(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2008/0187525 A1
`POrubcan
`(43) Pub. Date:
`Aug. 7, 2008
`
`US 20080187525A1
`
`(54) FORMULATIONS INCLUDING DIGESTIVE
`ENZYMES AND POLYSORBATE
`SURFACTANTS THAT ENHANCE THE
`COLONIZATION OF ADMINISTERED
`PROBOTCS MICROOGANISMS
`
`(76) Inventor:
`
`Randolph S. Porubcan, Victoria,
`MN (US)
`Correspondence Address:
`ERC P. MIRABEL
`35 TECHNOLOGY DRIVE, SUITE 100
`WARREN, NJ 07059
`
`(21) Appl. No.:
`
`12/022,380
`
`(22) Filed:
`
`Jan. 30, 2008
`
`Related U.S. Application Data
`(60) Provisional application No. 60/887,628, filed on Feb.
`1, 2007.
`
`O
`O
`Publication Classification
`
`(51) Int. Cl.
`A6 IK 38/46
`A6 IK 35/74
`
`(2006.01)
`(2006.01)
`
`A638/43
`A638/47
`A6IP3/02
`
`(2006.01)
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl. .................. 424/93.45; 424/94.63; 424/94.6;
`424/93.4; 424/94.1; 424/94.65; 424/94.66:
`424/94.61
`
`(57)
`
`ABSTRACT
`
`Disclosed a formulations for enhancing the 1. vivo coloni
`Zation of probiotic microorganisms that include digestive
`enzymes and probiotic microorganisms, and polysorbate Sur
`factants. The enzymes include lactogenic enzyme formula
`tions that promote growth of Lactobacillus probiotics, bifi
`dogenic enzyme formulations that promote growth of
`Bifidobacterium probiotics and combination formulations
`that benefit both types of probiotics. It has been discovered
`that certain polysorbate Surfactants, including polysorbate-60
`and polysorbate-80, further promote probiotic microorgan
`ism growth, when used with the enzyme formulations. The
`formulations are preferably compounded as dry powders, to
`avoid water R with t E. in SR formula
`tions. Such formulations can be contained incapsules, tablets,
`packets or bottles and administered orally, either sequentially
`or in one combined formulation.
`
`MYLAN - EXHIBIT 1019
`
`

`

`US 2008/O 187525 A1
`
`Aug. 7, 2008
`
`FORMULATIONS INCLUDING DIGESTIVE
`ENZYMES AND POLYSORBATE
`SURFACTANTS THAT ENHANCE THE
`COLONIZATION OF ADMINISTERED
`PROBOTCS MICROOGANISMS
`
`RELATED APPLICATIONS
`0001. This application claims priority to U.S. Provisional
`Application Ser. No. 60/887,628, filed Feb. 1, 2007, priority
`to which is hereby claimed.
`
`FIELD OF THE INVENTION
`0002 The invention relates to formulations for enhancing
`colonization in vivo of administered probiotic bacteria.
`
`BACKGROUND
`0003 Dietary supplements containing viable probiotic
`bacteria are increasing in popularity in the marketplace as
`their health benefits become recognized. Reported benefits
`range from alleviating constipation and diarrhea to reducing
`various intestinal infections such as those caused by rotavi
`ruses, pathogenic E. coli and Helicobacterpylorias discussed
`in U.S. Pat. Nos. 7,090,840 and 7,029,669. Beneficial species
`of Lactobacillus and bifidobacteria are among the widely
`recognized probiotics, and strains of these bacteria that are
`capable of colonizing the intestinal tract are advantageous
`(see U.S. Pat. Nos. 7,150,986 and 6,887.465).
`0004 Colonization may involve physical attachment to
`epithelial cell surfaces such as those of the microVilli in the
`ileum section of the Small intestine, or simple domination of
`the contents of the cecum, or adherence within the mucin
`layers of the colon. There is much to learn about how probi
`otics colonize the mammalian intestinal tract but it has been
`established that when colonization occurs, more probiotic
`microorganisms appear in the feces and this correlates with
`more probiotic microorganisms in both the proximal and
`distal sections of the intestinal tract. See Muralidhara et al.,
`1997, Journal of Food Protection Vol. 40, No. 5, Pages 288
`295. Young pigs were used to demonstrate the relationship
`between colonization and fecal probiotic counts in
`Muralidhara's work; the porcine intestinal tract is very similar
`in physiology to the human intestinal tract and similar studies
`with young pigs are presented in examples of the present
`invention to demonstrate its effectiveness.
