(cid:38)(cid:68)(cid:83)(cid:86)(cid:88)(cid:74)(cid:72)(cid:79)(cid:3)(cid:40)(cid:91)(cid:75)(cid:76)(cid:69)(cid:76)(cid:87)(cid:3)(cid:20)(cid:19)(cid:21)(cid:24)
`
`Page 1 of 15
`
`

`

`- U.S. Patent
`
`May 10, 1994
`
`Sheet 1 of 4
`
`5,310,555
`
`FIGURE 1
`
`Page 2 of 15
`
`Page 2 of 15
`
`
`

`

`U.S. Patent
`
`May 10, 1994
`
`Sheet 2 of 4
`
`5,310,555
`
` 'U
`50
`
`
`N E:
`
`3o
`
`H
`[u
`
`,g
`N
`
`ma
`
`:DoHa
`
`.
`
`no
`Q‘
`
`FIGURE2C
`
`
`
`W
`
`FIGURE2a
`
`Page 3 of 15
`
`Page 3 of 15
`
`

`

`US. Patent
`
`May 10, 1994
`
`Sheet 3 of 4 '
`
`5,310,555
`
`FIGURE2f
`
`51
`
`FIGURE2e
`
`Page4 of 15
`
`Page 4 of 15
`
`

`

`US. Patent
`
`May 10, 1994
`
`Sheet 4 of 4
`
`5,310,555
`
`VIABLB BACTERIAL COUNTS PER GRAN
`
`1.00E+10
`
`1.00E+09'
`
`g
`
`
`
`
`
`CPU/g(logarithmicscale)
`
`1.00E+08
`
`1.00E+07
`
`1.00E+O6
`
`1.00E+05
`
`1.00E+01
`
`1.00E+04
`
`1.00E+03
`
`1.00E+02
`
`1.00E+00
`
`Page 5 of 15
`
`Bolus #
`
`I MRS
`
`E] MRSO
`
`LBS
`
`FIGURE 3
`
`Page 5 of 15
`
`

