Note: Descriptions are shown in the official language in which they were submitted.
17. DIi'' ' 9$ r,c;lOl 14: 12 BUi,Nfc)N SEA RIA41N1 sy39 x:;541 78:'721 FS~~.
.
179P222CA
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
CA 02256256 1998-12-17
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
CA 02256256 1998-12-17
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
3
CA 02256256 1998-12-17
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
4
CA 02256256 1998-12-17
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
CA 02256256 1998-12-17
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
6
CA 02256256 1998-12-17
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
7
CA 02256256 1998-12-17
17. DIc: ' 9~~ !C:ICi) 14:20 BLIisNIiiTI SPA RIMINI OOpy io5erl 70=721 PAiI. .
4 9
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
CA 02256256 1998-12-17
l7. Ltl;~ ' 98 (o;l >) 14'21 Blic.:NlON sYA kIMINI iWJ'~9 W 41 W;s'21 FAC;.. =
9
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.
CA 02256256 1998-12-17
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.
CA 02256256 1998-12-17
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
CA 02256256 1998-12-17
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.
12
CA 02256256 1998-12-17
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 1 2 3 4 5
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
13
CA 02256256 1998-12-17
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 ~
14
CA 02256256 1998-12-17
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.
EXAMPLE 10
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
CA 02256256 1998-12-17
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.
EXAMPLE 13
' 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.
16
CA 02256256 1998-12-17