`0005 Colonization that results in the competitive exclu
`sion of pathogenic microorganisms is particularly beneficial
`and can occur when probiotics occupy most of the intestinal
`attachment sites and are encouraged to produce lactic acid
`and otherantimicrobial compounds. Effective intestinal colo
`nization by probiotics depends on the availability of proper
`microbial nutrition that must be provided by the diet. See
`Gibson et al 1995, Gastroenterology 106:975-982: Christlet
`al, 1992, Gut 33: 1234-1238 and Reid et al., 1990, Clin.
`Microbiol. Rev. 3: 335-344. However, normal diets do not
`provide nutrients that necessarily benefit probiotics so there is
`a need to fortify the diet with such nutrients. Prebiotics are
`one class of microbial nutrients that are currently popular in
`the marketplace. They are typically certain oligosaccharides
`that are not digested in the Small intestine but serve as nutri
`ents for select probiotic bacterial genera, e.g., bifidobacteria,
`when they arrive in the colon. An article titled “Prebiotics
`Enhance Gut Health' by Laura Brandt is available online at
`the nutrasolutions website; another titled "Prebiotics: A More
`
`Reliable Way to Increase Gut-Friendly Bacteria” by Dr.
`James Meschino is available at the chiroweb website, in the
`archival section. Today, a variety of functional foods are
`fortified with prebiotics such as fructooligosaccharides
`(FOS) and inulin, in an effort to provide probiotic stimulation
`in vivo. This practice adds cost to the foods being fortified and
`is not very effective in stimulating Lactobacillus probiotics.
`Thus, there is a need for reducing the cost of prebiotics while
`providing for both Lactobacillus and Bifidobacterium stimu
`lation.
`0006 Although enzymes have been used to generate pre
`biotics under laboratory conditions followed by subsequent
`feeding of the preformed prebiotics to achieve probiotic
`stimulation (see U.S. Pat. Nos. 6,791,015 and 6,730,502), no
`one has suggested using enzymes to generate these effects in
`vivo. U.S. Pat. No. 5,817,350 discloses the use of the prebi
`otic enzymes cellulose, amylase and hemicellulase, for use as
`dietary Supplements, but not use of these enzymes to stimu
`late administered probiotics, or enhancement of their prebi
`otic effect by addition of polysorbate compounds. U.S. patent
`application 20010031276 discloses the use of polysorbate
`compounds as feed additives for ruminant animals but does
`not relate to their use in combination with prebiotics and
`probiotics, or their use in non-ruminant animals.
`0007 If polysorbate surfactants are combined with
`enzymes, wateractivity (Aw) becomes important. High levels
`of Aw, e.g., Awe-0.04, can significantly destabilize the shelf
`life of certain enzymes. PolySorbates are viscous, Sticky, non
`aqueous liquids that are not available in dry form. Before
`incorporation into enzyme formulations, polysorbates must
`be rendered into dry powders and the Aw of any formulation
`containing them should be below 0.04.
`0008. “Internal Probiotic Culture' or IPC, as used herein,
`is the totality of viable probiotic microorganisms present in
`the intestinal tract at any given time: i.e., the Sum of probiotic
`microorganisms adhering to intestinal epithelial cells,
`mucous and mucin layers, ingested food and waste material.
`It is desirable to enhance the IPC of humans and mammals.
`IPC can be estimated from the total viable Lactobacillus
`and/or Bifidobacteria counts (colony forming units) present
`in fresh fecal matter.
`
`SUMMARY
`0009. Described herein are formulations of probiotics,
`enzymes to enhance or stimulate the growth of probiotics and
`food grade polysorbate surfactants that enhance the growth
`stimulating effect of the enzymes. The enzymes catalyze
`generation of microbial nutrients in vivo Such as cobiotics and
`prebiotics (cobiotics are nutrients utilized by both the host
`and the probiotics; prebiotics are substances utilized only by
`probiotics). It was discovered that certain digestive enzymes
`have the ability, upon reacting with food, to produce nutrient
`Substances in vivo that are stimulatory to some probiotics,
`including the Lactobacillus and Bifidobacterium species.