`

`5,310,555
`
`1
`
`ORAL NUTRITIONAL AND DIETARY
`COMPOSITION
`
`TECHNICAL FIELD
`
`2
`Another approach is to provide the bacteria them-
`selves as a pellet or bolus. Many such bolus products are
`commercially available.
`More recently. bolus or pellet formulations have been
`developed which include a combination of the bacteria
`and dry vitamin and trace mineral supplements. as
`nearly simultaneous administration in vivo of these
`components has been suggested as being highly benefi~
`cial to achieving the goals of appetite stimulation and
`bacterial population reestablishment. Many of these
`bolus formulations are available commercially. It has
`been found, however. that the supplements and bacteria
`are incompatible as the vitamin and mineral levels eff-
`cacious for livestock are toxic to the bacteria. As indi-
`cated previously, microorganisms are also sensitive to
`mixing abrasioa. aeration, compression and high tem-
`peratures. all of which occur during conventional hard
`bolus productiOn. Moreover.
`the bolus formulations
`also require binding. wetting and disintegrating agents,
`any or all of which may adversely affect the viability of
`the bacteria. Such bolus products. therefore. have lim-
`ited shelf stability in that the population of viable bac-
`teria is greatly reduced within about a week.
`largely
`Thus. a persistent and vexatious problem,
`unattended by the prior art, is the lack of a method for
`simultaneously delivering incompatible substances in
`vivo to animals, particularly when one of the substances
`is a viable microorganism culture.
`Various prior art methods of physical separation. e.g..
`encoating. encapsulation and microencapsulation. of
`nutritional supplements are known. however. none ade-
`quately address the preparation and storage require-
`ments of sensitive direct-fed microbial agents. For ex-
`ample. conventional microencapsulation subjects bac-
`teria to a number of potentially fatal packaging proce-
`dures and requires expensive materials. complex equip-
`ment. and carefully controlled environmental condi-
`tions. Polymeric microcapsules also require specific pH
`ranges or enzyme activities to effect release of their
`contents in vivo. These requirements often frustrate
`conventional laboratory assessment techniques and pre-
`vent effective nutrient release in animals whose rumen
`pull or enzyme balances have been disrupted by bacte-
`rial depopulation.
`U.S. Pat. No. 4.695.466 to Morishita discloses a multi-
`ple-encapsulation method. The Morishita process in-
`cludes successively encapsulating oil solutions or sus-
`pensions in soft capsules. Although the method of Mori-
`shita has potential for delivery of two components in a
`single vehicle,
`the use of oil carriers presents insur-
`mountable obstacles to the delivery of bacteria and
`vitamin supplement components.
`It
`is unlikely that
`Morishita‘s soft outer capsules will be able to withstand
`common shipping, storage and administration condi-
`tions and also is unlikely applicable to commonly avail-
`able microbial forms.
`Despite recognition of the knowit drawbacks of prior
`art products. the art has not adequately responded to
`date with a method for delivery in vivo of the incompat-
`ible components, namely. direct-fed microbials and
`nutrient supplements nearly simultaneously to cattle,
`sheep and goats.
`SUMMARY OF THE INVENTION
`
`The present invention responds specifically to the
`long-felt need heretofore unmet by the prior art, and
`especially with a view to overcoming the inherent inad-
`equacies of combination supplements and direct fed
`
`This invention relates generally to nutritional and
`dietary compositions for livestock. and specifically to
`compositions which combine incompatible substances
`such as nutrient supplements and bacteria. The inven-
`tion also relates to a method of preparing a shelf-stable
`composition of incompatible agents. The present inven-
`tion is particularly well suited to deliver in vivo nearly
`simultaneously, incompatible supplements such as vita-
`mins and minerals and bacteria to cattle. sheep and
`goats in the form of a capsule-in-a-capsule.
`BACKGROUND OF THE INVENTION
`
`In the past. the development of ell'ective treatments
`for feeding disorders in cattle, sheep and goats has been
`Spurred by a desire to maximize yields of meat and dairy
`products. Existing drug-based treatments (see, e.g., U.S.
`Pat. No. 4361.426 issued to Martin, et 21]., and US. Pat.
`No. 4,405,609 issued to Potter), however, have the seri—
`ous drawback of rendering products from treated ani-
`mals unsalable for long periods under laws designed to
`protect consumers from harmful drug residues. Farm-
`ers. unhappy with the need to choose between low
`yields or unsalable products, have long sought the de-
`velopment of alternative, drug-free dietary treatments.
`The goals of drug-free dietary treatments are generally,
`improved growth and performance, and specially. ap-
`petite stimulation and reestablishment of the rumen
`bacterial populations necessary for proper digestion.
`Much attention has been given in recent years to the
`use of certain microorganisms as dietary adjuncts in
`efforts to improve the growth and performance of live-
`stOck, and reestablishment of rumen bacterial popula-
`tions. Such dietary cultures are known as probiotics or
`direct-fed microbials. (Gilliland, S. E, 8th Im‘i. Biotech.
`Syn. Proc. Vol. 2. pp. 923—933 (1988)). Generally. the
`microorganisms of such probiotics are those that are
`expected to grow and/or function in the intestinal tract
`or rumen of the animal and can exert certain metabolic
`actions that influence the animal. Various microorgan-
`isms which have been considered for this type of usage
`include Lactobacillus acidophilus, Lactabccillus pion-
`tamm. Lacrobociflus fermentttm. Lacrobact'flus easel, and
`Streptococcus faeci‘um.
`To derive maximum benefit from use of probiotics.
`the microorganisms must survive and grow in the intes-
`tine. It
`is thus imperative that
`the probiotic contain
`viable and active microorganisms at the time of con—
`sumption. The bacteria used as probiotics.
`therefore,
`must be stable during preparation and during storage
`prior to consumption.
`The simplest approach to delivery of probiotics is to
`add cultures to animal feed. However, it appears that
`few direct-fed microbials are stable in feed for more
`than 3-5 days. (Aimutis, W. R.. Feeds Management.
`Vol. 42. pp. 26—32 (1991)). Moreover. some feed con-
`tains antibiotics which are contrary to microbials stabil-
`ity. Yet other feed is pelleted. and most Lactobacillus
`species. which are predominant and beneficial intestinal
`species. are susceptible to the high temperatures. com-
`pression. aeration and mixing abrasion to which they
`are exposed during the pelleting process.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`4o
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Page 6 of 15
`
`Page 6 of 15
`
`

