stahiliiy and preacrvabili.ty of feeds enriched with biologically active substances, for instance nutritional integrators, is all the greater, the higher the water content in the feeds. For example, liquid or semi-liquid mixtures with high water content, common in swine nutrition, arc unstable because they are subject to very pronounced fennentative or hydrolytic transformations.
In order to reduce the aforesaid drawbacks, the proposal has been advanced of encapsulating some biologically active substances for zootechnical use with films of pI-I-sensitive materials, able to withstand the gastric environment, for instance polymers based on polvvinylpyrrholidonc, vinyl polymers and copolymers, polyesters and polyamides, chemically rnoditicd cellulose, polypeptidic agents and starches, thereby obtaining a certain protecfiion of the active substances and their delayed release in the intestine ofthe animals.
Such solutions present some drawbacks in that the particles of biologically active substances so encapsulated occasionally have significant dimensions, and as such are subjected to the animal's mastication and, in the case of ruminants, to prolonged periods of permanence in the rumen. Moreover, the use of polymers as film producing-protective agents has high costs and, in the case of synthetic polymers, introduces non physiological substances in the animal diet.
It has also been proposed to protect some biologically active substances for aootechnical use, allowing their delayed release in the intestines of animals, incorporating the substances in oils (for instance hydrogenated soy or cottonseed oil, or coconut oil), fatty acids or triglycerides. However, solutions of this kind do not guarantee a sufficient resistance of the biologically active substances to the chcmicat en~ymatic attack whereto they are subjected in the animal orc:anism prior to reaching the intestine, which attack is particularly prolonged. in i.he case of ruminants.
'fhe applicant has now found that the incorporation of biologically active substances, having pharmacological and/or nutritional properties, in mixtures r. rmc .._, cc;t~m m:r~r F.m~rau:~r~ :FA r~murai _, o_,m f,.,_.,m r,cs.. . _.
comprising fatty acids or esters of fatty acids in combination with waxes allows a high resistance of the biologically active substances in the gastric system of the animals, as well as a controlled, delayed and calibrated release of the substances themselves. Such release, for instance in the case of ruminants, takes place mostly in the post-ruminal area, from the abomasal area to the small intestine, wherein the absorption of the non degraded administered substance thus takes place.
SUMMARY Or THE INVENTION
R subject ofthe present invention is therefore a composition for zootechnical use for the oral administration with controlled release of one or more biologically active substances having pharmacological and/or nutritional properties. 1n ha.rticnlax, the composition of the present invention is in the form of particles, preferably with dimensions front 400 to 2500 m, more preferably from 500 to l=100 m, which comprise IS a vehicle wherein said one or more biologically active substances are incorporated.. The vehicle preferably has a melting temperature of at least 40'C and it comprises:
- a component (A) constituted by one or more fatty acids and one or more esters of a fatty acid, and - a component (B) constituted by one or more waxes.
The component (A) is present in the composition in an amount equal to l0-90°i~
of weight, preferably 3U-$0°ro of weight, more preferably 35-75% of weight, with respect to the total weight of the vehicle. The component (T3) is present. in the composition in an amount equal to 10-90°ro in terms of weight, preferably 20-70°,~0 of weight, more preferably 25-65°io of 4veight, with respect to the total weight of the vehicle.
Some examples of components (A) are the following:
hydrogenated oils preferably saillr'attd, wrth IneltlIlg tC'111pP.1'atltCe from SO to 8:5°C and saponification number from.120 to 205;
- fatty acids, natural or hydrogenated or partially hydrogenated, having melting i temperatures from 57 to 70°C and saponitication number from 150 to 230;
- esters of fatty acids, of vegetable or animal origin, For instance mono-, di-and triglyceri<ies, with melting temperature from 45 to 70°C and saponii~ication number 17. ULC ' 98 (GLV) 1'1: 19 BUCNIUN SPA klMIN1 !!039 U5'll ?8721 F'AU~. . 1 from 17S to 205.
Preferably the component (A) of the vehicle comprises glycerides or long chained fatty acids (for instance C12-C22), which can be of vegetable origin (for example hydrogenated palm, soy, cottonseed, various seeds, olive, castor and sesame) or of animal origin. Hydrogenated oils of vegetable origin are preferred.
In U.S. Pharmacopoeia, hydrogenated oils of vegetable origin are classified according to their physical properties, regardless of their origin. Type I
fats are better indicated for application in the present invention, being characterized by a melting point ranging from S7 to 70°C, iodine value lower than 5 and saponi rcation number front 17>
lU to 20S according to the ASTM D1387 standard.
In the composition of the present invention the component (I3) is constituted by one or more natural or synthetic waxes. Natural waxes can be animal, vegetable or mineral. It is preferable to use natural waxes with melting point from 50 to 8E°C.
Particular examples of usable waxes according to the present invention are:
carnauba wax, beeswax, esparto wax, ceresine, ozoc.ertte (for example, not refined), paraffin waxes or micro-crystalline waxes.
DESC;RTPTION OF fHF PREFERRED EMBODIMENTS
In a preferred composition the component (B) of the vehicle is constituted by or comprises a micro-crystalline wax. Particularly preferred is a composition wherein the component (B) is constituted for at least SO% of its weight by one or more non-saponifrable micro-crystalline waxes with melting temperatures fror.n 54 to 10z°C, preferably from 90 to l00°C, and carnatiba. wax.
2S The term wax chemically means all substances essentially constituted. by saturated fatty acids, generally ra.nLing from. Cil2 to C30, by fatty alcohols, generally ranging from (,12 to CsO, by esters beriveen the aforementioned fatty alcohols and the fatl:v acids, by a mrnonty component of triglycerides, and by hydrocarbons. The latter are present in variat~l~ proportions in natural, animal and vegetable waxes, with length ranging from C19 to C31, whilst they constitute the dominant fraction of mineral w~ave~.
T3eeswax is formed by a mixture of linear monohydric alcohols with even Carbon number, front C'.26 to C36, such as cerilic alcohol (C26) and miricilic alcohol (CS6), S
17. Tile ' y8 (i;ICi) 14: 19 EtUc~NIpN SPA kIMINI 0039 0541 7~~3~21 Phu. . 2 9 cstcrified by li.ncar fatty acids with even Carbon number up to C36, w Jith presence of rieinoleie acid {C18 hydroxyaeid). Examples of such esters are traicontanol hexadecanoate and hexacosanol hexacosanoate. Such esters arc mixed with about 20°r~
by weight of linear hydrocarbons {paratfins) with odd Carbon number, from C? 1 to C33. Also present are about 6% of unidentified substances, in addition to propolis and pigments. L.r,S. Pha.rlnacopoeia describes yellow wax, obtained through primary refinement, and wherefrom white wax, useful in cosmetic applications, is obtained through bleaching with peroxides. 'Che melting paint of 'beeswax, regardless of its degree, varies from 62 to 65°C, the saponification number from 87 to 104 in accordance with ASTM D1387.
Carnauba wax comes from the exudate of the leaves of the palm. by the same name, classified as Copcrnicia Prunifcra (Muell), It contains waxy esters, i.e. esters of saturated fatty acids with saturated or hydroxylated fatty alcohols with average length of 12 Carhop atoms. The melting point of carnauba wax varies fro111 82 to 85.s°C, the saponification number from 78 t~ 89 in accordance with ASTM 171387.
k~sparto wax, dheroically constituted mostly by hydrocarbons {entriacontane C31 ) and by the classic waxy esters, is obtained as a by-product in paper processing.
Candelilla wax is obtained from the green parts of many I;uphorbiaceae and it is 11105111' constituted by hydrocarbons {47-57%), so it represents the natural component most 30 closely resembling mineral paraffin waxes.
Among waxes constituted mostly or completely by hydrocarbons, ozoccrite or ccresine are noted, which correspond to increasing levels of refinement of thz same material.
Micro-crystalline waxes, parall'ln waxes and carna.uha. wax are dcscribe.d in U.S.
?5 1'harlnacopoeia as excipients and as such allowed for human pharmaceutical use. They can be obtained by SL1CCCSS1VC; purilication from crude oi! or from lts hcay-frac(i~ns, or by pyrolysis of lignites or also by fischer-Tropseh synthesis from CO and F~1. Thev are classified according to.their chemical-physical. characteristics, with increasing lt7Cltlrlf~ point and hardness. To obtain products with high melting points, various 3() physical processes arc used: differential melting, fractionated crystallization, filtration 4vifh solvent mixtures, cte.1n the composition ofthe present invention the component {f3) is present i.n amounts ranlring from IO to 90% of the weight of the vehicle. 1-Iigh 17. DIC ' y~; !CaICi? 14:20 EUGNIuN SF'h RIMINI Op~9 p541 7; ;721 PF.i~. . J >
percentages of the component (R) allow greater protection of the biologically active substances contained in the composition. In the ease of low percentages of the component (B), it is advisable for the weight ratio between micro-cc~~stalline wax and the other natural waxes to be no lower than 2:1.