`Enzymes Such as proteases and amylases (that produce free
`amino acids and simple monosaccharides) primarily stimu
`late Lactobacillus probiotics and are referred to herein as
`lactogenic enzymes. Enzymes such as cellulase and hemicel
`lulase (that release prebiotic oligosaccharides or crack larger
`polysaccharides into oligosaccharides) primarily stimulate
`Bifidobacteria probiotics and are referred to herein as bifi
`dogenic enzymes.
`0010. In one administration embodiment, the probiotics
`are administered in advance of the enzymes, or in advance of
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`

`US 2008/O 187525 A1
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`Aug. 7, 2008
`
`both enzymes and polysorbates, by 2-72 hours to allow an
`internal probiotic culture (IPC) to develop in the intestinal
`tract. The IPC is enhanced, as demonstrated in vitro and in
`vivo in the examples provided below. Subsequent to the initial
`establishment of the IPC, which begins on day one of an
`administration program with the first dose of a probiotic
`formulation, e.g., with a dose of at least about 5 billion colony
`forming units (CFU), the probiotic, enzyme or enzyme plus
`polysorbate formulations are thereafter administered daily, or
`less frequently.
`0011
`Probiotic administration will preferably precede the
`enzyme or enzyme plus polysorbate administration by about
`4-12 hours. This delay allows the probiotics time to become
`established on the intestinal surfaces, and/or as an IPC, prior
`to action by the enzymes or their reaction products. This delay
`also limits direct interaction of the probiotics with the
`enzymes.
`0012. The result of administration of probiotics, enzymes
`and polysorbates as described herein is a Substantial increase
`in the Internal Probiotic Culture (IPC).
`
`DETAILED DESCRIPTION
`0013 Lactogenic enzymes are those that release cobiotics
`(free amino acids, short chain peptides, and monosaccharide
`Sugars) from ingested food. They stimulate the colonization
`and/or strength of the Internal Probiotic Culture (IPC) of
`Lactobacillus probiotics when used as described herein. Bifi
`dogenic enzymes are those that either release existing prebi
`otic oligosaccharides from food or enzymatically crack larger
`food borne polysaccharides into oligosaccharides that have
`prebiotic activity. They stimulate the colonization or IPC of
`bifidobacteria probiotics when used s described herein.
`Polysorbates such as Tween-60 and Tween-80 have been
`found to stimulate the effects of lactogenic and bifidogenic
`enzymes.
`0014. The various formulations herein can be packaged in
`capsules, tablets, packets or bottles and then into kits that
`provide all the necessary components for probiotic stimula
`tion in a consumer ready format. Although the various ingre
`dients could be utilized as liquids, dry formulations are pre
`ferred.
`
`Lactogenic Enzyme Formulations (LEF)
`0015 The lactogenic enzyme formulations of this inven
`tion contain protease and carbohydrase digestive enzymes.
`Exemplary lactogenic enzymes include the following pro
`teases: papain from Carica papaya (800 TU/mg), bromelain
`from Ananas comosus (2,000 GDU/g), fungal protease from
`Aspergillus oryzae (400,000 HU/g), acid protease from A.
`oryzae (500,000 HUT/g), bacterial protease from Bacillus
`subtilis (2,000,000 PC/g), and fungal peptidase from
`Aspergillus Oryzae (500 LAP/g); and the following carbohy
`drases: alpha-amylase from A. oryzae (100,000 SKB/g), glu
`coamylase from A. niger (1000AG/g), lactase from A. Oryzae
`(100,000 ALU/g), and invertase from Saccharomyces cerevi
`siae (200,000 Summer U/g). Activity units are shown in
`parenthesis for the stock enzyme concentrates that are
`obtained from a commercial source (and are defined below in
`Table I), but are not necessarily the activity for the final
`formulations. All enzymes are dry powders obtained from
`Bio-Cat, Inc., Troy, Va.
`0016 Many other enzymes such as pancreatin (a mixture
`of enzymes), trypsin, chymotrypsin and pepsin that are
`
`derived from animal tissue can be used to achieve some of the
`benefits of the present invention but may not exhibit signifi
`cant enzymatic activity deep in the intestinal tract as effec
`tively as the fungal enzymes indicated above. New to the
`market are enzymes Such as nattokinase and Serrapeptase that
`can also be used (though they were not approved for use in
`foods prior to October 1994).