`

`5,310,555
`
`5
`
`to
`
`20
`
`3
`microbials for oral delivery to animals. The composi-
`tion is a dietary adjunct or feedstuff, providing the con—
`venience and reliability of oral administration. while
`providing near simultaneous delivery in vivo of incom-
`patible substances. The composition is shelf stable, i.e.,
`allows substantially greater viability of microbials. and
`does not require binding. wetting and disintegrating
`agents necessary for pellet or bolus formulations.
`The foregoing. and other advantages of the present
`invention. are realized in one aspect thereof in an oral
`nutritional composition.
`i.e.. a dietary adjunct. useful
`for treating feeding disorders and improving feed effi-
`ciency in livestock, e.g., cattle, sheep and goats, espe-
`cially ruminants. The dietary adjunct composition com-
`prises a double capsule which includes live cultures of 15
`rumen bacteria in a first capsule which is enclosed with
`vitamin and mineral supplements in a second capsule.
`The capsules are preferably made of gelatin. The bac-
`teria and supplements may be combined with accept-
`able feed grade carriers.
`In another aspect. the invention is a method of simul-
`taneously delivering incompatible compounds to ani-
`mals in vivo. Such delivery is achieved by feeding an
`animal a double capsule containing a first substance in a
`first capsule, which is enclosed with a second substance. 25
`incompatible with the first substance, in a second larger
`capsule.
`this invention provides a
`In another embodiment.
`method for preparing shelf-stable compositions of in-
`compatible substances. which includes the use of multi- 30
`ple capsules of variable composition. Such method is
`accomplished manually or by machine.
`Other advantages and a fuller appreciation of the
`specific adaptations. compositional variations,
`and
`physical and chemical attributes of the present inven- 35
`tion will be gained upon an examination of the follow—
`ing detailed description of the invention. taken in com
`junction with the accompanying drawings and ap-
`pended claims.
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`The present invention will hereinafter be described in
`conjunction with the appended drawings. wherein like
`designations refer to like elements throughout and in
`which:
`
`FIG. 1 shows an enlarged sectional view of the cap-
`sule-in-a-capsule structure in accordance with the pres-
`ent invention;
`FIGS. Za—Zf illustrate a method by which each cap-
`sule-in-a-capsule is assembled; and
`FIG. 3 compares the efficacy of double capsules in
`accordance with the present invention and hard boluses
`in simultaneously delivering live microorganisms and
`incompatible nutrient supplements.
`DETAILED DESCRIPTION
`
`45
`
`50
`
`55
`
`The present invention relates broadly to nutritional
`supplements and dietary adjuncts for animals. such as
`cattle. sheep and goats. and specifically to compositions
`and nutrient delivery systems which permit delivery of 60
`incompatible substances. However, the composition of
`the present invention is most particularly adapted for
`use in oral supplementation formulations which com-
`bine nutrients, such as vitamins and minerals. and viable
`microbials, such as rumen bacteria. Accordingly.
`the 65
`present invention will now be described in detail with
`respect
`to such fields of endeavor: however.
`those
`skilled in the art will appreciate that such description of
`
`Page 7 of 15
`
`4
`the invention is meant to be exemplary only and should
`not be viewed as limitative of the full scope thereof.
`The present invention provides a nutritional composi-
`tiOn useful
`for ameliorating drug-induced.
`stress-
`related. and other feeding disorders in food—producing
`animals. e.g.. cattle. sheep and goats. The composition is
`particularly useful as a ruminant feedstufl' for improving
`feed efficiency and promoting growth. The composi-
`tion avoids the milk and slaughter withdrawal periods
`required after drug treatments and enhances the general
`nutritional status of the animal. Additionally. the com-
`position is shelf-stable and provides a general packaging
`system for incompatible materials, and is particularly
`useful for direct-fed microbial agents or probiotics.
`These attributes are achieved through a particular com-
`position meeting a special combination of physical pa-
`rameters.
`
`As used herein. the term “incompatible“ is meant to
`refer to substances which deleteriously react with one
`another when combined in desired levels or concentra-
`tions.
`In one embodiment. the invention provides a nutri-
`tional camposition whose cemponents are mutually
`incompatible. and which incompatible components are
`physically separated from each other until they reach
`their in vivo situs. In a preferred embodiment, the com—
`position includes components which are cultures of
`viable bacteria, and nutritional supplements. e.g.. vita-
`mins and minerals. As the cultures and nutritional sup-
`plements are incompatible, the microbial cultures are
`enclosed in a first capsule which is then enclosed with
`the vitamin and mineral supplements in a second such
`capsule. Le. a “capsule-in-a-capsule" structure. The
`bacteria of the first component serve the valuable func-
`tion of repopulating the rumen. thus enabling digestiOn
`to resume. producing digestive enzymes. and correcting
`acid imbalances which result from rumen bacterial de-
`population.
`The vitamins and minerals of the second component
`increase the nutritional status of animals laboring under
`COnditions of malnutrition caused by feeding disorders.
`Further, once dispersed throughout
`the rumen. these
`vitamins and minerals support the rapid growth of the
`bacteria of the first component. Oral administration of
`these vitamins and minerals contemporaneously with
`the administration of bacteria is preferable to separate
`or intravenous administration. Separate administration
`increases the risk that bacteria will not encounter dis-
`persed vitamins and minerals in the rumen and thus fail
`to exhibit their full growth potential.
`It has been found that the bacterial survival rate with
`the capsule-in-a-capsule structure of the present inven-
`tion after Up to six months storage, i.e.. six months after
`preparation. is nearly 500 times that of an admixture of
`the bacteria and nutrient supplements. An admixture of
`bacterial and nutrient supplements is typically a single
`capsule or a bolus formulation.
`FIG. 1 illustrates a capsule-in-a-capsule structure in
`accordance with the present invention and is generally
`designated as 20. Capsule-in-a-capsule 20 includes an
`inner capsule 22 and an outer capsule 24. Inner capsule
`22 contains viable bacteria 26 and outer capsule 24 con-
`tains vitamins and minerals generally designated as 28.
`Inner capsule 22 includes a top member 30 and a bOttom
`member 32 which is bigger than top member 30. Top
`member 30 and bottom member 32 are locked together
`after filling by a locking mechanism 33 which includes
`a groove 34 proximate the top 35 of bottom portion 32
`
`Page 7 of 15
`
`