S The biologically active substances that, according to the present invention, can be incorporated in the vehicle comprising the components (A) and (B), can be substances with pharmacological and/or nutritional properties, such as:
- essential amino acids, for instance Dl.-methionine and L,-lysine, or cholin~, or their salts;
- vitamins, for instance vitamins A, B1, B2, Bti, B12, C, E and PP, or similar precursors of metabolic intermediates;
- probiotic substances, for imtancc Lactobacilli and Bifidobacteria;
- antibiotic substances (for instance of the amphenicolic, tetraciclinic, quinolonic, t7uoroquinolonic, macrolidic or sulphotmmide type), antihelmintic, antiprotozoic, antidiarrhoic ar antimycotic substances.
The biologically active substances that are incorporated in the vehicle comprising the components (A) and (B) preferably have dimensions of 40-l0U m, more preferably of 50-70 m. Particularly advantageous results are reached by obtaining the desired dimensions by micronisation.
The one or more biologically active substances can be present in the composition of the present invention in a quantity that varies from 0.01 to 60°,'o by weight with respect to the weight of the vehicle. For example the duantity can be from 2 to 35% by weight with respect to the weight of the vehicle, or from 5 to 20% by weight with respect to the weight of the vehicle.
In the case of essential amino acids, such as DL-methionine or L-lysine, or in the case of choline or of their salts, the quantity is preferably from 20 to 50?i°, more preferably from 25 to 45°o by weight with respect to the weight of the vehicle.
In the case of vitamins, biotin, pantofhenic acid, nicotinic acid or nicotinamide.
the quantin~ is preferably from 0.01 to 35"/°, mare preferably from 1 to 2~°.% by weight with respect to the weight of the vehicle.
In the case of prohiotic substances, such as Lactobacilli or Bifidbt~acteria, the quantify is preferably from 2 to 30%, more preferably fmm 5 to 20% by weight with 17. DIc: ' 9~~ !C:ICi) 14:20 BLIisNIiiTI SPA RIMINI OOpy io5erl 70=721 PAiI. .
respect to the weight of the vehicle.
Tn addition, the composition of th.e present invention can be modified with conventional additives, preservatives and anti-oxidizers that are compatible with animal administration and are used in animal husbandry to increase the stability of feed formulations. Typical preservatives include: thyrnerosalt, chlorbutanol, methyl, ethyl, propyl or butyl parabene. 'typical anti-oxidi~,ers of the oily phase include:
alpha-tocopherol, alpha-tocopheryl acetate, B.H.T. and B.H.n.
The compositions according to the present invention can be prepared with a process that constitutes a further subject of the present invention and comprises the following operations:
(a) preparing a vehicle by melting a mixture comprising the component (n) and the component (B), (b) incorporating in the molten vehicle thus obtained one or more biologically active substances, preferably with the aforementioned dimensions, (c) subjecting to solidification and fragmentation the molten vehicle incorporating one or more biologically active substances, (d) subjecting the particles thus obtained to sifting.
Phase (c) can be eflected for instance by means of spray-cnaling ~r by means ~f atomization; in 1>Oth Ca.SeS, cooling can be obtained, for instance, with nitrogen counter flow. Particularly advantageous is ultrasound atomization.
With the aforesaid process, particles are obtained of preferably spheroidal shape and preferably with dimensions of 400-2500 rn, more preferably 500-14U0 m.
The compositions of the present invention are employed in the field of animal husha.ndry, since they can be used by adding them to feeds to obtain medicated and!or nutritionally integrated feeds.
The I:aeds thus prepared can he administered 4vith particularly advantageous results to ruminants since the biologically active substances contained therein are released in a controlled fashion (delayed and calibrated) in the post-ruminal system of the animals. The quantity of biologically active substances administered to the animal essentially reaches the post-ruminal system with no degradation and therefore it can be absorbed and used efFectivelf.
lvlorcovcr, the compositions of the present invention can be advantageously R
l7. Ltl;~ ' 98 (o;l >) 14'21 Blic.:NlON sYA kIMINI iWJ'~9 W 41 W;s'21 FAC;.. =
employed for the effective enrichment of feeds diluted in water (swills), as well as to improve the palatability of the medicated rations.
The examples that follow serve further to illustrate the invention and are not to be considered absolutely limiting.
17. DIO ' 98 !Gli>? 14:21 BlIC;NW )P7 9PA AIktINI 00=~ 0541 783121 PAI.x.. ~, EXA11~IYLE 1 (comparateve) SATURATED FATTI' AS'TD AND CRY~TAI,LINE METIIION1NL
l 20 g of stearic acid, melting point 69.b°C, iodine No. = U, acidity No. = 197.2 are melted at the temperature of 85°C and mixed with 80 g of crystalline DL-methionine> maintained under agi ation for 30 minutes until the mixture is homogenized. Spraying is then started, introducing a nitrogen counter flow for cooling.
The product is collected on a vibrating sieve composed of an upper net of 2200 m and a lower net of 600 m.
The particles obtained have the appearance of spheroida.l micro-granules, with grain size ranging from 1400 m (max top 1%) and S00 m (max top 5°ro) EXAMPLE 2 (comparative) SAT(1RATF. FATTY ACID AND MICRONESIA METAIONINF
Example l is repeated with the same reactants and operating procedures, with the exception that the DL-methionine is previously Micronesia to a grain size w~herc;by 100°,'o pass through the 63 m sieve, obtaining particles sized 1400-500 m.
EXE1MPLE 3 (comparative) ' Rt T)~'I'R~,yCFRIDT~ND IVIICItONFSIA METH10NINT' 120 g of hydrogenated palm oil, melting point 54-57°C.', iodine No. = 1 max, acidity no.
8 max, average fatty acid composition of 3% rninistic acid (C14;0), 26-30°r~ ~f"
palrnitic acid (Cl.fi;(1) and 58-68% of stearic acid (C 18;0) are melted at the temperature of 85°C and mixed rwith 80g ofMicronesia DL-methioninc, maintained under agitation for 30 minutes until mixing is complete. Spraying is effected in nitrogen counter flow and the product is collected on a vibrating sieve composed of two nets of 2200 and 600 m. The particles appear as spheroidal micro-granules of grain size ranging from 1400 m (max top 1°ro) and 500 m (rt~ax top 5°/,).
EXAMPLE ~
SATIJRA'Ti~'D'TRI rI,YC~~tIphr WffN N.ATL1RAL. WAXI~.S ANO ~TICRONI?~IA
METF1IONINE , Example 3 is repeated under the same conditions, with the difference that the quantiy of hydrogenated paten oil is 108 g and that said oil is mixed while heated with 12 g of carnauha wax, ohtaining, a'f'ter treatments identical to those of h'xample 3, particles si~cd 1400-SU0 m.
17. h10 9~; ri_~li)? 11:21 BIJi_aNIiiN :>FA RI)\4IIJ1 V0~9 0541 7c;'~'721 FAu:~. . , 1!.XAMPLE 5 ~1IC.RO-CRYSTALLINF~ WAX'~Vl'fII SA'TL)RATI;T) TRTGLYCERID1; ~1NU
11~IICRONESIA M1~'TF(IONINE
lUU g of micro-crystalline wax, melting point R4-R7°C, mixed with 20g of the S same hydrogenated palm oil used in ~xarnple 3, are treated according to the procedures already set out in the previous examples (iron ~ 2 to 4), obtaining particles containing 40°,~ of DL-methionine by weight.
EXAMP1.~E G
IJE'fERMINING h~~'HiQNINF 'TITEPt 'T'he mcthionine content of Cxalnples 1 through 5, and similarly for the subseduent Examples, is determined by titration in non aqueous environment after dissolving the particle in non aqueous acid environment and at the temperature of 85°C.
The dissolution of the sample (containing about 140 mg of rnethionine) is effected with SU mI of glacial acetic acid, whereto are added about 3 ml of formic acid, heating to 85°C'., Then titration is performed cvitlz 0.1 N perchloric acid with potentiometer, using the following computational formula:
Melhionine (°-o) = 1 UU x ( 149.2 x 0.1 x A) / Pc where 149.2 - molecular weight of methionine j 20 U. 1 - normality of perchloric acid A - ml of U.1 N perchlori.c acid consumed Pc - weight of the sample in mg >~or examples 1-5, values around 40% arc obtained. The method is also applied to the examples that follow to measure the quantity of residual DL-tnethionine in resistance tests iyi vitrw and in vivo.