`0017 Specific formulations are made by combining one or
`more lactogenic enzyme with MCC (microcrystalline cellu
`lose available as Avicel PH112 from FMC, Philadelphia, Pa.)
`used as an inert carrier to standardize enzyme strength (activ
`ity units). MCC may comprise 10-80% of the formulation
`depending on the quantity of other ingredients used. In addi
`tion, food-grade silica such as Syloid 63 FP (W.R. Grace,
`Columbia, Md.) can be added at 2% by weight to improve dry
`flow characteristics and reduce water activity (Aw). Option
`ally, up to 2% by weight pharmaceutical grade magnesium
`stearate is added if the formulation is to be filled into capsules
`or tableted. All ingredients may be blended in a Paterson
`Kelly type twin-cone blender to achieve a uniform mixture,
`typically requiring 10-15 minutes mixing at 50 rpm. For
`purposes of reducing Aw and enhancing shelf-life, all opera
`tions should be conducted in a dry, humidity controlled facil
`ity having a relative humidity of 20-40%. The final formula
`tion can be filled directly into hard capsules made of gelatin,
`cellulose, HPMC or any suitable capsule material, or into
`hermetically sealed packages or bottles.
`0018. The lactogenic formulations of this invention pro
`mote growth of a number of Lactobacillus probiotics includ
`ing Lactobacillus acidophilus LA-1. Lactobacillus paracasei
`F-19 and Lactobacillus rhamnosus HNO01 and Lr-32, as
`exemplified herein, and may also be used with other Lacto
`bacillus probiotic species, including: L. casei, L. bulgaricus,
`L. fermentum, L. plantarum, L. delbrukei, L. salivarius, L.
`jensenii, L. gaserii, L. reuteri, L. helveticus, L. lactis, L.
`brevis, and L. johnsonii. The mode of action may be due to the
`generation of monoaccharides and free amino acids and/or
`short chain peptides at or near the intestinal colonization sites
`of the lactobacilli (which are predominantly in the small
`intestine). These same monosaccharides and amino acids
`stimulate bifidobacteria in vitro but, in practice, due to their
`rapid absorption and utilization in the Small intestine, rarely
`make it to the colon where the bifidobacteria reside. There
`fore, one preferred embodiment of the lactogenic formula
`tions includes one or more protease enzymes as well as car
`bohydrase enzymes, e.g., alpha-amylase and glucoamylase.
`Each enzyme is Supplied at the minimum activity specified in
`Table 1. Another embodiment includes the aforementioned
`enzymes plus the additional carbohydrase enzymes, lactase
`and invertase, that split the disaccharides lactose and Sucrose,
`respectively, releasing the monosaccharides glucose, galac
`tose and fructose. The diet of the person or animal being
`treated dictates which embodiments to utilize; for example, a
`diet lacking lactose would not require inclusion of lactase.
`Bifidogenic Enzyme Formulations (BEF)
`0019. The bifidogenic formulations herein promote
`growth of Bifidobacterium probiotics such as Bifidobacte
`rium lactis HNO19, BL-04 and Bifidobacterium bifidum
`BB-12 as exemplified herein, but can also be used to promote
`growth of other Bifidobacterium probiotic species, e.g., B.
`animalis, B. breve, B. longum, and B. infantis. The mode of
`action may involve two separate actions: 1) The release of
`performed prebiotics (such as fructooligosaccharides or
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`US 2008/O 187525 A1
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`Aug. 7, 2008
`
`FOS) present in fruits, vegetables and whole grains, that are
`otherwise trapped in a cellulose matrix that makes them less
`bioavailable: and 2) The enzymatic cracking of hemicellulose
`and other non-cellulose polysaccharides into oligosaccha
`rides that have prebiotic activity. Therefore, one embodiment
`of the bifidogenic formulations includes at least one cellulase
`enzyme and hemicellulase in amounts (activity units) speci
`fied in Table 1, where the cellulase releases existing prebiotics
`while the hemicellulase cracks non-cellulose polysaccha
`rides, both of which are bifidogenic effects. Another embodi
`ment includes addition of pectinase and beta-glucanase, in
`amounts shown in Table 1, to this formulation. Again, the diet
`is controlling, in that diets rich in pectins and beta-glucans
`would benefit from the inclusion of pectinase and beta-glu
`canase. Exemplary fiber-digesting enzymes in the bifidogenic
`enzyme formulation include the following: cellulase-TL
`from Trichoderma longibrachiatum (150,000 CU/g), cellu
`lase-AN from Aspergillus niger (50,000 CU/g), beta-gluca
`nase from T. longibrachiatum (3,000 BGU/g), hemicellulase
`from A. niger (400,000 HCU/g), pectinase from A. niger
`(500,000 AJDU/g), and Xylanase from T. longibrachiatum
`(150,000 XU/g). Activity units in parenthesis are those of the
`stock enzyme concentrates, purchased commercially, and are
`not necessarily the activities in the final formulations. All
`enzymes are dry powders available from Bio-Cat, Inc., Troy,
`Va.