`

`5
`and a complementary ridge 36 substantially about the
`midpottion 37 of top portion 30, forming a circumferen-
`tially nested ridge and groove. Similarly, outer capsule
`24 has a top member 33 and a bottom member 40 in
`which top member 38 is locked to bottom member 40
`with a groove 42 and a ridge 44. The capsules are pref-
`erably made of gelatin. Capsule shells are, however,
`easily reformulated to meet a myriad of size. transporta-
`tion, storage, and administration requirements. e.g..
`excessive heat or cold, vibration, humidity, compres-
`sion or impact, aeration. or ultraviolet light.
`The bacteria of the first component,
`i.e., of inner
`capsule 22. include one or more of the indigenous intes-
`tinal bacteria selected from Lucrobact'lius acidopln'lus.
`Lactcbact'flus Men's. Lactobact'llus easel) Streptococcus
`faecium. and Pediacoccus cerevisiae. The bacteria can be
`processed in accordance with conventional methods of
`bacteriology to produce direct-fed microbial agents
`suitable for encapsulation in gelatin-shelled capsules and
`administration to cattle, sheep and goats. The in vitro
`viability of the bacteria of the inner capsule is deter-
`mined by counting the co10ny-forming units per gram
`(CPU/g) of' the culture administered. according to stan-
`dard feed industry protocols. At time of preparation,
`the bacterial count in the inner capsule in accordance
`with the present
`invention is suitably about 20><l09
`CFU/g.
`The bacteria can be employed in admixtures with
`conventional excipients. e.g., acceptable feed grade
`carriers suitable for enteral (cg. oral) administration
`which do not deleteriously react with the bacteria.
`Suitable feed grade carriers include, but are not limited
`to, calcium carbonate, nonhygroscopic whey, rice bulls,
`and sucrose.
`
`The bacterial preparations can also be mixed with
`auxiliary agents. e.g., whole dried milk. dextrose, en-
`zymes, plasma proteins or amino acids to promote the
`growth and nutritional status of the animal and the
`bacteria in vivo.
`The vitamins and minerals of the second component,
`i.e., outer capsule, are selected from one or more vita-
`mins. namely. A, Big, C. D, E, and K, niacin, thiamine,
`choline, biotin, folic acid, riboflavin, pantothenic acid,
`and one or more minerals, namely. cobalt, copper. iron,
`manganese, selenium and zinc.
`The vitamins and minerals of'the outer capsule 28 can
`be processed in accordance with conventional methods
`of pharmacy to produce agents suitable for encapsula-
`tion in gelatin-shelled capsules and administration to
`cattle. sheep and goats. For example, the vitamins and
`minerals can be administered in alternative sulfate. ox-
`ide, chelated, or other chemical forms to promote effi-
`cient dissolution and absorption in vivo.
`The vitamins and minerals can also be employed in
`admixtures with conventional excipients, e.g., accept-
`able feed grade carriers which do not deleteriously
`react with them. Suitable carriers include, but are not
`limited to, antioxidants,
`flavoring agents. cellulose,
`grain by-products, or other inert vegetable materials.
`Nutrient-rich, dried organic materials. such as kelp, are
`highly preferred carrier materials, as they contribute
`significantly to the vitamin and mineral status of the
`animal and rumen bacteria.
`The capsule-in-a-capsules for adult cattle are suitably
`prepared in 15 g double capsules and capsule-in-a—cap—
`sules for calves are suitably prepared as 4.5 g double
`capsules. Both sizes of capsules are orally administered
`daily as 1-2 capsule-in—a—capsule for three consecutive
`
`Page 8 of 15
`
`5,310,555
`
`6
`days at freshening or other signs of stress or feed dis»