EXAMPI ,E 7 I~ESISTAN E TESTS - IN VITxc~ ~,~ ~'rH~n I
' The degree of protection obtained incorporating the biologically active principle in the component (A) (fat) and/or in the component {I3) (wax) is determined by 3U measuring the decrease in the concentration of the active ingredient (methioninc) after 8 hours of permanence in a buffer solution with pl-16.8 at the temperature of 39°C. In a glass bottle, 2 g of particles obtained incorporating the active ingredient in the 17. DIC ' 9'c,' r.GIU! 14:22 RUGNIUN SPA A.IMINI l10=9 ii541 78_;721 PP_i:;. .
a, I , component (A) andlor in the component (B) are added. During the 8 hours of the test, taken as arbih~ary~ reference to simulate an average permanence in the rumen, moderate agitation is applied 2-3 times, maintaining the indicated temperature with double boi ler.
At the end the solution is filtered on nylon filter, then bottle and ~Iter are washed with 250 ml of buffer solution. Lastly, the quantity of active ingredient is determined, by means of potentiometric titration as described in Example 6.
The degree of protection expresses the ratio between the titer of the sample at the end of the test, washed and dried in a stove, with respect to the initial tiler at time 0:
Degree of protection (%) = 100 x Tb / Ta where Tb -- active ingredient (methionine) titer after 8 hours in pH 6.R solution at 39°C
Ta - initial active ingredient (methionine) titer.
The particles of the Examples frolll 1 to 5 were considered, obtaining a good correlation W th the data obtained from in vtvv tests. Thus it is evident that an excellent simulation of gastric protection can be obtained by means of non invasive tesis, easily accessed and inexpensive.
f;XAMP~,E 8 RE:SISTAN ~ TZST~ - IN VII~O METHOD
'Testing is conducted in accordance with the guidelines set out in the publication by various authors in Zoot. Nutr, Anim. ( 1994) 20;281-91. The test consists of the nylon hag technique which entails the abomasal insertion oI' th.e sample to be tested. The details are described in the guidelines provided by the Proteins in Polygastric. Feeding commission of the ASPA (1994) which follows the French PDI system (NRA, 1985), ' 2S partly modified based on appropriate indications of the literature (Susmel and Stef'anon, 1987; Susmel et al., 199.'ib).
For lhc test, 3 Italian Frisian hovincs were used, provided. with ruminal bstula in dry physiological state. .
Incubaiior~ times were U, 2, 4, 6, 8, 12, 16, 20, 3H, 48 and 96 hours. The dry content (Table I) is measured by weighing the hag previously dried in stove according to standardised procedure, whilst the methionine content (Table 2) was determined according to the method described in Lxarnplc 6.
17. LiIC ' ~,, !;lei) 14:1'1 P,II~;NIUiN ~:F'A kI1,41N1 ~iii~;~nr-~4l '-,~
~iGl F'P.~:;. . .
The results are shown in Tables 1 and 2.
Example 1 Example 2 Example 3 Eaan~ple 4 Example 5 Fable 1 - total dry substance content ffat and/or wax -r DL-meth_ionine Example Examffle Example Example Example 1 ?. 3 4 5 '~ (%> 9~7 4,2 6,3 5,1 6,0 b {%) 31,5 29,3 41,3 51,2 29,55 i a-t-b d1,2 33,5 ~47,b 56,3 35,9 {%) ..._ c {o~) ?~,ZS- 7'Ll 2~5 1,9 2,2 . . __ .-._ .__ 17C~ (Ef%h);5,1~ . ,.9,0 . c 20,1.x._ L)G (b%h)33,6~ 1 f,0 15,5 ~~ 14,5 ~n 12;1 F, B ~
fable 2 - DL-methionine content Example )~llLll~>It:Exanyle Example L:~ampie i _ _ a (oi) 193~ 11,5 ~ 14,2 t' ~ 9,~1 9,5 x _. _._ ~ _~ ~' f .
h(%) 70,1 B 6~'1 ~~ 6~~~ D 69,7 ~ 49,5 ~
- _-_.--._..
-~b (~ SO,d~ 76,Ef 78,9 F' 79,1 ~ 59,0 C
o) a _ a, . ___ .
S'2 ~; '1~~,_~.~4'2 ~ 'y c DG (b ...~2'2~1'1S'f _ 37,9 n 28,3 E
oh)..l __.. ~ 'l;~ 1 _ ... 0 __ ncr (&roh)7'1,1~ ~a,2 ~' 3S,E ~ 3;,2'~ 'o;~l . ~:
a (r) = immediately degradable fraction b (%) = slowly d~,gradable fraction a+b (%) --- total degradable fraction c (%m) - hourly degradation rate ' DG (6%/h ) = effective deLradability with kp = 6%/h DG (8%;'h ) = effective degradability i with kp = 8~o,~h 1?. GIC: ' ~~. !C,IG) 14 25 BUGNIU)tI 8PA AfMINI V03~J 0541 ?;33721 PAG. I C, Statistical analysis was performed by means of GLM and the differences were compared by means of Schoffe's test (SAS, 1994).
A = significance U
B = significance 1 C = significance 2 D = significance 3 = significance 4 F = significance 5 The immediately degradable fraction, a (%) corresponding to time (), is obtained by washing the sample in cold water for 15 minutes, followed by centrifuging.
The slowly degradable fraction, b (%) is obtained from the difference between the final asymptotic weight (placed at 72 hours) and the immediately degradable fraction a (°ro). T'he value expressed a.s c (%lh) in practice corresponds to a percent degradation rate as hourly average.
l 5 1'he kinetic degradation parameters are defined with a, b and c, whilst the constant k -= O.OG or k = 0.08 corresponds to high speeds of ruminal transits equal to 6°i°Ih or 8°~o/tt, typical o~ concentrates.1n other words, kp = b% refers to an average transit of l6-2g llULirs, whereas kp = 8%lh refers to a ruminal transit corresponding to 13-14 hours.
The value of degraded portion (DG) is obtained using the formula:
2U DG--a+(bxc)/(c+k) To high T~Cr values corresponds a low rumina.l degradation protection. '~fhe duantity bypassing the rumen can be assumed equal to 100 - DG. analyzing the values I
obtained in the tests, valid indications are obtained on the protective capacity of the substances used io incorporate the biologically active substance.
.
COMI'OSl'1'lON WffHCON'CROI.LI~DREL,FAS~ Of BIOI,OCrICAl,I,Y ACTIVE?
SI1BSTANCES FOR ZOOTECHNLC.A(, USR.
B~CKC;ROj 1Nn OF 'fl-1F? INVENTION
The present invention relates to a composition for zootechnical use for the oral administration with controlled release of biologically active substances having pharmacological and Jor nutritional properties.
The present invention further relates to a method for the preparation of said Q composition, as well as the use of said composition as an additive to feeds, in order to obtain medicated andlor nutritionally integrated feeds.
Biologically active substances having nutritional properties ( i.c. food integrators) and/or pharmacological properties are commonly administered to animals orally, generally added to the feeds.
15 In such administra.ticrns the biologically active substances undergo a chemical-enzymatic debn~adation during the transit time through the animal organism prior to reaching the intestine. Such degradation is dune severe in the case ofruminanis, since ' transit time in the ruminant system is very high and due to the presence of the microbic.
flora.
The biologically active substances may also undergo a degradation during the feed enrichment phase and during the storage of the enriched feeds prior to their administration to the animals.
It is also important to mask the taste of the biologically active substances, particularly those with pharmacological properties, in order to make rnctre attractive the 25 medicated portions of feed to be administered to the animals.
F?xamples of substances commonly added to feeds are some vitamins. Vltamrns are prone to >7eing degraded prior to reaching the intestine of the animal.
Such degradation is also caused by interference with the microllora which is present in the organs of the animals (and especially in the ruminal system of ruminants) which are 30 traversed hef~re the intestine is reached.
For instance, in the case of vitamin C, the rate of degradation during permanence in the rumen can even exceed ~)0%. More or less severe degradation is also experienced l 17. Dli..' ' ~; !yIG! 14: 1. BIJi;NIcSN SPA. R_ItetlNl ii'j ,= 'i541 ';
:._~7G1 F':~.c-:. . .~ , with other vitamins, suctr as vitamin A, vitamin B6, vitamin 1312, vitamin D
and vitamin L. Particularly high is the degradation of the free form of vitamin >r (ILL-alpha-tocophrrrol)less high is the degradation of the esterified form (DI~ alpha-t~copherylacet~te).
Other examples of substances that are added to feeds and arc subject to degradation a.re ribof7a.vin, folic acid, niacin a.nd chOlrllC. The latter plays an essential role in large lactiferous animals during the first phase of lactation, where a considerable mobilization of reserve fats taken place t.o compe.nsate for energy shortages.
The fatty acids accumulated in the liver are therein transformed into triglycerides, which in turn give rise to pathological hepatic steazosis. Choline is able to obviate such accumulation favoring the phospholipidic metabolism, and thus the lipoproteins that carry out the function of triglyceride transport and removal. 'fhe proper administration ohcholinc improves the animal's hepatic functionality and productivity. However, unprotected choline is degraded into trimethyla.rnine, an inactive substance that gives unpleasant I S tlavor and odor to the milk. Such degradation can reach levels of about '70% of the quantity initially administered.