`0020 Standardization, mixing and packaging for BEF for
`mulations are essentially the same as noted above for LEF.
`Lactogenic and bifidogenic formulations generally will both
`be included in the final formulation since most commercial
`probiotic formulations contain both Lactobacillus and bifi
`dobacteria.
`
`Polysorbate Formulations
`0021. The polysorbate formulations of this invention con
`tain food grade polysorbate surfactants: Polysorbate-60,
`polysorbate-80 or any polysorbate with an HLB>12, where
`HLB is the hydrophile-lipophile balance, designated from 1
`to 20. Surfactants having HLB values greater than 12 are
`more hydrophilic than lipophilic and produce oil in water
`emulsions. The chemical name for Polysorbate 60 is poly
`oxyethylene sorbitan monostearate, having an HLB of 14.9.
`polysorbate-80 is polyoxyethylene sorbitan monooleate, hav
`ing an HLB of 15.0. A source for suitable polysorbates are the
`Tweens, specifically Tween-60 and Tween-80, available from
`ICI Specialties, Wilmington, Del.
`0022 Polysorbates are oily, sticky, non-aqueous liquids
`that are not compatible with dry enzymes. Mixing them
`directly with dry enzymes destabilizes the enzymes resulting
`in shortened shelf life as exemplified herein. Therefore, dry
`polysorbate products that will result in shelf stable formula
`tions when mixed with dry enzymes are preferred. Polysor
`bates such as Tween-60 or Tween-80, are not available com
`mercially in dry form and it is not possible to dry liquid
`polysorbates by heating them, as heat causes decomposition.
`It has been discovered that polysorbates can be dried by
`absorbing them into powdered silicates, e.g., alumnosilicates,
`silicas, food Starches, or combinations thereof, which
`involves mixing a liquid polysorbate directly into the pow
`dered material.
`0023 Drying Polysorbates
`0024. Although a variety of absorbent substances such as
`clays, starches, and hydrocolloid gums can be used to pro
`duce dry-absorbed polysorbate products suitable for formu
`
`lations herein, one formulation is advantageous, as it results
`in a dry powder with a polysorbate concentration of 35% or
`more. That formulation is made as follows: Polysorbate in the
`form of Tween-80 or Tween-60 is slowly poured into calcium
`silicate (Hubersorb-600 from J.M. Huber Corp., Harve de
`Grace, Md.) that is under constant agitation in a Hobert type,
`double-action, rotary mixer. Seventy (70) grams of polysor
`bate is added per 30 grams of Hubersorb-600 over a period of
`30 minutes while mixing at 100 RPM; resulting in a mixture
`that is dry but somewhat lumpy. To each 100 grams of this
`Polysorbate-Hubersorb mixture 20 grams of silica (Syloid 63
`FP. W.R. Grace, Columbia, Md.) and 80 grams of potato
`starch (Perfectamyl D6, Avebe) are slowly added with con
`stant mixing, typically done in the same mixer, and requiring
`about 30 minutes at 100 RPM. The final formulation contains
`per 100 grams: 35 grams Tween-80, 40 grams potato starch,
`15 grams Hubersorb-600 and 10 grams silica. The potato
`starch is dried under an infrared lamp prior to use for 7 hours
`at 220 F to remove approximately 14% of the moisture.
`0025. The resulting dry powder has a polysorbate concen
`tration of 35% and a water activity of less than 0.04 and is
`referred to herein as DP35 (Dry Polysorbate 35%). DP35 and
`can be blended with the enzyme formulations of the present
`invention without adversely affecting shelf life at room tem
`perature (65-85 F), or can be administered separately, in
`capsules for example, in a kit containing the enzyme and
`probiotic formulations.