`tress.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`65
`
`50
`
`55
`
`It will be appreciated, hOwever, that the actual pre-
`ferred amounts of the compounds in the inner and outer
`capsules will vary according to the age, weight and
`species of animal being treated, and the particular feed-
`ing disorder of interest. For example. the amounts of
`vitamins and minerals in outer capsules for calves is
`suitably one-third of that in outer capsules for adult
`cattle. Feed guidelines can be determined by means of
`an appropriate conventional dietary protocol.
`In another embodiment.
`the invention provides a
`method for simultaneously delivering incompatible sub-
`stances to livestock in vivo. Specifically, the method
`includes oral administration of a capsule—in-a-capsule
`which structure includes a first gelatin-shelled capsule
`containing a first substance and a second gelatin-shelled
`capsule encapsulating the first capsule that contains a
`substance incompatible with the first. When the first
`substance is a bacterial culture of rumen bacteria, such
`method delivers at least 3X 109 CFU/capsule-in-a-cap-
`sule.
`
`The method in accordance with the present inventiOn
`advantageously preserves the activity of' mutually reac-
`tive or otherwise incompatible substances by physically
`separating them during production. storage and admin-
`istration. When live microorganisms are administered to
`animals simultaneously with vitamins and minerals in
`bolus or single capsule formulations. the microorgan-
`isms often are rendered nonviable. The present method
`advantageously facilitates the consolidation of multi—
`step therapies into easily administered,.single-step thera-
`pies. Such efficient
`administration eliminates
`the
`stresses induced by the sequential administration of
`multi-phase treatment components and ensures the de-
`livery of' correct unit doses. The capsule-in-a-capsule
`vehicle is suitably delivered by hand or balling gun to
`cattle. sheep and goats.
`In another aspect, the invention is an oral nutritional
`supplement delivery system for livestock. The system is
`a two-component system which effects near simulta-
`neously delivery of the two components. The system
`comprises a first capsule containing at least one live
`microorganism and a second capsule enclosing both the
`first capsule and an admixture of vitamins and mineralsI
`incompatible with live microorganisms. The capsules
`physically separate the incompatible microorganism
`cultures and nutrient supplements, thereby eliminating
`the need for separate administration of the microorgan»
`isms and the vitamin and mineral admixture.
`To fabricate a capsule-in-a-capsule in accordance
`with the present invention, a first capsule, typically a
`gelatin capsule.
`is
`filled with a first substance and
`capped. This first capsule is placed inside the bottom
`member of a second, larger capsule, typically also a
`gelatin capsule. and the second capsule is then filled
`with a second substance.
`incompatible with the first
`substance, and capped. This method for efficiently and
`inexpensively preparing a shelf-stable composition of
`incompatible substances constitutes another aspect of
`the invention. The steps may be performed manually Or
`by machine. This method of fabrication has certain
`production economics compared to production of hard
`boluses.
`Reference is now made to FIGS. Za-Zf which illus-
`trate a method of preparing the capsule-in-a-capsule
`formulatiou of the present invention. Specifically, the
`larger bottom members 32 of the inner. smaller capsules
`
`Page 8 of 15
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`