Other substances that are added to feeds and are subject to degradation prior to reaching the intestine of ruminants are amino acids. Enrichment with amino acids is important since milk production is particularly sensitive to fhe required duantity of plasmatic amino acids, essential for de nnvu synthesis of milk proteins at the mammary gland level. nll amino acids necessary for the metabolism of Illllk proterns Illust be present in the blood in the quantity necessary for the proper "assembly" of the proteinaceous -cascous component.
Maximum proteinaceous eft7ciency is often compromised by the lack of two essential amino acids: L-lysine and DL-methionine. The massive administration of protein sources is not always able to obviate this problem. A protein excess can set off forms of competition upon absorption and/or tissue and cell utilisation, for instance i competition between L,-triptophane and branched amino acids or between h-lysine and L-arginine. Moreover, the excess of nitrogenous substances, if not carlihratccf. entails an overload of ammonia defecation, aggravating environmental impact and hygienic conditions inside the stock farm, particularly if enclosed.
Furthermore. a shortage of amino acids, especially L7L-methionine, limits lhc t~. vtc ' ~_, rc;~o> »: i su<::r;tor~ ,<.~Fw emmrm _~e~~ ~~~~~i ?c;~;li r~;~:;.
growth, productivity and fertility of the animals.
Examples of pharmacologically active substances added to feeds and administered to animals are some antibiotics, both synthetic and fermentativc; in many eases, the antibiotics so added, especially macrolidic or quinolinic ones, in addition to being S degraded, also modify the taste of the feeds making thorn unattractive.
'fhe need to improve the stahiliiy and preacrvabili.ty of feeds enriched with biologically active substances, for instance nutritional integrators, is all the greater, the higher the water content in the feeds. For example, liquid or semi-liquid mixtures with high water content, common in swine nutrition, arc unstable because they are subject to very pronounced fennentative or hydrolytic transformations.
In order to reduce the aforesaid drawbacks, the proposal has been advanced of encapsulating some biologically active substances for zootechnical use with films of pI-I-sensitive materials, able to withstand the gastric environment, for instance polymers based on polvvinylpyrrholidonc, vinyl polymers and copolymers, polyesters and polyamides, chemically rnoditicd cellulose, polypeptidic agents and starches, thereby obtaining a certain protecfiion of the active substances and their delayed release in the intestine ofthe animals.
Such solutions present some drawbacks in that the particles of biologically active substances so encapsulated occasionally have significant dimensions, and as such are subjected to the animal's mastication and, in the case of ruminants, to prolonged periods of permanence in the rumen. Moreover, the use of polymers as film producing-protective agents has high costs and, in the case of synthetic polymers, introduces non physiological substances in the animal diet.
It has also been proposed to protect some biologically active substances for aootechnical use, allowing their delayed release in the intestines of animals, incorporating the substances in oils (for instance hydrogenated soy or cottonseed oil, or coconut oil), fatty acids or triglycerides. However, solutions of this kind do not guarantee a sufficient resistance of the biologically active substances to the chcmicat en~ymatic attack whereto they are subjected in the animal orc:anism prior to reaching the intestine, which attack is particularly prolonged. in i.he case of ruminants.
'fhe applicant has now found that the incorporation of biologically active substances, having pharmacological and/or nutritional properties, in mixtures r. rmc .._, cc;t~m m:r~r F.m~rau:~r~ :FA r~murai _, o_,m f,.,_.,m r,cs.. . _.
comprising fatty acids or esters of fatty acids in combination with waxes allows a high resistance of the biologically active substances in the gastric system of the animals, as well as a controlled, delayed and calibrated release of the substances themselves. Such release, for instance in the case of ruminants, takes place mostly in the post-ruminal area, from the abomasal area to the small intestine, wherein the absorption of the non degraded administered substance thus takes place.
SUMMARY Or THE INVENTION
R subject ofthe present invention is therefore a composition for zootechnical use for the oral administration with controlled release of one or more biologically active substances having pharmacological and/or nutritional properties. 1n ha.rticnlax, the composition of the present invention is in the form of particles, preferably with dimensions front 400 to 2500 m, more preferably from 500 to l=100 m, which comprise IS a vehicle wherein said one or more biologically active substances are incorporated.. The vehicle preferably has a melting temperature of at least 40'C and it comprises:
- a component (A) constituted by one or more fatty acids and one or more esters of a fatty acid, and - a component (B) constituted by one or more waxes.
The component (A) is present in the composition in an amount equal to l0-90°i~
of weight, preferably 3U-$0°ro of weight, more preferably 35-75% of weight, with respect to the total weight of the vehicle. The component (T3) is present. in the composition in an amount equal to 10-90°ro in terms of weight, preferably 20-70°,~0 of weight, more preferably 25-65°io of 4veight, with respect to the total weight of the vehicle.
Some examples of components (A) are the following:
hydrogenated oils preferably saillr'attd, wrth IneltlIlg tC'111pP.1'atltCe from SO to 8:5°C and saponification number from.120 to 205;
- fatty acids, natural or hydrogenated or partially hydrogenated, having melting i temperatures from 57 to 70°C and saponitication number from 150 to 230;
- esters of fatty acids, of vegetable or animal origin, For instance mono-, di-and triglyceri<ies, with melting temperature from 45 to 70°C and saponii~ication number 17. ULC ' 98 (GLV) 1'1: 19 BUCNIUN SPA klMIN1 !!039 U5'll ?8721 F'AU~. . 1 from 17S to 205.
Preferably the component (A) of the vehicle comprises glycerides or long chained fatty acids (for instance C12-C22), which can be of vegetable origin (for example hydrogenated palm, soy, cottonseed, various seeds, olive, castor and sesame) or of animal origin. Hydrogenated oils of vegetable origin are preferred.
In U.S. Pharmacopoeia, hydrogenated oils of vegetable origin are classified according to their physical properties, regardless of their origin. Type I
fats are better indicated for application in the present invention, being characterized by a melting point ranging from S7 to 70°C, iodine value lower than 5 and saponi rcation number front 17>
lU to 20S according to the ASTM D1387 standard.
In the composition of the present invention the component (I3) is constituted by one or more natural or synthetic waxes. Natural waxes can be animal, vegetable or mineral. It is preferable to use natural waxes with melting point from 50 to 8E°C.
Particular examples of usable waxes according to the present invention are:
carnauba wax, beeswax, esparto wax, ceresine, ozoc.ertte (for example, not refined), paraffin waxes or micro-crystalline waxes.
DESC;RTPTION OF fHF PREFERRED EMBODIMENTS
In a preferred composition the component (B) of the vehicle is constituted by or comprises a micro-crystalline wax. Particularly preferred is a composition wherein the component (B) is constituted for at least SO% of its weight by one or more non-saponifrable micro-crystalline waxes with melting temperatures fror.n 54 to 10z°C, preferably from 90 to l00°C, and carnatiba. wax.
2S The term wax chemically means all substances essentially constituted. by saturated fatty acids, generally ra.nLing from. Cil2 to C30, by fatty alcohols, generally ranging from (,12 to CsO, by esters beriveen the aforementioned fatty alcohols and the fatl:v acids, by a mrnonty component of triglycerides, and by hydrocarbons. The latter are present in variat~l~ proportions in natural, animal and vegetable waxes, with length ranging from C19 to C31, whilst they constitute the dominant fraction of mineral w~ave~.
T3eeswax is formed by a mixture of linear monohydric alcohols with even Carbon number, front C'.26 to C36, such as cerilic alcohol (C26) and miricilic alcohol (CS6), S
17. Tile ' y8 (i;ICi) 14: 19 EtUc~NIpN SPA kIMINI 0039 0541 7~~3~21 Phu. . 2 9 cstcrified by li.ncar fatty acids with even Carbon number up to C36, w Jith presence of rieinoleie acid {C18 hydroxyaeid). Examples of such esters are traicontanol hexadecanoate and hexacosanol hexacosanoate. Such esters arc mixed with about 20°r~
by weight of linear hydrocarbons {paratfins) with odd Carbon number, from C? 1 to C33. Also present are about 6% of unidentified substances, in addition to propolis and pigments. L.r,S. Pha.rlnacopoeia describes yellow wax, obtained through primary refinement, and wherefrom white wax, useful in cosmetic applications, is obtained through bleaching with peroxides. 'Che melting paint of 'beeswax, regardless of its degree, varies from 62 to 65°C, the saponification number from 87 to 104 in accordance with ASTM D1387.