`0026. The concentration of polysorbate required to effect
`an enhancement of enzyme activity that concomitantly
`results in an enhancement of probiotic colonization or IPC
`ranges from 0.02 to 0.2% of the weight of the food matrix
`(food or test diet) being consumed, and is equivalent to 0.057
`to 0.57% DP35. The DP35 is either mixed with the enzyme
`formulations at 10-80% by weight, or is supplied separately.
`0027. The advantages that polysorbates confer can be real
`ized by orally ingesting liquid polysorbates; for example, by
`adding polysorbates to fruit juice or Some similar beverage, or
`by encapsulating the polysorbates in sealed pharmaceutical
`grade capsules Suitable for liquids. However, when adminis
`tered separately, they may cause digestive upset or an
`unpleasant after taste.
`(0028. Enzyme Strength in LEF and BEF Formulations
`0029. Each enzyme must have a certain minimum activity,
`measured as activity units per administered dose, in order to
`achieve significant probiotic enhancement. This holds true
`whether the enzymes are administered individually or incom
`bination with other enzymes, however, the activities can be
`additive. Thus, when compounding enzyme mixtures, for
`example, a lactogenic enzyme formulation containing three
`protease enzymes all at the minimum recommended activity
`per dose (see Table 1) will generate more protein digestion
`than any one of the proteases used alone at its recommended
`minimum activity. Thus, formulations can be developed uti
`lizing in vitro probiotic models, where a given formulation
`can be designed to lit a desired dose program. For example, a
`formulation containing three protease enzymes may not need
`to be administered as frequently as one containing only one
`protease. In addition, there are other situations where a par
`ticular combination of enzymes is required to maximize pro
`biotic enhancement; for example, to release monosaccharides
`from starch, a combination of alpha-amylase and glucoamy
`lase is preferred. In this case, the minimum activity is speci
`fied for glucoamylase in Table 1. Although it will release
`monosaccharides to some extent when administered alone,
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`US 2008/O 187525 A1
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`
`since pancreatic alpha-amylase is present in the intestinal
`tract, it is is more effective when administered in combination
`with alpha-amylase.
`0030 Table 1 discloses the minimum activity required for
`each enzyme per formulation dose, where, one dose is the
`amount of formulation ingested in one oral administration.
`The frequency of oral administrations (doses) can range from
`one to four times daily, to once daily, to less frequently Such
`as once every-other-day or once or twice weekly, or other
`wise. When more than one enzyme is present in a LEF, BEF
`ora joint formulation, each enzyme should meet or exceed the
`minimum activity per dose indicated in Table 1. The activity
`units specified in Table 1 are those used by the commercial
`suppliers for these enzymes; however, there are different
`activity units and different assay procedures which can be
`used to measure the activity. The assay used often depends on
`the intended application and/or the manufacturer of the
`enzymes. However, equivalent biological activity can be
`determined by laboratory analysis. Venture Laboratories, Inc.
`in Lexington, Ky., provides enzyme analysis by different
`methods.
`
`TABLE 1.
`
`Enzyme (Minimum Activity Required Dose)
`Papain (10,000 TU = Tyrosine Units)
`Bromelain (50 GDU = Gelatin Dissolving Units)
`Fungal Protease (1,000 HU = Hemoglobin Units)
`Acid Protease (50 SAP)
`Fungal Peptidase (10 LAP)
`Alpha-Amylase (1,000 SKB = Sandstedt, Kneen and Blish Units)
`Glucoamylase (2.5 AG = Amyloglucosidase Units)
`Lactase (100 ALU = Acid Lactase Units)
`Invertase (100 SU = Sumner Units)
`Cellulase-TL (1,000 CU = Cellulase Units)
`Cellulase-AN (500 CU = Cellulase Units)
`Hemicellulase (1,000 HCU = Hemicellulase Units)
`Pectinase (1,000 AJDU = Apple Juice Depectinizing Units)
`Beta-Glucanase (50 BGU = Beta-Glucanase Units)
`Xylanase (1,000 XU = Xylanase Units)
`
`0031 Probiotic Formulations
`0032. The probiotic formulations containing Lactobacil
`lus or Bifidobacterium species or a combination thereof, have
`total viable plate counts within the range of 500 million to 1
`trillion CFU/g (colony forming units/gram). A minimum
`viable plate count per dose is about 5 billion CFUs. Viable
`plate counts are determined by procedures outlined in Stan
`dard Methods for the Examination of Dairy Products (16"
`ed.) using a modified De Man Rogosa and Sharpe agar(MRS)
`to which L-cysteine HCL is added. The counts are reported in
`colony forming units, either per gram or per dose.