`

`5,310,555
`
`7
`22 are first placed in openings 46 in an assemblyr board
`43 in a housing 50. Each bottom shell 32 is then filled
`with the bacterial culture 26. Top member 30 is then
`placed on each bottom member 32 and "locked" in
`place by using a mechanism which is the same or similar
`to locking mechanism 33 described hereinbefore by
`gentle pressure from pressboard 49 to form a sealed
`capsule 5]. Filled sealed capsules 5] are ejected from
`the board 48. and the board 48 is removed from the
`housing 50.
`A second assembly board 56, having holes 54. corre-
`sponding to the bottom members 40 of larger capsules
`24. is placed in the machine housing 50. The bottom
`members 4|] of the larger capSules 24 are placed in the
`board 56. and one sealed capsule 51 containing the bac-
`teria is placed within each. The remaining volume of
`each bottom member 40 of capsule 14 is then filled with
`the vitamin and mineral admixture 28. A top member 38
`is then placed over each bottom member 40 and locked
`as described previously in place with gentle pressure
`from the pressboard 52. The resulting capsule-in-acap-
`sule is then ejected from the second board 56.
`Capsule-in-a-capsules can also be fabricated by ma-
`chine using, e.g.. a Torpac Capsule Filling Machine
`commercially available.
`This fabrication method can be used to produce sig-
`nificantly more stable probiotics than conventional
`bolus or single-capsule delivery systems which combine
`sensitive bacteria with toxic levels of vitamins and trace
`minerals. The use of a multiple capsule also facilitates
`the administration of higher. more efficacious doses of
`vitamins and minerals than are delivered by conven-
`tional boluses. Further, gelatin-shelled capsules are effi-
`ciently and inexpensively assembled. thereby incurring
`reduced production costs relative to microencapsula-
`tion or hard bolus vehicles. The contents of such cap-
`sules also are not subjected to extremes oftemperature,
`pressure or abrasion during their manufacture, thereby
`facilitating the consolidation of multi-step therapies
`utilizing 00mponents sensitive to such conditions.
`Finally. capsule shells which are easily reformulated
`to provide particular protection to enclosed materials
`(cg. different sized capsules or ultra-violet light filter-
`ing capsules) may be used interchangeably and in con-
`cert with other capsule shells of standard feed grade
`composition.
`In contrast, microencapsulation equip-
`ment cannot accommodate frequent coat or nutrient
`composition reformulations without costly equipment
`changes.
`The present invention is further explained by way of
`the following examples which are to be construed as
`merely illustrative, and not limitative, of the remainder
`of the disclosure in any way whatsoever. 1n the follow-
`ing examples. all temperatures are expressed in degrees
`Celsius. All test capsules were randomly selected for
`analysis, and microorganisms were enumerated using
`the National Feed Ingredient Association's Standard
`Practice for the Enumeration of Microorganisms from
`Direct-Fed Microbials and Silage lnnoculants. Bacte-
`rial viability is expressed in colony-forming units per
`gram of source material {CFU/g) or CFU per capsule.
`EXAMPLE 1
`
`Preparation of Capsule-ima-Capsule
`
`Capsule-in-a—capsule vehicles appropriated for ad-
`ministration to adult cattle were prepared by the
`method as described hereinbefore. The bacterial cul-