Carnauba wax comes from the exudate of the leaves of the palm. by the same name, classified as Copcrnicia Prunifcra (Muell), It contains waxy esters, i.e. esters of saturated fatty acids with saturated or hydroxylated fatty alcohols with average length of 12 Carhop atoms. The melting point of carnauba wax varies fro111 82 to 85.s°C, the saponification number from 78 t~ 89 in accordance with ASTM 171387.
k~sparto wax, dheroically constituted mostly by hydrocarbons {entriacontane C31 ) and by the classic waxy esters, is obtained as a by-product in paper processing.
Candelilla wax is obtained from the green parts of many I;uphorbiaceae and it is 11105111' constituted by hydrocarbons {47-57%), so it represents the natural component most 30 closely resembling mineral paraffin waxes.
Among waxes constituted mostly or completely by hydrocarbons, ozoccrite or ccresine are noted, which correspond to increasing levels of refinement of thz same material.
Micro-crystalline waxes, parall'ln waxes and carna.uha. wax are dcscribe.d in U.S.
?5 1'harlnacopoeia as excipients and as such allowed for human pharmaceutical use. They can be obtained by SL1CCCSS1VC; purilication from crude oi! or from lts hcay-frac(i~ns, or by pyrolysis of lignites or also by fischer-Tropseh synthesis from CO and F~1. Thev are classified according to.their chemical-physical. characteristics, with increasing lt7Cltlrlf~ point and hardness. To obtain products with high melting points, various 3() physical processes arc used: differential melting, fractionated crystallization, filtration 4vifh solvent mixtures, cte.1n the composition ofthe present invention the component {f3) is present i.n amounts ranlring from IO to 90% of the weight of the vehicle. 1-Iigh 17. DIC ' y~; !CaICi? 14:20 EUGNIuN SF'h RIMINI Op~9 p541 7; ;721 PF.i~. . J >
percentages of the component (R) allow greater protection of the biologically active substances contained in the composition. In the ease of low percentages of the component (B), it is advisable for the weight ratio between micro-cc~~stalline wax and the other natural waxes to be no lower than 2:1.
S The biologically active substances that, according to the present invention, can be incorporated in the vehicle comprising the components (A) and (B), can be substances with pharmacological and/or nutritional properties, such as:
- essential amino acids, for instance Dl.-methionine and L,-lysine, or cholin~, or their salts;
- vitamins, for instance vitamins A, B1, B2, Bti, B12, C, E and PP, or similar precursors of metabolic intermediates;
- probiotic substances, for imtancc Lactobacilli and Bifidobacteria;
- antibiotic substances (for instance of the amphenicolic, tetraciclinic, quinolonic, t7uoroquinolonic, macrolidic or sulphotmmide type), antihelmintic, antiprotozoic, antidiarrhoic ar antimycotic substances.
The biologically active substances that are incorporated in the vehicle comprising the components (A) and (B) preferably have dimensions of 40-l0U m, more preferably of 50-70 m. Particularly advantageous results are reached by obtaining the desired dimensions by micronisation.
The one or more biologically active substances can be present in the composition of the present invention in a quantity that varies from 0.01 to 60°,'o by weight with respect to the weight of the vehicle. For example the duantity can be from 2 to 35% by weight with respect to the weight of the vehicle, or from 5 to 20% by weight with respect to the weight of the vehicle.
In the case of essential amino acids, such as DL-methionine or L-lysine, or in the case of choline or of their salts, the quantity is preferably from 20 to 50?i°, more preferably from 25 to 45°o by weight with respect to the weight of the vehicle.
In the case of vitamins, biotin, pantofhenic acid, nicotinic acid or nicotinamide.
the quantin~ is preferably from 0.01 to 35"/°, mare preferably from 1 to 2~°.% by weight with respect to the weight of the vehicle.
In the case of prohiotic substances, such as Lactobacilli or Bifidbt~acteria, the quantify is preferably from 2 to 30%, more preferably fmm 5 to 20% by weight with 17. DIc: ' 9~~ !C:ICi) 14:20 BLIisNIiiTI SPA RIMINI OOpy io5erl 70=721 PAiI. .
respect to the weight of the vehicle.
Tn addition, the composition of th.e present invention can be modified with conventional additives, preservatives and anti-oxidizers that are compatible with animal administration and are used in animal husbandry to increase the stability of feed formulations. Typical preservatives include: thyrnerosalt, chlorbutanol, methyl, ethyl, propyl or butyl parabene. 'typical anti-oxidi~,ers of the oily phase include:
alpha-tocopherol, alpha-tocopheryl acetate, B.H.T. and B.H.n.
The compositions according to the present invention can be prepared with a process that constitutes a further subject of the present invention and comprises the following operations:
(a) preparing a vehicle by melting a mixture comprising the component (n) and the component (B), (b) incorporating in the molten vehicle thus obtained one or more biologically active substances, preferably with the aforementioned dimensions, (c) subjecting to solidification and fragmentation the molten vehicle incorporating one or more biologically active substances, (d) subjecting the particles thus obtained to sifting.
Phase (c) can be eflected for instance by means of spray-cnaling ~r by means ~f atomization; in 1>Oth Ca.SeS, cooling can be obtained, for instance, with nitrogen counter flow. Particularly advantageous is ultrasound atomization.
With the aforesaid process, particles are obtained of preferably spheroidal shape and preferably with dimensions of 400-2500 rn, more preferably 500-14U0 m.
The compositions of the present invention are employed in the field of animal husha.ndry, since they can be used by adding them to feeds to obtain medicated and!or nutritionally integrated feeds.
The I:aeds thus prepared can he administered 4vith particularly advantageous results to ruminants since the biologically active substances contained therein are released in a controlled fashion (delayed and calibrated) in the post-ruminal system of the animals. The quantity of biologically active substances administered to the animal essentially reaches the post-ruminal system with no degradation and therefore it can be absorbed and used efFectivelf.
lvlorcovcr, the compositions of the present invention can be advantageously R
l7. Ltl;~ ' 98 (o;l >) 14'21 Blic.:NlON sYA kIMINI iWJ'~9 W 41 W;s'21 FAC;.. =
employed for the effective enrichment of feeds diluted in water (swills), as well as to improve the palatability of the medicated rations.
The examples that follow serve further to illustrate the invention and are not to be considered absolutely limiting.
17. DIO ' 98 !Gli>? 14:21 BlIC;NW )P7 9PA AIktINI 00=~ 0541 783121 PAI.x.. ~, EXA11~IYLE 1 (comparateve) SATURATED FATTI' AS'TD AND CRY~TAI,LINE METIIION1NL
l 20 g of stearic acid, melting point 69.b°C, iodine No. = U, acidity No. = 197.2 are melted at the temperature of 85°C and mixed with 80 g of crystalline DL-methionine> maintained under agi ation for 30 minutes until the mixture is homogenized. Spraying is then started, introducing a nitrogen counter flow for cooling.
The product is collected on a vibrating sieve composed of an upper net of 2200 m and a lower net of 600 m.
The particles obtained have the appearance of spheroida.l micro-granules, with grain size ranging from 1400 m (max top 1%) and S00 m (max top 5°ro) EXAMPLE 2 (comparative) SAT(1RATF. FATTY ACID AND MICRONESIA METAIONINF
Example l is repeated with the same reactants and operating procedures, with the exception that the DL-methionine is previously Micronesia to a grain size w~herc;by 100°,'o pass through the 63 m sieve, obtaining particles sized 1400-500 m.
EXE1MPLE 3 (comparative) ' Rt T)~'I'R~,yCFRIDT~ND IVIICItONFSIA METH10NINT' 120 g of hydrogenated palm oil, melting point 54-57°C.', iodine No. = 1 max, acidity no.
8 max, average fatty acid composition of 3% rninistic acid (C14;0), 26-30°r~ ~f"
palrnitic acid (Cl.fi;(1) and 58-68% of stearic acid (C 18;0) are melted at the temperature of 85°C and mixed rwith 80g ofMicronesia DL-methioninc, maintained under agitation for 30 minutes until mixing is complete. Spraying is effected in nitrogen counter flow and the product is collected on a vibrating sieve composed of two nets of 2200 and 600 m. The particles appear as spheroidal micro-granules of grain size ranging from 1400 m (max top 1°ro) and 500 m (rt~ax top 5°/,).
EXAMPLE ~
SATIJRA'Ti~'D'TRI rI,YC~~tIphr WffN N.ATL1RAL. WAXI~.S ANO ~TICRONI?~IA
METF1IONINE , Example 3 is repeated under the same conditions, with the difference that the quantiy of hydrogenated paten oil is 108 g and that said oil is mixed while heated with 12 g of carnauha wax, ohtaining, a'f'ter treatments identical to those of h'xample 3, particles si~cd 1400-SU0 m.