`0033 Exemplary Lactobacillus species suitable for the
`formulations herein include: L. Casei, L. bulgaricus, L. fer
`mentum, L. plantarum, L. delbrukei, L. Salivarius, L. jens
`enii, L. gaserii, L. reuteri, L. helveticus, L. lactis, L. brevis, L.
`johnsonii, L. acidophilus, L. paracasei, and L. rhamnosus.
`0034 Exemplary Bifidobacterium species include: B. ani
`malis, B. breve, B. longum, B. infantis, B. lactis and B. bifi
`dum.
`0035. Formulations are generally compounded from
`freeze-dried concentrates of probiotic species from commer
`cial Sources, where the strain designations for each are indi
`cated. The following strains are suitable for use: Lactobacil
`lus acidophilus LA-1. Lactobacillus rhamnosus HNO01,
`Lactobacillus rhamnosus Lr-32, Lactobacillus casei 163,
`
`Bifidobacterium lactis HNO19 and Bifidobacterium lactis
`BL-04 from Danisco USA, Inc. Madison, Wis. Bifidobacte
`rium bifidum BB-12 from Chr. Hansen, West Allis, Wis. and
`Lactobacillus paracasei F-19 from Medipharm USA, Des
`Moines, Iowa). The probiotics are blended with microcrys
`talline cellulose (Avicel PH 112, FMC) and silica (Syloid 63
`FP. W.R. Grace) to achieve the desired CFU/g. The amount of
`silica generally should not exceed 2% by weight. Blending is
`preferably carried out under dry conditions (relative humidity
`about 20%) in a Patterson Kelly type, twin-cone mixer until a
`uniform mixture is obtained (usually requires 10-15 minutes
`at 100 RPM). The wateractivity of the blend should be below
`Aw-0.03 for optimum shelf life. Magnesium stearate, phar
`maceutical grade, is added at 2% by weight to formulations
`which are intended for filling into hard capsules or tableting.
`0036 Water activity (Aw) is equivalent to the relative
`humidity generated in a closed container containing the
`sample multiplied by 0.01, or 1.0% relative humidity=0.01
`Aw. Measurement of Aw in the examples herein was con
`ducted using a Rotronic Hygrometer Model A2 from
`Rotronic Instrument Corp., Huntington. N.Y.
`0037. The following examples will further illustrate the
`present invention but are not intended to limit the scope of the
`invention.
`
`EXAMPLE NO. 1
`
`0038 I) Lactobacillus acidophilus LA-1 was inoculated at
`one (1) million CFU/ml into 500 ml of sterile broth
`medium containing 2% casein, 2% corn starch, 0.1% cal
`cium carbonate, 0.1% magnesium carbonate, and 0.05%
`yeast: extract (Difico); a medium in which it is difficult to
`achieve significant Lactobacillus growth but which con
`tains typical nutrients found in human diets. The inoculated
`broth (contained in 1L Erlenmeyer flasks) was incubated at
`37 C for 24 hours and then plated on MRS agar to deter
`mine CFU/ml. Result (average of triplicate samples): 12
`million CFU/ml.
`0039 II) The test in (I) was repeated where the medium
`also contained 0.1% Tween-80. Result: 14 million CFU/
`ml.
`III) The test in (I) was repeated where the medium
`004.0
`was pre-treated with digestive enzymes for 24 hours prior
`to sterilization at 121 C for 20 minutes at 15 psi. The
`pre-treatment involved adding 250 mg of a lactogenic
`enzyme mixture containing papain (10,000 TU), brome
`lain (50 GDU), alpha-amylase (1,000 SKB) and glucoamy
`lase (2.5 AG). Result: 87 million CFU/ml.
`0041 IV) The test in (III) was repeated where 0.1%
`Tween-80 (as DP35) was added during the enzyme pre
`treatment procedure. Result: 128 million CFU/ml.