`tures were commercial formulations of dormant Lacto-
`
`Page 9 of 15
`
`8
`bacillus acidopht‘lus. Lactobcct‘lhts lat-it's, Lactobact'llus
`caret. Streptococcus faecr'um, and Pedtococcus cerew'rt‘ae
`having a viability of 20.0)(109 CFU/g at the time of
`preparation. The vitamin and mineral admixture con-
`tained the following in the amounts indicated in paren-
`theses: vitamin A (5x105 IU). vitamin D (7.5x 10‘ lU).
`vitamin E (750 IU). vitamin Bu (2000 pg). niacin
`(3 x 103 pg). pantothenic acid (15 pg), choline (750 pg).
`biotin (75 pg), cobalt (20 pg), c0pper (none), iron (30
`|ug). manganese (30 pg), zinc (75 pg). and trace quanti-
`ties of riboflavin. thiamine. folic acid, vitamin K, vita—
`min C. and selenium. Each capsule-in-a~capsule con-
`tained approximately 0.25 g of bacterial culture and 12.5
`g of the vitamin and mineral admixture,
`the above-
`enumerated vitamins and minerals having been com-
`bined with kelp, an acceptable and nutrient-rich feed
`grade carrier.
`Capsule-in-a-capsule vehicles appropriate for admin-
`istration to calves were prepared by an identical
`method, excepting that the vitamin and mineral admix-
`ture contained approximately one-third of the amounts
`of the vitamins and minerals enumerated above for use
`
`in the adult cattle capsule-in-a-capsules. Each calf-size
`capsule-in-a-capsule coutained approximately 0.25 g of
`bacteria culture and 3.5 g of the vitamin and mineral
`admixture. the vitamins and minerals having also been
`combined with kelp. Capsule-in-a-capsule vehicles iden-
`tical to those of Example 1 were used in the following
`tests.
`
`EXAMPLE 2
`
`Determination of the Efficacy of Capsule-In-A-Capsule
`for Preserving the Viability of Anaerobic Bacterial
`Colony Forming Units
`
`Capsule-in-a-capsule vehicles appropriate for admin-
`istration to adult cattle as described in Example 1 were
`variously tested against single capsule forms with an
`admixture of cultures and nutrient supplements as used
`in the adult cattle capsule-in-a-capsule form. All cap-
`sules were stored at room temperature for one week
`following their preparation. The capsule-in-a-capsules
`were randomly apportioned to two experimental
`groups. A and B. The single capsules enclosing admixed
`microbes and nutrient supplements comprised group C,
`and group D (control) comprised an unencapsulated
`sample of the microbial culture.
`Inner capsule contents of four group A samples. outer
`capsule contents of one group B sample (to test the
`microbial content of the nutrient mixture). capsule con-
`tents of one group C sample. and nine of the group D
`control culture were randomly selected for comparison.
`Each test sample was individually serially diluted in
`autoclaved 6.25 mM phosphate buffer at pH=T.2. One
`(I) ml aliquots of each dilution were transferred into
`separate sterile petri plates. Twenty {20} ml of sterile
`LMRS (Lactobacilli Man Rugosa Sharpe Agar). cooled
`to 44° C. to 46‘ C.. were then added to each petri plate
`with swirling. The plates were covered and cooled to
`room temperature before being inverted and placed into
`GasPak TM anaerobic jars. The plates were incubated
`at 35" C. until colonies were readily discernable. ap-
`proximately two to three days. Colonies were then
`counted using a Quebec colon) counter.
`These data and the bacterial survival rate are given in
`Table 1 below.
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`45
`
`50
`
`55
`
`65
`
`Page 9 of 15
`
`