17. h10 9~; ri_~li)? 11:21 BIJi_aNIiiN :>FA RI)\4IIJ1 V0~9 0541 7c;'~'721 FAu:~. . , 1!.XAMPLE 5 ~1IC.RO-CRYSTALLINF~ WAX'~Vl'fII SA'TL)RATI;T) TRTGLYCERID1; ~1NU
11~IICRONESIA M1~'TF(IONINE
lUU g of micro-crystalline wax, melting point R4-R7°C, mixed with 20g of the S same hydrogenated palm oil used in ~xarnple 3, are treated according to the procedures already set out in the previous examples (iron ~ 2 to 4), obtaining particles containing 40°,~ of DL-methionine by weight.
EXAMP1.~E G
IJE'fERMINING h~~'HiQNINF 'TITEPt 'T'he mcthionine content of Cxalnples 1 through 5, and similarly for the subseduent Examples, is determined by titration in non aqueous environment after dissolving the particle in non aqueous acid environment and at the temperature of 85°C.
The dissolution of the sample (containing about 140 mg of rnethionine) is effected with SU mI of glacial acetic acid, whereto are added about 3 ml of formic acid, heating to 85°C'., Then titration is performed cvitlz 0.1 N perchloric acid with potentiometer, using the following computational formula:
Melhionine (°-o) = 1 UU x ( 149.2 x 0.1 x A) / Pc where 149.2 - molecular weight of methionine j 20 U. 1 - normality of perchloric acid A - ml of U.1 N perchlori.c acid consumed Pc - weight of the sample in mg >~or examples 1-5, values around 40% arc obtained. The method is also applied to the examples that follow to measure the quantity of residual DL-tnethionine in resistance tests iyi vitrw and in vivo.
EXAMPI ,E 7 I~ESISTAN E TESTS - IN VITxc~ ~,~ ~'rH~n I
' The degree of protection obtained incorporating the biologically active principle in the component (A) (fat) and/or in the component {I3) (wax) is determined by 3U measuring the decrease in the concentration of the active ingredient (methioninc) after 8 hours of permanence in a buffer solution with pl-16.8 at the temperature of 39°C. In a glass bottle, 2 g of particles obtained incorporating the active ingredient in the 17. DIC ' 9'c,' r.GIU! 14:22 RUGNIUN SPA A.IMINI l10=9 ii541 78_;721 PP_i:;. .
a, I , component (A) andlor in the component (B) are added. During the 8 hours of the test, taken as arbih~ary~ reference to simulate an average permanence in the rumen, moderate agitation is applied 2-3 times, maintaining the indicated temperature with double boi ler.
At the end the solution is filtered on nylon filter, then bottle and ~Iter are washed with 250 ml of buffer solution. Lastly, the quantity of active ingredient is determined, by means of potentiometric titration as described in Example 6.
The degree of protection expresses the ratio between the titer of the sample at the end of the test, washed and dried in a stove, with respect to the initial tiler at time 0:
Degree of protection (%) = 100 x Tb / Ta where Tb -- active ingredient (methionine) titer after 8 hours in pH 6.R solution at 39°C
Ta - initial active ingredient (methionine) titer.
The particles of the Examples frolll 1 to 5 were considered, obtaining a good correlation W th the data obtained from in vtvv tests. Thus it is evident that an excellent simulation of gastric protection can be obtained by means of non invasive tesis, easily accessed and inexpensive.
f;XAMP~,E 8 RE:SISTAN ~ TZST~ - IN VII~O METHOD
'Testing is conducted in accordance with the guidelines set out in the publication by various authors in Zoot. Nutr, Anim. ( 1994) 20;281-91. The test consists of the nylon hag technique which entails the abomasal insertion oI' th.e sample to be tested. The details are described in the guidelines provided by the Proteins in Polygastric. Feeding commission of the ASPA (1994) which follows the French PDI system (NRA, 1985), ' 2S partly modified based on appropriate indications of the literature (Susmel and Stef'anon, 1987; Susmel et al., 199.'ib).
For lhc test, 3 Italian Frisian hovincs were used, provided. with ruminal bstula in dry physiological state. .
Incubaiior~ times were U, 2, 4, 6, 8, 12, 16, 20, 3H, 48 and 96 hours. The dry content (Table I) is measured by weighing the hag previously dried in stove according to standardised procedure, whilst the methionine content (Table 2) was determined according to the method described in Lxarnplc 6.
17. LiIC ' ~,, !;lei) 14:1'1 P,II~;NIUiN ~:F'A kI1,41N1 ~iii~;~nr-~4l '-,~
~iGl F'P.~:;. . .
The results are shown in Tables 1 and 2.
Example 1 Example 2 Example 3 Eaan~ple 4 Example 5 Fable 1 - total dry substance content ffat and/or wax -r DL-meth_ionine Example Examffle Example Example Example 1 ?. 3 4 5 '~ (%> 9~7 4,2 6,3 5,1 6,0 b {%) 31,5 29,3 41,3 51,2 29,55 i a-t-b d1,2 33,5 ~47,b 56,3 35,9 {%) ..._ c {o~) ?~,ZS- 7'Ll 2~5 1,9 2,2 . . __ .-._ .__ 17C~ (Ef%h);5,1~ . ,.9,0 . c 20,1.x._ L)G (b%h)33,6~ 1 f,0 15,5 ~~ 14,5 ~n 12;1 F, B ~
fable 2 - DL-methionine content Example )~llLll~>It:Exanyle Example L:~ampie i _ _ a (oi) 193~ 11,5 ~ 14,2 t' ~ 9,~1 9,5 x _. _._ ~ _~ ~' f .
h(%) 70,1 B 6~'1 ~~ 6~~~ D 69,7 ~ 49,5 ~
- _-_.--._..
-~b (~ SO,d~ 76,Ef 78,9 F' 79,1 ~ 59,0 C
o) a _ a, . ___ .
S'2 ~; '1~~,_~.~4'2 ~ 'y c DG (b ...~2'2~1'1S'f _ 37,9 n 28,3 E
oh)..l __.. ~ 'l;~ 1 _ ... 0 __ ncr (&roh)7'1,1~ ~a,2 ~' 3S,E ~ 3;,2'~ 'o;~l . ~:
a (r) = immediately degradable fraction b (%) = slowly d~,gradable fraction a+b (%) --- total degradable fraction c (%m) - hourly degradation rate ' DG (6%/h ) = effective deLradability with kp = 6%/h DG (8%;'h ) = effective degradability i with kp = 8~o,~h 1?. GIC: ' ~~. !C,IG) 14 25 BUGNIU)tI 8PA AfMINI V03~J 0541 ?;33721 PAG. I C, Statistical analysis was performed by means of GLM and the differences were compared by means of Schoffe's test (SAS, 1994).
A = significance U
B = significance 1 C = significance 2 D = significance 3 = significance 4 F = significance 5 The immediately degradable fraction, a (%) corresponding to time (), is obtained by washing the sample in cold water for 15 minutes, followed by centrifuging.
The slowly degradable fraction, b (%) is obtained from the difference between the final asymptotic weight (placed at 72 hours) and the immediately degradable fraction a (°ro). T'he value expressed a.s c (%lh) in practice corresponds to a percent degradation rate as hourly average.
l 5 1'he kinetic degradation parameters are defined with a, b and c, whilst the constant k -= O.OG or k = 0.08 corresponds to high speeds of ruminal transits equal to 6°i°Ih or 8°~o/tt, typical o~ concentrates.1n other words, kp = b% refers to an average transit of l6-2g llULirs, whereas kp = 8%lh refers to a ruminal transit corresponding to 13-14 hours.
The value of degraded portion (DG) is obtained using the formula:
2U DG--a+(bxc)/(c+k) To high T~Cr values corresponds a low rumina.l degradation protection. '~fhe duantity bypassing the rumen can be assumed equal to 100 - DG. analyzing the values I
obtained in the tests, valid indications are obtained on the protective capacity of the substances used io incorporate the biologically active substance.
2~ The data in 'fable 1 refer to the total duantity of particles (vehicle -~-act.ive ingredient) not degraded during permanence i.n the rumen, They show poor resistance of the fatty acid (examples 1. and 2) and of the saturated glyceride (example:
3) not used in combination with waxes. Greater rcaistanee is observed with saturated triglyceridc employed in combination with carnauba wax (example 4). Rven greater rfaistance is 30 observed with the saturated triglyceride used in combination with micro-crystalline wax (example 5).
rxAmlel_.r ~
17. I>It' ' yg (f:ls~l 14:21; E'oJC~NI<oN sFA RIMINI 009 0541 715721 FAG. . ~
F
PREI~PARA~TION IiY MF.A~S OFATO1VIIZATIOj~
236 kg of hydrogenated palm oil, melting point 54-57°C, iodine No. -- 1 mar, acidity No, _ $ max, with average fatty acid composition of 3% mirislic acid (C14;0j, 26-30°,% palmitic acid (C16;0) and 58-68% stearic acid (C18;0), are loaded in reactor with 97.4 kg of carnauba wax and with 240 kg oi~ Sasolwaks C 80 micro-crystalline mineral wax, then heated with steam jacket to the temperature of 85°C
and mixed in the molten state.