`0042 Conclusion: Lactogenic enzyme pre-treatment sig
`nificantly stimulates the growth of L. acidophilus LA-1 and
`enzyme pre-treatment plus Tween-80 yields even signifi
`cantly stimulation.
`
`EXAMPLE NO. 2
`
`0043. The procedures of Example 1 were repeated with
`Lactobacillus rhamnosus HNO01 substituted for Lactobacil
`lus acidophilus LA-1. The results are as follows: Test I): 6
`million CFU/ml. Test II): 7.5 million CFU/ml. Test III): 62
`million CFU/ml. Test IV): 106 million CFU/ml.
`
`

`

`US 2008/O 187525 A1
`
`Aug. 7, 2008
`
`0044 Conclusion: Lactogenic enzyme pre-treatment sig
`nificantly stimulates the growth of L. rhamnosus HNO01 and
`enzyme pre-treatment plus Tween-80 yields significantly
`greater stimulation.
`
`EXAMPLE NO. 3
`0045. The procedures of Example 1 were repeated with
`Lactobacillus paracasei F-19 substituted for Lactobacillus
`acidophilus LA-1. The results are as follows: Test I): 14
`million CFU/ml. Test II): 19 million CFU/ml. Test III): 88
`million CFU/ml. Test IV): 148 million CFU/ml.
`0046 Conclusion: Lactogenic enzyme pre-treatment sig
`nificantly stimulates the growth of L. paracasei F-19 and
`enzyme pre-treatment plus Tween-80 yields significantly
`greater stimulation.
`
`EXAMPLE NO. 4
`0047 V) The procedures of Example 1 were repeated with
`Lactobacillus acidophilus LA-1 except that the enzyme
`pre-treatment step was as follows: The pre-treatment
`involved adding 250 mg of a lactogenic enzyme mixture
`containing fungal protease (1,500 HU), acid-protease (50
`SAP), fungal peptidase (10 LAP), alpha-amylase (1,000
`SKB) and glucoamylase (2.5 AG). In addition, Tween-60
`was used in place of Tween-80. The results are as follows:
`Test I): 10 million CFU/ml. Test II):9 million CFU/ml. Test
`III): 74 million CFU/ml. Test IV): 108 million CFU/ml.
`0048 Conclusion: This different lactogenic enzyme pre
`treatment also significantly stimulates the growth of L. aci
`dophilus LA-1 and enzyme pre-treatment plus Tween-60
`yields significantly greater stimulation.
`
`EXAMPLE NO. 5
`0049 I) Bifidobacterium lactis BL04 was inoculated at
`two (2) million CFU/ml into 500 ml of sterile broth
`medium containing 2% casein, 1% rye flour, 1% asparagus
`powder (ground freeze-dried asparagus), 1% barley flour,
`0.1% calcium carbonate, 0.1% magnesium carbonate, and
`0.05% yeast extract (Difco); a medium in which it is gen
`erally difficult to achieve significant Bifidobacteria growth
`but contains typical nutrients found in human diets. The
`inoculated broth (contained in 1 L Erlenmeyer flasks) was
`incubated anaerobically at 37 C for 24 hours and then
`plated on MRS agar plus L-cysteine HCL to determine
`CFU/ml (plates were incubated anaerobically). Result (av
`erage of triplicate samples): 8 million CFU/ml.
`0050 II) The test in I) was repeated where the medium
`also contained 0.025% Tween-80. Result: 3 million CFU/
`ml.
`III) The test in I) was repeated where the medium
`0051
`was pre-treated with digestive enzymes for 24 hours prior
`to sterilization at 121 C for 20 minutes at 15 psi. The
`pre-treatment involved adding 250 mg of a bifidogenic
`enzyme mixture containing cellulase-TL (1,000 CU),
`hemicellulase (1,000 HCU), beta-glucanase (50 BGU),
`papain (10,000 TU) and bromelain (50 GDU). Result: 74
`million CFU/ml.
`0052 IV) The test in III) was repeated where 0.025%
`Tween-80 was added during the enzyme pre-treatment pro
`cedure. Result: 98 million CFU/ml.
`0053 Conclusion: Bifidogenic enzyme pre-treatment of a
`medium containing largely prebiotic carbohydrate Sub
`stances significantly stimulates