`

`9
`
`TABLE 1
`
`5,310,555
`
`10
`
`EXAM PLE 4
`
`Bacterial Viability of CapsuieJn-A-Capsule
`Theoretical
`Bacterial Count
`Bacterial Count.
`Test
`At Preparation.
`CPU/g, 1 Week
`Bacteria
`
`Group
`CPU};
`After Preparation
`Survival. 'fi'
`a
`23 x 109
`23 x 109
`100
`B
`—
`s x 102
`-—
`1:
`22 x 109
`<106
`<21
`
`23 x 109D(cor|troI) 100 23 x 10“
`
`
`
`These data indicate that the contents of intact. gelatin-
`sheiled capsule-in-a-capsules successfully retain 100%
`of their colony-forming activity after one week of stor-
`age at room temperature. Conversely, capsules which
`do not separate microbial cultures from nutrient supple-
`ment materials are not successful in protecting bacterial
`viability. and their contents lose 99.79% of their colony-
`forming activity within one week.
`EXAMPLE 3
`
`Determination of the Shelf Stability of Anaerobic
`Bacterial Colony Forming Units Packaged in
`Capsule-In-A-Capsules During Long-Term Storage at
`Room Temperature
`
`Capsule-in-a—capsules suitable for administration to
`adult cattle were fabricated as described above in Ex-
`
`ample l and stored at room temperature. At preselected
`intervals after their manufacture, i.e, one, two, three.
`four. five and six months, inner capsules containing the
`bacterial cultures were removed from randomly se-
`lected capsule—in-a-capsules. The contents of each inner
`capsule were serially diluted in autoclaved 6.25 rnM
`phosphate buffer, pH=7.2. and l m] aliquots of each
`dilution were transferred into sterile petri plates.
`Twenty (20] m] of sterile LMRS Agar cooled to 44" C.
`to 46" C.. were then added to each petri plate with
`swirling. The plates were covered and cooled to room
`temperature before being inverted and placed into Gas—
`Pak TM anaerobic jars. The plates were incubated at 35°
`C. until colonies were readily discernable. approxi-
`mately two to three days. Colonies were then counted
`using a Quebec colony counter. These data and the
`bacterial survival rates are given in Table 2 below.
`
`TABLE 2
`
`Shelf Stabilitv of Capsule-ln-A-Capsule
`Time After
`Bacterial
`Bacterial
`
`Manufacture. mos.
`Count. CFUr'g
`Survival. 9t:
`
`1
`2
`2
`4
`5
`s
`
`‘
`
`22.5 x 10"
`20.0 x 10"
`21.2 x 10°
`15.1 x 109
`13.0 x 109
`12.0 x 10"
`
`100
`39
`. 94
`22
`53
`53
`
`These data indicate that sufficient bacteria remain via-
`
`ble in the capsule-in-a-capsule vehicle to repopulate the
`rumen after six months of storage under typical field
`conditions. In contrast. the number of CFU/g of can-
`ventional bolusas approaches zero approximately three
`weeks after manufacture under similar conditions.
`
`Page 10 of15
`
`Determination of the Shelf Stability of Anaerobic
`Bacterial Colony Forming Units Combined with
`Vitamin and Mineral Supplements During Long-Term
`Storage at Room Temperature
`
`The shelf stability of single capsule formulations iden-
`tical to the adult cattle capsule-in—a-capsule formula-
`tions of Example 1 were determined. Single gelatin-
`shelled capsules were filled with an admixture of the
`bacterial culture and vitamin and mineral supplement in
`a 1:39.55 gram:gram ratio present in the capsule-in-a-
`capsule vehicle of Example 1. The capsules were
`packed in ice for two days prior to testing; thereafter all
`capsules were stored at room temperature. A pure sam-
`ple of the microbial culture was reserved on ice as a
`control.
`
`to
`
`is
`
`At 2, 3, 4, 5, 8, 12, 16, 20, 25. and 32 days after manu-
`facture, the contents of three randomly-selected cap-
`sules were nonabrasivaly combined. and samples there-
`from were serially diluted in autoclaved 6.25 mM phos-
`phate buffer at pH=7.2. During the initial analysis, a
`control sample from the microbial culture control was
`also diluted and analyzed. One (1) ml aliquots of each
`dilution were transferred in duplicate into separate ster-
`' ile petri plates. Twenty (20) ml of sterile Lactobacillus
`30
`MRS Agar, cooled to 44° C. to 46° C.. were then added
`to each petri plate with swirling. The plates were cov-
`ered and cooled to room temperature before being in-
`verted and placed into GasPak TM anaerobic jars. The
`plates were incubated at 35' C. until colonies were
`readily discernable, approximately two to three days.
`Colonies were then counted using a Quebec colony
`counter.
`
`20
`
`25
`
`35
`
`40
`
`These data and bacterial survival rates are given in
`Table 3 below.
`
`
`
`TABLE 3
`
`45
`
`50
`
`55
`
`60
`
`65
`
`Shelf Stability of Single Capsules
`Eguivalent to Capsule-In-A~Capsule
`T