These components, maintained under agitation at the temperature of RS°C, are enriched with 1.6 kg )3HT, and then ~.vith 42S kg of Micronesia DL-methionine with grain size 63 m for 30 minutes.
The mixture obtained is then introduced in an atomizer, fed with a constant flow of 15 kg/min, with nitrogen counter .flow equal to 0.1 - 0.2. l/kg at the external temperature of 5-12°C', and equal to 0.5-0.7 llkg at the external temperature of 20-30°C.
The particles are lastly collected for the analysis of the composition, which shows a concentration of Dla-methionine equal to about 40'% of weight. Average particle dimensions are retained in the range between 2200 and 600 m by means of sifting on sieves having the dimensions indicated above.
PRC.PARATION BY ME~,1NS OF SONICATION filL'TRASOL1NDS1 A heat-mixed mixture containing the cluanlity of ingredients in Example 9 and prepared with identical procedure constitutes the constant supply for an ultrasound atomizer (Tecnea, Castelguelfo - Bologna] adjusted to the intensity of 50 kHz, power 1800 Watts.
Composition is similar to that of Cxarnple 9 and average particle sire is retained in the range from 2200 to 600 m by means of sifting on sieves of the dimcnsiona indicated above.
EXAMPLr, 11 COMPOSITION ~~~TTAININCx VITAMIN PP
100 kg ~f hydrogenated palm oil and 250 kg of stearic acid, with the characteristics described in >xamplc 9, are loaded in reactor with 139 kg of carnauba wax and with 200 kg of Sasolwaks C 80 micro-crystalline wax, and heated with steam jacket to the temperature of 85°C and Then mixed. The mixture thus obtained in the lS
17. L»:~ ' 9~0 (t:la) 14:26 P1.IC~NIC1N sFA RlMINI iic)9 ~~=41 "_,_721 FAc:~.
. = '.
molten state is enriched with I.0 kg of BIiT and 2.0 kg of Kavarom L. (I-I. &
R. -Haarman and Reimer - Germany), and then with 408 kg of Micronesia vitamin PP, with grain size 63 rn for 3U minutes.
The particles are then obtained with similar procedure to that of Example 9, thus with similar morphololry.
>FxAI~~rL», COMPOSITION CONT,~ING E13~'T~IROMYf:IN TH10 YA~1ATE
519 kg of monoglyceride GMS (Fact S:p.A. - Carasco - Genoa) and 38U kg of carnauba wax are loaded in reactor and heated with steam jacket to the temperature of I O 85°C and then mixed. The mixture thus obtained in the molten state is enriched with 1.U
kg ofBl-1T and then with lU0 kg activity of ery~lln-omycin thiocyanate, and mixed for 30 mi.n utes.
' Particles are lastly produced with a procedure similar to that of Example 9.
' 15 ~ MPQ~ITION CONTAINI~fG PROBIOTjCS
400 kg of oil of steari.c acid, 25U kg of carnauba wax and 240 ky of type lI
paraftin wax are loaded in reactor, then heated with steam jacket to the temperature of 65°C and mixed in the molten state.
Thcsc components, maintained under agitation at the temperature of 85°G, are 20 enriched with 1.0 kg of BHT, mined for 3U minutes, and then enriched in the molten state with I00 kg of Lactobacillus acidophilus and mixed for 30 minutes.
Particles are lastly produced with a Procedure similar to that of Example 9.
rxAmlel_.r ~
17. I>It' ' yg (f:ls~l 14:21; E'oJC~NI<oN sFA RIMINI 009 0541 715721 FAG. . ~
F
PREI~PARA~TION IiY MF.A~S OFATO1VIIZATIOj~
236 kg of hydrogenated palm oil, melting point 54-57°C, iodine No. -- 1 mar, acidity No, _ $ max, with average fatty acid composition of 3% mirislic acid (C14;0j, 26-30°,% palmitic acid (C16;0) and 58-68% stearic acid (C18;0), are loaded in reactor with 97.4 kg of carnauba wax and with 240 kg oi~ Sasolwaks C 80 micro-crystalline mineral wax, then heated with steam jacket to the temperature of 85°C
and mixed in the molten state.
These components, maintained under agitation at the temperature of RS°C, are enriched with 1.6 kg )3HT, and then ~.vith 42S kg of Micronesia DL-methionine with grain size 63 m for 30 minutes.
The mixture obtained is then introduced in an atomizer, fed with a constant flow of 15 kg/min, with nitrogen counter .flow equal to 0.1 - 0.2. l/kg at the external temperature of 5-12°C', and equal to 0.5-0.7 llkg at the external temperature of 20-30°C.
The particles are lastly collected for the analysis of the composition, which shows a concentration of Dla-methionine equal to about 40'% of weight. Average particle dimensions are retained in the range between 2200 and 600 m by means of sifting on sieves having the dimensions indicated above.
PRC.PARATION BY ME~,1NS OF SONICATION filL'TRASOL1NDS1 A heat-mixed mixture containing the cluanlity of ingredients in Example 9 and prepared with identical procedure constitutes the constant supply for an ultrasound atomizer (Tecnea, Castelguelfo - Bologna] adjusted to the intensity of 50 kHz, power 1800 Watts.
Composition is similar to that of Cxarnple 9 and average particle sire is retained in the range from 2200 to 600 m by means of sifting on sieves of the dimcnsiona indicated above.
EXAMPLr, 11 COMPOSITION ~~~TTAININCx VITAMIN PP
100 kg ~f hydrogenated palm oil and 250 kg of stearic acid, with the characteristics described in >xamplc 9, are loaded in reactor with 139 kg of carnauba wax and with 200 kg of Sasolwaks C 80 micro-crystalline wax, and heated with steam jacket to the temperature of 85°C and Then mixed. The mixture thus obtained in the lS
17. L»:~ ' 9~0 (t:la) 14:26 P1.IC~NIC1N sFA RlMINI iic)9 ~~=41 "_,_721 FAc:~.
. = '.
molten state is enriched with I.0 kg of BIiT and 2.0 kg of Kavarom L. (I-I. &
R. -Haarman and Reimer - Germany), and then with 408 kg of Micronesia vitamin PP, with grain size 63 rn for 3U minutes.
The particles are then obtained with similar procedure to that of Example 9, thus with similar morphololry.
>FxAI~~rL», COMPOSITION CONT,~ING E13~'T~IROMYf:IN TH10 YA~1ATE
519 kg of monoglyceride GMS (Fact S:p.A. - Carasco - Genoa) and 38U kg of carnauba wax are loaded in reactor and heated with steam jacket to the temperature of I O 85°C and then mixed. The mixture thus obtained in the molten state is enriched with 1.U
kg ofBl-1T and then with lU0 kg activity of ery~lln-omycin thiocyanate, and mixed for 30 mi.n utes.
' Particles are lastly produced with a procedure similar to that of Example 9.
' 15 ~ MPQ~ITION CONTAINI~fG PROBIOTjCS
400 kg of oil of steari.c acid, 25U kg of carnauba wax and 240 ky of type lI
paraftin wax are loaded in reactor, then heated with steam jacket to the temperature of 65°C and mixed in the molten state.
Thcsc components, maintained under agitation at the temperature of 85°G, are 20 enriched with 1.0 kg of BHT, mined for 3U minutes, and then enriched in the molten state with I00 kg of Lactobacillus acidophilus and mixed for 30 minutes.
Particles are lastly produced with a Procedure similar to that of Example 9.
Claims (18)
1. Composition for zootechnical use for the oral administration with controlled release of one or more biologically active substances, having pharmacological and/or nutritional properties, said composition being in the form of particles comprising a vehicle in which said one or more biologically active substances are incorporated, said vehicle comprising:
- a component (A) constituted by one or more fatty acids and one or more esters of a fatty acid and, - a component (B) constituted by one or more waxes, each of the components (A) and (R) being present in quantities equal to 10-90%
toy weight with respect to the total weight of the vehicle.
- a component (A) constituted by one or more fatty acids and one or more esters of a fatty acid and, - a component (B) constituted by one or more waxes, each of the components (A) and (R) being present in quantities equal to 10-90%
toy weight with respect to the total weight of the vehicle.
2. Composition according to claim 1, in the form of particles with dimensions from 400 to 2500 m.
3. Composition according to claim 1 and/or 2, wherein the vehicle has a melting temperature of at least 40°C.
4. Composition according to one or mare of the previous claims, wherein the vehicle is constituted for 30-80% of its weight by the component (A) and for 20-70% by the component (B).
5. Composition according to one or more of the previous claims, wherein the component (A) is a prevalently saturated hydrogenated oil, having melting temperature from 50 to 85°C and saponification number form 120 to 205.
6. Composition according to one or more of the claims from 1 to 4, wherein the component (A) is a fatty acid having melting temperature from 57 to 70°C and saponification number from 150 to 230.
7. Composition according to one or more of the claims from 1 to 4, wherein the component (A) is an ester of a fatty acid, said ester having a melting temperature from 45 to 70°C and saponification number from 175 to 205.
8. Composition according to claim 7, wherein the component (A) is a mono-, di-or triglyceride.
9. Composition according to one or more of the previous claims, wherein the component (B) is a natural wax having melting temperature from 50 to 86°C.
10. Composition according to one or more of the claims from 1 to 8, wherein the component (B) is a wax chosen from among carnauba wax, beeswax, esparto wax, ceresine, ozocerite, paraffin wax and micro-crystalline wax.
11. Composition according to claim 10, wherein the component (B) is constituted for at least 50% of its weight by one or more micro-crystalline waxes.
12. Composition according to one or more of the previous claims, wherein the one or more biologically active substances are chosen from among DL-methionine, L-lysine, choline and their salts.
13. Composition according to one or more of the claims from 1 to 11, wherein the one or more biologically active substances are vitamins or probiotic substances.
14. Composition according to one or more of the claims from 1 to 11, wherein the one or more biologically active substances are antibiotic, antihelmintic, antiprotozoic, antidiarrhoic or antimycotic substances.
15. Method for the preparation of a composition according to one or more of the previous claims, comprising the following operations:
(a) preparing a vehicle by subjecting to melting a mixture comprising a component (A) constituted by one or more fatty acids and one or more esters of a fatty acid, and a component (B) constituted by one or more waxes, (b) incorporating in the molten vehicle thus obtained one or more biologically active substances, (c) subjecting to solidification and fragmentation the molten vehicle incorporating one or more biologically active substances and, (d) subjecting to sifting the particles thus obtained.
(a) preparing a vehicle by subjecting to melting a mixture comprising a component (A) constituted by one or more fatty acids and one or more esters of a fatty acid, and a component (B) constituted by one or more waxes, (b) incorporating in the molten vehicle thus obtained one or more biologically active substances, (c) subjecting to solidification and fragmentation the molten vehicle incorporating one or more biologically active substances and, (d) subjecting to sifting the particles thus obtained.
16. Method according to claim 15, wherein phase (c) is effected by means of spray-cooling or by means of atomization.
17. Method according to claim 16, wherein phase (c) is effected by means of ultrasound atomization.
18. Use of a composition according to one or more of the claims from 1 to 14 for addition to feeds for the purpose of obtaining medicated and/or nutritionally integrated feeds.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT97BO000383A IT1292636B1 (en) | 1997-06-20 | 1997-06-20 | CONTROLLED RELEASE COMPOSITION OF BIOLOGICALLY ACTIVE SUBSTANCES FOR ZOOTECHNICAL USE. |
| CA002256256A CA2256256A1 (en) | 1997-06-20 | 1998-12-17 | Composition with controlled release of biologically active substances for zootechnical use |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IT97BO000383A IT1292636B1 (en) | 1997-06-20 | 1997-06-20 | CONTROLLED RELEASE COMPOSITION OF BIOLOGICALLY ACTIVE SUBSTANCES FOR ZOOTECHNICAL USE. |
| CA002256256A CA2256256A1 (en) | 1997-06-20 | 1998-12-17 | Composition with controlled release of biologically active substances for zootechnical use |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2256256A1 true CA2256256A1 (en) | 2000-06-17 |
Family
ID=32031343
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002256256A Abandoned CA2256256A1 (en) | 1997-06-20 | 1998-12-17 | Composition with controlled release of biologically active substances for zootechnical use |
Country Status (2)
| Country | Link |
|---|---|
| CA (1) | CA2256256A1 (en) |
| IT (1) | IT1292636B1 (en) |
Cited By (18)
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| WO2005006876A1 (en) * | 2003-07-21 | 2005-01-27 | Ascor Chimici S.R.L. | A composition of matter comprising particles which contain choline chloride to be administered in a rumen protect and post-ruminally effective form |
| WO2008015203A2 (en) * | 2006-08-04 | 2008-02-07 | Eurhema S.R.L. | Compositions of microparticles and granules for oral controlled release of substances for veterinary use |
| US7785635B1 (en) | 2003-12-19 | 2010-08-31 | The Procter & Gamble Company | Methods of use of probiotic lactobacilli for companion animals |
| US7906112B2 (en) | 2003-12-19 | 2011-03-15 | The Procter & Gamble Company | Canine probiotic Lactobacilli |
| US7923033B2 (en) | 2003-07-21 | 2011-04-12 | Valentini S.R.L. | Composition of matter comprising particles which contain choline chloride to be administered in a rumen protected and post-ruminally effective form |
| US7998473B2 (en) | 2003-12-19 | 2011-08-16 | The Procter & Gamble Company | Methods of treatment or prevention of gastrointestinal disorders using canine probiotic bifidobacterium |
| US8034601B2 (en) | 2005-05-31 | 2011-10-11 | The Procter & Gamble Company | Feline probiotic bifidobacteria |
| US8563522B2 (en) | 1997-07-08 | 2013-10-22 | The Iams Company | Method of maintaining and/or attenuating a decline in quality of life |
| US8679546B2 (en) | 2004-09-24 | 2014-03-25 | Valentini S.R.L. | Ruminant feed pellet composition with controlled release of physiologically active substances, and procedure for preparation and use thereof |
| US8809035B2 (en) | 2003-12-19 | 2014-08-19 | The Iams Company | Canine probiotic Bifidobacterium |
| US8877178B2 (en) | 2003-12-19 | 2014-11-04 | The Iams Company | Methods of use of probiotic bifidobacteria for companion animals |
| US9192177B2 (en) | 2005-05-31 | 2015-11-24 | The Iams Company | Feline probiotic Lactobacilli |
| WO2016007173A1 (en) * | 2014-07-11 | 2016-01-14 | Benemilk Oy | Ruminant feed compositions for reducing methane generation |
| US9415083B2 (en) | 2004-05-10 | 2016-08-16 | Mars, Incorporated | Method for decreasing inflammation and stress in a mammal |
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-
1997
- 1997-06-20 IT IT97BO000383A patent/IT1292636B1/en active IP Right Grant
-
1998
- 1998-12-17 CA CA002256256A patent/CA2256256A1/en not_active Abandoned
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| US8563522B2 (en) | 1997-07-08 | 2013-10-22 | The Iams Company | Method of maintaining and/or attenuating a decline in quality of life |
| US7923033B2 (en) | 2003-07-21 | 2011-04-12 | Valentini S.R.L. | Composition of matter comprising particles which contain choline chloride to be administered in a rumen protected and post-ruminally effective form |
| WO2005006876A1 (en) * | 2003-07-21 | 2005-01-27 | Ascor Chimici S.R.L. | A composition of matter comprising particles which contain choline chloride to be administered in a rumen protect and post-ruminally effective form |
| US7906112B2 (en) | 2003-12-19 | 2011-03-15 | The Procter & Gamble Company | Canine probiotic Lactobacilli |
| US8802158B2 (en) | 2003-12-19 | 2014-08-12 | The Iams Company | Methods of use of probiotic Lactobacilli for companion animals |
| US7785635B1 (en) | 2003-12-19 | 2010-08-31 | The Procter & Gamble Company | Methods of use of probiotic lactobacilli for companion animals |
| US7998473B2 (en) | 2003-12-19 | 2011-08-16 | The Procter & Gamble Company | Methods of treatment or prevention of gastrointestinal disorders using canine probiotic bifidobacterium |
| US9580680B2 (en) | 2003-12-19 | 2017-02-28 | Mars, Incorporated | Canine probiotic bifidobacterium pseudolongum |
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| US9771199B2 (en) | 2008-07-07 | 2017-09-26 | Mars, Incorporated | Probiotic supplement, process for making, and packaging |
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| US10104903B2 (en) | 2009-07-31 | 2018-10-23 | Mars, Incorporated | Animal food and its appearance |
| WO2016007173A1 (en) * | 2014-07-11 | 2016-01-14 | Benemilk Oy | Ruminant feed compositions for reducing methane generation |
| WO2017048859A1 (en) * | 2015-09-16 | 2017-03-23 | Corr-Jensen, Inc. | Multi-phase release of sports nutrition and energy drink compositions utilizing lipid particulates |
| IT201900013449A1 (en) * | 2019-07-31 | 2021-01-31 | Vetagro Int S R L | Composition comprising amino acids and a lipid matrix for non-mammalian monogastric animals and its use |
Also Published As
| Publication number | Publication date |
|---|---|
| ITBO970383A0 (en) | 1997-06-20 |
| IT1292636B1 (en) | 1999-02-08 |
| ITBO970383A1 (en) | 1998-12-20 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |