This application comprises a divisional out of the parent Indian Patent Application No. 2859/DELNP/2004.
This invention relates to compositions containing bioactive substances and methods for administiation thereof.
Background
Bioactive substances, which may have a powerful physiological effect, are frequently very sensitive to hostile environments such as the mammalian gastric environment, a high temperature environment, or a high-humidity environment. This frequently limits their use and means they cannot be conveniently administered orally, or they can not be conveniently stored, or they can not be convenientiy applied on a warm product line.
The bioactive substances may be selected from the group including growth promoters, antlneoplastic agents, oral vaccines, lnhaiants, living microorganisms ;for example protobiotics such as Lactobacillus spp}, peptides, poiypeptides, nuvieotldes, poiynucleotldes, nucsecssdes, proteins, g'vcoproieins, sugars and complex carbohydrates. anti-infectants, antirnicrobia-s, disinfectants, antiseptics, anildepressants, psycnoactive agents, genetically modified organisms and infectious agents used as vectors for other bioactive substances eg bacteria! vectors (including E. coii, Salmonella, Vibrio, LactobacilH, Bacilius, Mycobacteria, Shlgelia), viral vectors (Including Adenovirus, Poxvirus, BacculovJrus, Herpesvirus, Enterovirus, Paramyxovirus and Orthomyxovirus), plant vectors (including tobacco, potato and banana), yeast vectors, immunoglobuiins or affinity purified immunoglobulins including antibodies directed against diseases and disease causing agents (for example Heiicobocter pylori, E. coii, Bacillus spp, pathogenic Yersinia spp., and al'erqens} and fragments, -derivatives and complexes containing any of the above,
Examples of hositile regions which compromise the ''unction of bioactive substances include highty acid or hiqhty akaline regions. regicns which contain

proteolytic enzymes, regions in which desiccation occurs, regions of high humidity, regions of high temperature, regions in which elevated pressure leads to denaturation eg a tableting machine and regions containing DNA-ases.
A particularly hostile region for compromising the function of bioactive substances is a mammalian gastric environment, which contains areas of high acid conditions, elevated temperature, moisture, high concentrations of proteolytic enzymes and carbohydrate digesting enzymes.
A particular application of this invention is to the preservation of bioactive agents whose biological function is compromised in a mammalian gastric environment.
A number of methods to preserve bioactive function in a mammalian gastric environment are known to the art. These include enteric coatings, buffering, formalin stabilisation, addition of antisecretory agents, giving the substance in conjunction with a meal and altering the immunochemistry of cows to cause the secretion of bioactive materials into colostrum.
Freichel OL and Lippold BC (International Journal of Pharmacology, 2001, March 23; 216(1-2): 165-9) suggest the use of an enteric coating comprising methylhydroxy ethyiceltulose and hydroxypropyl rnethlyceilulose acetate succinate to provide a coating that protects its contents in mammalian stomachs.
Jacket C et al (The New England Journal of Medicine,1986, May 12, 1240-1243) used a buffering solution of bicarbonate of soda to decrease proteolysis in the human stomach.
Paliwal R and London E (Biochemistry 1996, Feb 20; 35 (7): 2374-9) describe the use of formalin treatment to stabilise the conformation of diphtheria toxoid.

Asad M et a\ (Life Science 2001, Nov 21; 70(1):17-24) used oxytocin and rantinide respectively to decrease acid secretion in the stomach of mammals to increase gastric ulcer healing rates.
McClead and Gregory, Infections and Immunity 44: 474-478 have shown that specific proteins secreted by cows into colostral milk are more stable in the gastric environment than would be expected when proteins of the same class are produced by alternative processes This principle of preserving bioactive function has been utilised by Abbot US Patent 5260037, who immunised cows prior to harvesting of colostral milk. The immunisation leads to the secretion of specific antibodies (eg directed against Helicobacter pylori} into colostral milk (this is called hyperimmune colostrum).
Hyperimmune bovine colostrum has also been described in International Application PCT/AU94/00562 to be stable in terms of biological function in high pressure environments for example in a tabieting machine. The use of hyperimmune bovine colostrum has also been taught by:
» Mitra et al, Acta Paediatrics, 84: 996-1001, 1995. Hyper immune cow colostrum reduces diarrhoea due to rotavirus: a double blind, controlled clinical study.
» Nord et al, AIDS 4, 581-584, 1990. Treatment with bovine hyperimmune colostrum of cryptosporidial diarrhea in AIDS patients.
• Tacket et al, New England Journal of Medicine. Ma7 12, 1988.
Protection by milk immunoglobulin concentrate against oral challenge
with enterotoxigenic E. coll
* Tacket et al, American Journal of Tropical Medical Hygiene. 47 (3), 276-
283. 1992. Efficacy of Bovine milk immunoglobulin concentrate in
preventing illness after Shigella flexeri challenge.
In the above prior art the hyperimmune colostrum was the source .of bioactive material and subsequent operations (if any) involved the removal of undesirable components eg water, fat, cellular material, bacteria and lactose. There is no teaching that the addition of colostrum or colostrum extract or

colostrum components leads to enhanced protection of the function of the bioactive substance in a hostile environment.
Problems associated with the above methods for preserving bioactive function in a mammalian gastric environment include the following:
The strategy of using enteric coatings is inappropriate for delivering agents which act in the stomach. This is a particular limitation with respect to the control of Helicobacter pylon infection (which is a significant cause of gastritis) by oral vaccines and/or the use of antibodies directed against H. pylori in a passive immunity approach.
The strategy of using buffer solutions .to decrease proteolysis in a mammalian stomach is inappropriate when the loss of function in the bioactive material is occasioned by the action of enzymes eg peptidases, amylases. Such1 enzymes are frequently associated with loss of function in protein therapeutics and in therapeutics based on living organisms. Furthermore, the use of buffers such as bicarbonate on a regular basis is undesirable.
The use of formalin treatment (or other cross-linking strategies) to preserve bioactive function in a hostile environment is frequently ineffective when the cross-linking reaction destroys the structure and function of the substance. For this reason this technique is rarely used except present a surface antigen to stimulate an immune reaction (eg Diphtheria toxin as discussed by Petre et al Developmental Biologicai Standards 1996; 87: 125-34).
The method of encouraging the secretion of bioactive material by cows into colostra! milk has a number of limitations.
Some bioactive materials are difficult or impossible for cows to secrete into colostrum.
The amount of bioactive material is variable and this is frequently unacceptable in quality control terms.

The maximum amount of specific bioactive material is also low and typically less than 5% of immunoglobulins in colostrum. The acceptable maximum volume in a regimen of tablets is 0.5 cubic centimetre per tablet (2 to 3 tablets per day is. acceptable). Low concentrations of specific antibodies severely limit therapeutic options.
Furthermore the immunised cows that produce colostrum also produce milk for human consumption. This presents difficulties in terms of regulatory and food safety issues.
Furthermore, colostrum is harvested only once a year from a cow at calving and this causes problems in logistics and costs of production, particularly if the bioactive ingredient is secreted by the cow into the colostrum.
Summary
We have made the surprising discovery that it is possible to preserve the function of a labile bioactive substance by combining the labife bioactive substance with mammalian colostrum or components thereof in an in vitro (outside the body) mixing operation. The mammalian colostrum may be a processed form of mammalian colostrum.
The invention accordingly provides a composition for administration of a labile bioactive substance comprising a mixture of the labile bioactive substance and a mammalian colostrum.
A labile bioactive substance is a bioactive substance which is functionally impaired in its environment of use particularly in the case of hostile environment such as a mammalian stomach or rumen, a high temperature environment or high humidity environment.
In a further aspect the invention provides a method of administering a labile bioactive substance (for example a bioactive substance which is functionally

impaired in the mammalian stomach or other hostile environment) comprising forming a mixture of the bioactive substance and mammalian colostrum and applying the composition to an environment under which the bioactive substance is otherwise labile such as a gastric environment or other hostile environment.
In one preferment the composition is administered orally.
The invention further provides a method for using a bioactive substance which is labile, that is functionally impaired, in the mammalian stomach or other hostile environment, in the manufacture of an ingestible medicament for treating or preventing disease, comprising mixing the therapeutic substance with mammalian colostrum or components thereof.
Although greater than expected stability of substances secreted by cows into colostrum has been noted by many workers, there has been no suggestion that colostrum or processed colostrum can be added to bioactive materials to preserve their function in the stomach or rumen or other hostile environment.
Preferably the bioactive substance is a substance capable of causing a measureable change in a physiological or pharmaceutical parameter of an organism.
in one particularly preferred embodiment the bioactive substance is an antibiotic. Accordingly the invention provides an antibiotic composition comprising an antibiotic and mammalian colostrum and optionally excipients.
In a further aspect of the invention we provide a probiotic composition comprising a probiotic bacterium such as Lactobacillus spp. and mammalian colostrum.

The invention is preferably used with a bioactive substance which shows at least 20% diminution in function after the substance has been incubated for 60 minutes at 37°C in a liquor comprising a 0.32% solution of porcine pepsin in 0.03 M NaCI and adjusted to pH 1.2 with HCI.
Preferably the mammalian colostrum is bovine colostrum retained from the first 4 days post parturition, more preferably bovine colostrum retained from the first 2 days post parturition, even more preferably bovine colostrum retained from the first day post parturition, and most preferably bovine colostrum retained from the first milking post parturition.
The term colostrum where used herein includes colostral milk; processed colostra! milk such as colostral milk processed to partly or completely-remove one or more of fat, cellular debris, lactose and casein; and colostral milk or processed colostral mi!k which has been dried by for example, freeze drying, spray drying or other methods of drying known in the art. Coiostral milk is generally taken from a mamma! such as a cow within five days after parturition.
Preferably the mammalian colostrum has been processed using a defatting operation, more preferably using a defatting operation and an operation to remove cellular debris, more preferably a defatting operation, an operation to remove cellular debris and an operation to remove salts, sugars, other low molecular weight entities and some water.
The colostrum and or bioactive material may be in dried form. The components may be intimately mixed before, during or after the drying process.
Preferably the bioactive substances and mammalian colostrum are mixed, and the mixture is an aqueous mixture which may be dried preferably by lyophilization.

In one preferment a mixture of the bioactive substance and colostrum extract is lyophilised and at least half of the lyophilised material by weight comprises added colostrum or processed colostrum. In another preferment at least three quarters of the lyophilised material by weight comprises colostrum or processed colostrum.
Preferably the bovine colostrum collected from the cow comprises at least 4% total protein (weight %), more preferably 5%, more preferably at least 8%, more preferably at least 10%.
Preferably the ratio of IgG to total protein of the colostrum collected from the cow is at least 10%, more preferably 20%.
Preferably the bioactive substance is selected from the group consisting of growth promoters, antineoplastic agents, oral vaccines, inhalants, living microorganisms (for example protobiotics such as Lactobaciilus spp), peptides, polypeptides, nucleotides, polynucleotides, nucleosides, proteins, glycoproteins, sugars and complex carbohydrates, anti-infectants, antimicrobials, disinfectants, antiseptics, antidepressants, psychoactive agents, genetically modified organisms and infectious agents used as vectors for other bioactive substances eg bacterial vectors (including E. coli, Salmonella, Vibrio, Lactobacilli, Bacillus, Mycobacteria, Shigella), viral vectors (including Adenovirus, Poxvirus, Bacculovims, Herpesvirus, Enterovirus, Pararnyxovirus and Qrthomyxovirus), plant vectors (including tobacco, potato and banana), yeast vectors, immunoglobulins, affinity purified immunoglobulins including antibodies directed against diseases and disease causing agents (for example Heticobacter pylori, E. coli, Bacillus spp, pathogenic Yersinia spp., and allergens) and fragments, derivatives and complexes containing any of the above.
It is particularly preferred that the bioactive material comprises monoclonal or polyclonal immunoglobulins or chimeric monoclonal antibodies or humanised monoclonal antibodies or dendrimer presented immunoactive fragments or immunoactive fragments such as F(ab) and F(ab)2 fragments or recombinant immunoactive fragments, or affinity purified immunoglobulins or immunoactive

fragments thereof. These immunoglobulins or fragments thereof may bind to pathogenic organisms including Helicohacter pylori, Enterotoxigenic E.coti, Yersinia pestis, entemvirus 71.
The proportions of colostrum and bioactive substance in the compositions of the invention wiil depend on the nature of the active substance and the degree to which it is sensitive to conditions in the stomach. Typically the weight ratio of colostrum to active substance is greater than 0.5:1, more preferably greater than 2;1, even more preferably greater than 5:1. The upper limit of the added colostrum component is constrained by the practical limit of the amount of therapeutic substance which can be conveniently administered.
The composition of the invention may be administered in a range of forms such as capsule, powder tablets, aerosol spray, syrup, liquid or other form known in the art. The composition may further comprise carriers or excipients suitable for gastrointestinal administration. Examples of carriers and excipients include: silica, talc, titanium dioxide, alumina, starch, kaolin, powdered cellulose, microcrystailine cellulose, Arnylopectin N, sucrose, lactose, dextrose, polyvinylpyrroitdone, hydroxypropyl cellulose, methyl cellulose, hydroxyethy! cellulose, carboxymethyl cellulose, citric acid, sodium bicarbonate, magnesium stearate, shellac, cellulose acetate, cetyl alcohol, triethyl citrate, polyethylene glycol.
In another particular preferment the bioactive material comprises irnmunogenic proteins, glycoproteins or other components of a vaccine or organisms which express components of a vaccine. The vaccine may be directed at a pathogenic organism for example Helicobacter pylori, Enterotoxigenic E.coli, Yersinia pestis..
In a particularly preferred embodiment the bioactive substance is selected from
the group consisting of:
(a) antibodies and fragments thereof against the disease organisms
H.pylori, enterotoxic E. colt (ETEC), pscorna viruses especially
Enteroviruses rotavirus anthrax and Yersinia pestis;

(b) antibodies and fragments thereof against toxins produced by H. pyion,
ETEC, anthrax, Yersinia pestis and antibodies against ricin; and
(c) oral vaccines and inhalation vaccines against H. pylori, ETEC, picorna
viruses especially Enteroviruses, rotavirus, anthrax, Yersinia pestis.
In a particularly preferred embodiment the invention provides a method of treatment or prophylaxis of gastrointestinal disease in a patient comprising administering to the patient a composition comprising (a) a vaccine directed against a pathogenic microorganism selected from H.pylori and coliforms and (b) mammalian colostrum. The colostrum will generally be present in an amount sufficient to enhance the activity of the vaccine in the gastric environment.
In another particularly preferred embodiment the invention provides a method of treatment or prophylaxis of gastric ulcers or H. pylori infection in. a patient comprising administering to the patient a composition comprising a binding protein directed to H. pylori and further comprising marnmaiian colostrum.
in one preferment the immunoglobuSins or fragments thereof are derived from eggs, preferably from hens immunised with killed vaccines against pathogens.
The invention is particularly useful for delivering bioactive agents which act in the stomach. For example agents against He//cobacferpy/o/7 infection. However even bioactive substances used to control respiratory pathogens in the form of an inhaled spray may lose activity when placed in simulated gastric fluid or other hostile environment. The colostrum or processed colostrum may protect against this loss of function, and this protective effect is expected to be beneficial when the bioactive substance is delivered as an inhaled spray.
The invention is useful for protecting bioactive agents from proteoiysis occasioned by enzyme action as well as proteoiysis occasioned by low pH conditions.

The field of probiotics has developed rapidly over the last 10 years. Probiotics comprise beneficial bacteria that may out complete harmful bacteria in the gastrointestinal tract. The indigestion of friendly bacteria discourage the presence of Lactobacillus spp, arid Bifidobacter spp are important bacteria used in commercial probiotics sold in Australia. Lactic acid production in these bacteria discourage the harmful bacteria which do not thrive in acid environments, Lactobaciiius may also produce specific antibiotics. For example Lactobacillus acidophiius produces acidophiline and L.bugaricus produced bulgarican. Bifidobacterium can also be used as a probiotic organism. It has been reported that probiotics aid in digestion, assist in cleaning the gut and promoting regular bowel movements, contribute to the destruction of mould, viruses and parasites and balance intestinal pH.
Probiotics are also useful in treatment or prophylaxis of acid gut syndrome resulting from accumulation of acid and production of endotoxin in the gastrointestinal tract.
Administration of probiotics orally can result in significant loss of activity as a result of the probiotic passing through the severe conditions in the stomach. We have found that a greater level of activity of the probiotic is preserved when administered orally in accordance with the invention.
The probiotic may contain excipients and adjuvants and may be in tablet, powder, capsule or liquid form. The invention further provides a method of treatment or propylaxis of gastrointestinal dysfunction comprising administering a composition comprising a probiotic bacteria such as Lactobacillus spp. and mammalian colostrum.
The dose of bacterium may depend on the condition of the patient and nature of the dysfunction but is typically at least 105 CFU Lactobacillus per day.
The invention is useful for preserving the function of non-cross-linked bioactive materials.

The invention enables the formulation of therapeutic agents which are significantly superior to hyperimmune colostrum in the following respects:
> The amount of bioactive material can be controlled to within tight
limits
> The amount of bioactive material, which is prepared separately to
the colostrum, is no longer subject to variable production of
imrnunoactive material by the cows. This is highly desirable from a
quality control point of view.
The amount of bioactive material that can be presented in a given
volume can be increased over that possible with hyperimmune
colostrum by a factor greater than 4. For example hyperimmune
immunoglobulin from avian eggs particularly an egg yolk from an
immunised hen can be affinity purified and F(ab}2 fragments
made. This provides significantly expanded therapeutical options.
The invention allows the production of therapeutics without
compromising the integrity of dairy production for human
consumptions. For example the bioactive active substance can be
Manufactured in the laboratory and the colostrum harvested from
normal cows.
Bovine Colostrum extract is a substance listed by the Therapeutic
Goods Authority for use as a complementary medicine in Australia
so that utilisation of colostrum in oral therapeutics is facilitated.
The invention will now be described with reference to the following examples. It is to be understood that the examples are provided by way of illustration of the invention and that they are in no way limiting to the scope of the invention.
Many of the examples are discussed with reference to results shown in the attached drawings. In the drawings:

Figure 1 is a chart showing the colostrum protection of urease activity reported in Example 1;
Figure 2 is a chart showing the colostrum protection of antibody activity reported in Example 3;
Figure 3 is a chart showing colostrum protection of Lactobacillus plantarum viability reported in Example 4;
Figure 4 is a chart showing colostrum protection of erythromycin activity reported in Example 6;
Figure 5 is a chart showing colostrum protection of Lactobacillus casei shirota viability reported in Example 7;
Figure 6 is a chart showing colostrum protection of chlolera toxin activity
reported in Example 8;
Figure 7 is a chart showing the colostrum protection of L. piantarum reported in Example 9.
EXAMPLES
Example 1;
Protection by colostrum of the activity of an enzyme within the stomach environment as an example of protection of a bioactive protein
1ME5QDUCIIJDN:
The role of bovine colostrum as a protectant of bioactive protein activity in a gastric environment was investigated using the urease enzyme. To investigate the protective properties of colostrum, the effect of simulated gastric fluid on urease activity was compared in the presence and absence of bovine colostrum.

MATERIALS AND METHODS: Reagents
Urease: Sigma Jack Bean Urease Type III (Cat No U-1500) was used as the source of the enzyme. The freeze-dried urease (quoted activity of 16 units per mg; one unit liberates LOμrnole of ammonia from urea per min at pH 7.0 and 25 °G) was dissolved in MilliQ water to'0.3units/(al (approx. 21 rng/ml) and stored on ice until used in the study.
Simulated Gastric Fluid(SGF): (adapted from Hilger et al, 2001). A 0.32% solution of Sigma porcine pepsin (Cat No P-7012 from stomach mucosa) was prepared in 0.03M NaCI and adjusted to pH 1.2 with HCI.
Colostrum: Defatted, freeze dried Bovine Colostrum Extract (Anadis Limited "Gastran" Batch G01) was sourced from non-immunised cows and a stock preparation was reconstituted at 300mg/ml in MilliQ water. See Example 2 for method of production of Bovine Colostrum Extract.
Treatment of Urease with Simulated Gastric Fluid (SGF)
Incubation mixtures were prepared in quadruplicate by combining aliquots of the urease solution with an equal volume of either the reconstituted colostrum or MilliQ water. A control mixture of the colostrum preparation and water was also
prepared (in duplicate).
Incubation mixtures 1 • 4:1QOi.il of urease + 100μLof MilliQ water Incubation mixtures 5 - 8:100f.il of urease + 100μL of colostrum Incubation mixtures 9 -10: 100(^1 of colostrum + 100μLof MilliQ water
Mixtures were preheated in a 37°C water bath for 5 minutes before starting treatment by the addition of 50)il of SGF to tubes 1, 2, 5, 6 and 9. Mock treatments were initiated by the addition of 50μl of 0.03M NaCI to tubes 3, 4, 7, 8 and 10. All samples were incubated for 15 minutes at 37°C before reactions were stopped by the addition of 0.25 volumes of chilled 160mM Na2CO3 to each tube. Tubes were then centrifuged for 3 minutes rat 20,800g in an Epperidorf

centrifuge 5417C and stored on ice. Supernatants were assayed for urease activity.
Urease activity was assayed using a coupled enzyme assay for increased sensitivity (modified from Kaltwasser and Schlegel, 1966). In this reaction, the urease enzyme catalyses the hydrolysis of urea:
Urea + H20 + 2H+ =* 2NH4+ + CO2
which is measured by coupling ammonia production to a glutamic dehydrogenase reaction:
2NH4' + 2 α~ketoglutarate + 2 NADH =» 2 glutamate + 2NAD+ + 2H2O
The reaction is followed by the oxidation of NADH to NAD.
The final assay volume of 1ml contained final concentrations of 1.6mM α-ketoglutarate (Boehringer Mannheim Cat No. 127 205), 1.5mM NADH (Sigma p-NADH Cat No. N-8129), 15 units/ml of L-glutamic dehydrogenase (Sigma Cat No. G-4387), 10mM Urea (Boehringer Mannheim Cat No. 100 164) and 1mM sodium sulphide (Sigma Cat No S-4766) in 50mM Tris-HCI buffer (pH 8.0). These reagents were mixed in 1cm path length polystyrene cuvettes (Sarstedt Cat No 67.742) and then allowed to equilibrate for several minutes to room temperature in a Beckman DU70 recording spectrophotometer. The speetrophotometer was zeroed using the above mixture, before the addition of the sample.
A ten-microlttre sample of the supernatant of each incubation mixture was added to the assay mixture to start the assay. The reaction rate was recorded every 10 seconds at 340nm for up to 4 min at RT. Reaction rate was calculated from the linear portion of curve (generally after first 1-2 mins) and urease activity was then expressed as μmole of urea hydrolysed per min per mg of protein,

RESULTS
(Table Removed)
As shown in the table of results and figure 1, the addition of simulated gastric fluid to a diluted urease solution resulted in the irreversible denaturation of the enzyme with subsequent loss of activity. The remaining low level of urease activity after SGF was equivalent to that found in the colostrum alone (no urease) controls, NOTE: "MOCK" TREATMENT is a treatment where saline rather than simulated gastric fluid (SGF) is used.
In contrast, the addition of colostrum to the urease solution provided protection of the urease from the effect of the pepsin and acid, in the presence of colostrum, the protected urease retained approximately 80% of the activity of the mock treated urease/colostrum mixture, Using this enzyme model, bovine colostrum has been shown to provide protection of protein activity in a simulated gastric environment.
REFERENCES
Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Hentges F (2001), Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. Clin Exp Immunol, 123:387-394.
Kaltwasser H and Schlegel HG (1966). NADH-dependent coupled enzyme assay for urease and other ammonia-producing systems. Analytical Siochem, 16:132-138.

Scott DR, Weeks D, Hong C, Postius S, Melchers K and sacns a (1998). me role of internal urease in acid resistance of Helicobacter pylori. Gastroenterology, 114: 58-70.
Example 2:
Method of preparation of processed bovine colostrum powder
The following diagram shows the principles used to take colostrum and convert it to a processed form.
(Diagram Removed)

The raw colostrum is collected from dairy cows most preferably at the first milking after calving. The colostrum is stored at 4°C on farm and then transported either for ionger term storage at -20°C or sent directly to wet manufacturing.
The raw colostrum is warmed to approximately 37°C and then skimmed with a rotary milk separator to remove fat. The resultant liquid may be pasteurised or microfiltered with a 7-10 micron ceramic filter system (to remove bacteria and debris. The liquid is then Ultrafiitered (for example in a Abcor 10m2 Uitrafiltration plant) to remove a majority of the water, lactose and electrolytes leaving a high protein concentrate. The resultant high protein concentrate is further processed preferably by lyophilization (freeze-drying) or spray-drying.

The above method yield a processed bovine colostrum powder with the specifications as below. This product as defined below is listed as a substance suitable for inclusion in therapeutic goods by the Therapeutic Goods Authority of Australia.
Appearance: Free-flowing, pale yellow powder.
Properties : Dispersible in water. Mild odour of milk when contacted with moisture.
Moisture Range 2 to 5 % m/m AS 2300.1.1 (1988) Fat Range 1 to 4 % m/m AS 2300.1.3 (1988) Ash(@550°C) Not more than 8 % m/m AS 2300.1.5 (1988) Total Nitrogen (TN) For information** AS 2300.1.2 (1991) Non-protein nltrog'en(NPN) For information** AS 2300.1.2.2 (1988) True protein Not less than 60 % rn/m (TN-NPN)% x 6.38 Protein Not less than 60 % m/m AS 2300.1.2 (1991)
Lactose (monohydrate) Not more than 15 % rn/m
UV assay following enzymatic hydrolysis and oxidation
(Boehringer Mannheim)
Total immunoglobuiins Not less than 20 % m/m Radial immunodiffusion assay
(m/m means mass of component by total mass of composition)
Microbial limits Complies with TGA guidelines
Residues: Heavy metals Agricultural and Veterinary chemicals
Subject to ANZFA Food Standards Code for daisy products. Where there is no applicable Food Standard, the BP test for heavy metals applies (2 ppm calculated as lead) and also She BP requirements for pesticide residues.
** Used to calculate the value for true protein

'AS' refers to document of the Australian Standards Organisation series of 'Australian Standards' - in this case referring to standardised methods of quality and component testing for dairy products.
Example 3
Colostrum protection of antibody activity during acid/pepsin treatment
The role of bovine colostrum as a protectant of protein activity in a gastric environment was investigated using an antibody model. To investigate the protective properties of colostrum, the effect of simulated gastric fluid on the bioactivity of an influenza virus specific monoclonal antibody was compared in the presence and absence of bovine colostrum.
MATERIALS AND METHODS .Reagents
Influenza Virus and Specific Monoclonal Antibody:The TYPEA influenza virus -
used in this study was Mem7lH-BelN (H3N3). The antibody used was a Mem7lH-BelN specific !gG2a monoclonal (Mab 36/2) diluted 1:10 in phosphate
buffered saline.
Simulated Gastric Fluid (SGF): (adapted from Hilger et al, 2001). A 0.32% solution of Sigma porcine pepsin (Cat No P-7012 from stomach mucosa) was prepared in 0.03M NaCi and adjusted to pH 1.2 with HCI.
CplQs.tr.unx Defatted, freeze dried Bovine Colostrum Extract (Anadis Limited "Gastran" Batch (301) was sourced from non-immunised cows and a stock preparation was reconstituted at 300mg/mL in MilliQ water.
TrejitjiienX^
Incubation mixtures were prepared by combining aliquots of antibody diluted 1:10 in phosphate buffered saline (PBS) with an equal volume of either the reconstitute*'! colostrum or MilliQ water. A control mixture of the colostrum preparation and water was also prepared.

incubation mixtures 1-2: 100μ1 of Mab 36/2 +100μl of MiiliQ water Incubation mixtures 3-4: 100μ1 of Mab 36/2 + 100μl of colostrum Incubation mixtures 5--6: 100μ.l of colostrum + 100μl of MiltiQ water
Mixtures were preheated in a 37°C water bath for 5 minutes before starting treatment by the addition of 50μl of SGF to tubes 1, 3 and 5. Mock treatments were initiated by the addition of 50μl of 0.03M NaCI to tubes 2,4 and 6. All samples were incubated for 15 minutes at 37°C before reactions were stopped by the addition of 0,25 volumes of chilled 160mM Na2CO3 to each tube. Tubes were then centrifuged for 3 minutes at 16,100g in an Eppendorf centrifuge 5415D and stored on ice. Supernatants were assayed for antibody activity.
Haemagglutination lnhibition Assay
Antibody activity was assayed using the haemagglutination inhibition (Hi) assay. Haemagglutination titrations and HI assays were performed by standard procedures with 96 well U-bottorned rnicrotitre plates (Sarstedt Group, SA, Australia) and 1% (vol/vol) chicken eryihrocytes. The haemagglutination assay was used to quantitate the haemagglutinating units (HAU) of the virus used for the HI assay. Each incubation mixture was diluted 1:3.2 in PBS which would result in a starting concentration of 1:100 of Mab 36/2. incubation mixtures were then serially diluted two-fold in duplicate wells across the microtitre plate for a final volume of 25 μL per well. An equal volume of virus at a concentration of 4 HAU was added to each well and antibody and virus incubated for 30 minutes at room temperature. After 30 minutes 25 uL of chicken eryihrocytes (1% vol/vol in PBS) was added to each well and incubated for 30 minutes. The HI titre was taken as the reciprocal of the highest antibody titre inhibiting 4 HAU of virus. The assay was repeated twice in duplicate.

RESULTS
(Table Removed)

As shown in the table of results and Figure 2, the treatment of Mab 36/2 with simulated gastric fluid resulted in a 6-foid reduction in the haemagglutination inhibition activity of the antibody.
In contrast, the addition of colostrum to the antibody preparation provided protection of the antibody from the "effect of the pepsin and acid. In the presence of colostrum, the protected antibody showed no reduction of activity.
Using this antibody model, bovine colostrum has been shown to provide protection of antibody activity in a simulated gastric environment.
REFERENCES
Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Hentges F (2001). Differentia! binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. Clin Exp Immunol, 123:387-394.
Deliyannis G, Jackson D, Dyer w, Bates J, Coulter A, Harling-McNabb L, Brown L, (1998), Immunopotentiation of humoral and cellular responses to inactivated influenza vaccines by two different adjuvants with potential for human use. Vaccine, 16:2058-2068.
Anders EM, Hartley C, Jackson D (1990). Bovine and mouse serurn ji inhibitors of influenza A viruses are mannose-binding lectins. Proc Natl Acad USA,
87:4485-4489,

Example 4:
Colostrum protection of lactobacillus plantarum viability during acid/pepsin treatment
INTRODUCTION:
The role of bovine colostrum as a protectant of bacterial viability in a gastric environment was investigated using a Lactobacillus viable plate count assay. To investigate the protective properties of colostrum, the effect of simulated gastric fluid on Lactobacillus plantarum was compared in the presence and absence of bovine colostrum.
MATERIALS AND METHODS:
: The Lactobacillus plantarum strain used in this study is a strain used in probiotic and wfjs -taken from the culture collection of the Microbial Research Unit Royal Childrens Hospital, Parkviile, Victoria and typed by 16S sequencing (sequencing of DNA from ribosornal su bun its. The strain was cultured on horse blood agar (HBA) plates in a 37°C CO?, incubator for 48 to 72 hours. An inoculum of Lactobacillus was prepared by picking at least 2 colonies from a HBA plate and inoculating 2 ml of saline to reach a turbidity equivalent to a 0,5 McFarland standard. This inoculum was then used for all treatments.
Simulated Gastric Fluid.(SGF}: (adapted from Hiiger et al, 2001). A 0.32% solution of Sigrna porcine pepsin (Cat No P-7012 from stomach mucosa) was prepared in 0.03M NaCI and adjusted to pH 1.2 with HCI.
Colostrum Defatted, freeze dried Bovine Colostrum Extract (Anadis Limited "Gastran" Batch G01) was sourced from non-immunised cows and irradiated. A stock preparation was reconstituted at 300mg/mL in MilliQ water.

incubation mixtures were prepared by combining aiiquots of the L. plantarum inoculum with an equal volume of either the reconstituted colostrum or MilliQ water. A control mixture of the colostrum preparation and water was also prepared.
Incubation mixtures 1-2: 100μl of Lactobacillus •'• 100,μl of MiiliQ water Incubation mixtures 3-4: 10Gμl of Lactobscillus + 100μl of colostrum Incubation mixtures 5-6: 100μl of colostrum + 1 00μlof MilliQ water
Mixtures were preheated in a 37°C water bath for 5 minutes before starting treatment by the addition of 50μl of SGF to tubes 1 , 3 and 5. Mock treatments were initiated by the addition of 50i.il of 0.03M NaCl to tubes 2,4 and 6. All samples were incubated for 15 minutes at 37°C before reactions were stopped by the addition of 0.25 volumes of chilled 1 GOmM Na2CO3 to each tube.
Lactohacillus survival after treatment was assayed using a viable plate count technique. Ten-fold dilutions of the incubation mixtures were prepared in saline immediately after treatment. 100 μL of each dilution was then spread onto duplicate piates, Plates were incubated for 48 hours and the number of colonies per plate counted. The number of colony forming units (cfu) per mL in the incubation mixture was then calculated by multiplying the number of cfu/mL of diluted suspension by the dilution factor. The assay was done two times in duplicate.

RESULTS
(Table Removed)

As shown in the table of results and Figure 3, the addition of simulated gastric fluid to an inoculum of Lactobacillus resulted in a 1000 fold reduction in Lactobacillus viability.
Sn contrast, the addition of colostrum to the LactobaoHlus inoculum provided protection of the bacteria from the effect of the pepsin and acid. In the presence of colostrum, the protected Lactobacilli showed no reduction in viability. Using this viable count technique, bovine colostrum has been shown to provide protection of probiotic viability in a simulated gastric environment.
REFERENCES
Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Hentges F (2001). Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. Cltri Exp Immunol, 123:387-394.
Miles A, Misra S (1938). The estimation of the bactericidal power of the blood. J Hyg, 38:732-749.
Exampl 6:
COLOSTRUM PROTECTION OF ERYTHROMYC1N ACTIVITY DURING ACID
PEPSIN TREATMENT •
INTRODUCTION:
The role of bovine colostrum as a protectant of activity of a bioactive moiety in a gastric environment was investigated using an antibiotic model. To investigate the protective properties of colostrum, the effect of simulated gastric fluid on erythromycin activity was compared in the presence and absence of bovine colostrum.
MATERIALS AND METHODS:
Erythomycin Boehringer Mannheim hrythrornycin (C37H57NO13) was used as the source of the antibiotic, dissolved in 95% ethanol at a stock concentration of 1mg/mL and stored at 4°C. For this study the erythromycin stock was diluted in MilliQ water to 100 μg/mL and stored on ice until use.
Simulated Gastric Fluid (SGF) (adapted from Hilger at al, 2001). A 0.32% solution of Sigma porcine pepsin (Cat No P-7012 from stomach mucosa) was prepared in 0.03M NaCI and adjusted to pH 1,2 with HCI.
Colostrum: Defatted, feeze dried Bovine Coiostrurn Extract (Anadis Limited "Gastran" Batch 601} was sourced from non-immunised cows and a stock preparation was reconstituted at 300mg/mL in MilliQ water.
Treatment of Erythromycinwith SGF
Incubation mixtures were prepared by combining aliquots of the erythromycin
solution with an equal volume of either the reconstituted colostrum or MilliQ water. A control mixture of the colostrum preparation and water was also
prepared,
Incubation mixtures 1-2: 100μl of erythromycin + 100μl of MilliQ water Incubation mixtures 3-4: l00μl of erythromycin + l00μl of colostrum incubation mixtures 5-6: 100μl of colostrum * 100μl of MilliQ water

Mixtures were preheated in a 37°C water bath for 5 minutes before starting treatment by the addition of 50μ1 of SGF to tubes 1, 3 and 5. Mock treatments were initiated by the addition of 50μl of 0.03M NaCI to tubes 2,4 and 6. Ali samples were incubated for 15 minutes at 37°C besfore reactions were stopped by the addition of 0.25 volumes of chilled 160mM Na2CO3 to each tube. Tubes were then centrifuged for 3 minutes at 16,100g in an Eppendorf centrifuge 5415D and stored on ice. Supernatants were assayed for erythromycin activity.
Erythromycin Susceptibility Assay
Erythromycin activity was assayed -using a Bacillus subtilis disc diffusion susceptibility test. An inoculum of B. suhtilis (ATCC 6633} was prepared by picking at least 2 colonies from an overnight culture grown on horse blood agar (NBA) and inoculating 2 ml of saline to reach a turbidity equivalent to a 0.5 McFarland standard. NBA plates for the assay were then inoculated by streaking a sterile swab, dipped into the standardised solution, evenly in three directions over the entire surface of the plate to obtain a uniform inoculum. Plates were then allowed to dry for 3 to 5 minutes before the discs were applied.
Each treatment was serially diluted 1:2 with MiHQ water, resulting in six dilutions for each. 20 uL of each dilution was then loaded onto duplicate blank susceptibility discs (Oxoid, Hampshire, England). These discs were allowed to dry for at least 30 minutes before being placed onto duplicate plates. Each plate contained six evenly placed discs corresponding to the six dilutions of a single treatment. Untreated erythromycin diluted in MilliQ water to the equivalent concentration of the treated samples was also used as a control to obtain a standard curve. Plates were incubated for 16-18 hours at 37°C.
After 16-18 hours incubation the susceptibility of B. subtilis to erythromycin was determined by measuring the diameter of the zones of inhibition which appear around the discs. These zones result from the diffusion of the antibiotic from the disc into the surrounding agar. A standard curve was generated using the diameters of zones resulting from the aerially diluted untreated erythromycin control. Diameters for the test samples were then used to obtain the

percentage of erythromycin activity remaining compared with the untreated control. The assay was repeated three times in duplicate.
RESULTS
(Table Removed)

As shown in the table of results and Figure 4, the addition of simulated gastric fluid to a 100 μg/mL solution resulted in a 92% reduction in erythromycin
activity.
in contrast, the addition of colostrum to the erythromycin solution provided protection of the erythromycin from the effect of the pepsin and acid. In the presence of colostrum, the protected erythromycin retained 100% activity and actually showed improved activity when compared with the untreated control (see figures 1 and 2). This enhanced activity was also evident for the solutions of erythromycin and colostrum that were mock treated. Colostrum alone showed no background antibiotic activity so the enhanced activity seen with added colostrum cannot be explained by background erythromycin present in the colostrum. Using this antibiotic model, bovine colostrum has been shown to provide protection of antibiotic activity in a simulated gastric environment.
REFERENCES
Hilger 0, Grigioni F, De Beaufort C, Michel G, Freiiinger J and Hentges F (2001). Differential binding of IgG and IgA antibodies to antigenic determinants of bovine serum albumin. Clio Exp Immuno!, 123:387-394.
Barry AL and Thornsherry C (1991). Susceptibility tests: diffusion test procedures. In Balos A, Mauser WJ, Herman KL, Isenberg HD, and Shadomy HJ. Manual of Clinical Microbiology 5tjl Edition, American Society for Microbiology, Washington, pp 1117-1125,
The British Society for Antimicrobial Chemotherapy (1991) A Guide to Sensitivity Testing. The British Society for Antimicrobial Chemotherapy, San Diego.
Example 7:
RECOLOSTRUM PROTECTION OF LACTOBACILLUS CASE/ SHIROTA VIABILITY DURING ACID / PEPSIN TREATMENT
The role of bovine colostrum as a protectant of probiotic viability in a gastric environment was investigated using a Lactohacillus viable plate count assay. To investigate the protective properties of colostrum, the effect of simulated gastric fluid on LactohaciHus easel shirota isolated from Yakult fermented liquor .was compared in the presence and absence of bovine colostrum.
Reagents
Lactobacillus casei shirota The LactohaciHus easel shirota strain used in this study was isolated from the probiotic product - Yakult. The strain was cultured on horse blood agar (HBA) plates in a 37°C C02 incubator for 48 to 72 hours. An inoculum of Lactobacillus was prepared by picking at least 2 colonies from a HBA place and inoculating 2 ml of saline to reach a turbidity equivalent to a 0.5 McFariand standard, This inoculum was then used for all treatments.
Simulated GastricFluid (SGF): (adapted front Hilger et al, 2001). A 0.32% solution of Sigma porcine pepsin (Cat No P-7012 from stomach mucosa) was prepared in O.G3M NaCI and adjusted to pH 12 with HCl

Colostrum: Defatted, freeze dried Bovine Colostrum Extract (Anadis Limited "Gastran" Batch G01) was sou reed from non-immunised cows and irradiated. A stock preparation was reconstituted at 300mg/mL in MilliQ water.
'Treatment of Lactobacillus with SGF.
Incubation mixtures were prepared by combining aliquots of the L, easel shirota
inoculum with an equal volume of either the reconstituted colostrum or MilliQ
water. A control mixture of the colostrum preparation and water was also
prepared.
Incubation mixtures 1-2: 100μl of Lactobacillus + 1QGμL of MiHiQ water Incubation mixtures 3-4: 100μ1 of Lactobacillus + 100μL of coiostmm Incubation mixtures 5-6: 100μl of colostrum +100μL of MilliQ water
Mixtures were preheated in a 37°C water bath for 5 minutes before starting treatment by the addition of 50μL of SGF to tubes 1, 3 and 5, Mock treatments were initiated by the addition of 50μL of Q.03M NaCI to tubes 2,4 and 6. Ali samples were incubated for 15 minutes at 37°C before reactions were stopped by the addition of 0.25 volumes of chilled 160mM Na2CC3 to each tube.
Lactobacillus Viable Plante Counts
LactohaciHus survival after treatment was assayed using a viable plate count technique. Ten-fold dilutions of the incubation mixtures were prepared in saline immediately after treatment. 100 μL of each dilution was then spread onto duplicate plates. Piatos were incubated for 48 hours and the number of colonies per plate counted. The number of colony forming units (cfu) per ml. in the incubation mixture was then calculated by multiplying the number of cfu/mL of diluted suspension by the dilution factor. The assay was done two times in duplicate.

RESULTS
(Table Removed)

As shown in the table of results and Figure 5, the addition of simulated gastric fluid to an inoculum of Lactohacillus easel shirota resulted in at least a 10,000 fold reduction in Lactobacillus viability,
In contrast, the addition of colostrum to the Lactobacillus inoculum provided protection of the probiotic bacteria from the effect of the pepsin and acid. In the presence of colostrum, the protected Lactobacilli showed no reduction in viabifity. Using this viable count technique, bovine colostrum has been shown to provide protection of probiotic viability in a simulated gastric environment.
REFERENCES
Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Heniges F (2001). Differential binding of IgG and [g/\ antibodies to antigenic determinants of bovine serum albumin, Ciin Exp Imrnunol, 123:387-394.
Miles A, Misra S (1938). The estimation of the bactericidal power of the blood, J Hyg, 36:732-749.
Example B:
COLOSTRUM PROTECTION OF CHOLERA TOXIN ACTIVITY DURING ACID/PEPSIN TREATMENT

INIRODUCTION
The role of bovine colostrum as a protectant of adjuvant activity in a gastric environment was investigated using a mucosal adjuvant. To investigate the protective properties of colostrum, the effect of simulated gastric fluid on the activity of cholera toxin was compared in the presence and absence of bovine colostrum.
MATERIALS AND METHODS: Reagents
Cholera toxin and Y1adrenal cells Sigma Vibrio cholorae Cholera Toxin (Cat No C-8QS2) was used as the source of the adjuvant. The cholera toxin was diluted in MilliQ water to a concentration of 3.12 μg/mL and stored on ice until use. Y1 mouse adrenal cells were seeded in 96-weil microtitre plates at a concentraion of 2 X 104 cells per well in DMEM growth medium supplemented with 10% foetal calf serum (PCS) and gentamictn.
Simulated Gastric Fluid .(SGF): (adapted from Hifger et al, 2001). A 0.32%
solution of Sigma porcine pepsin (Cat No P-7012 from stomach mucosa) was prepared in 0.03M NaCI and adjusted to pH 1.2 with HCI.
liQlQstn.jm: Defatted, freeze dried Bovine Colostrum Extract (Anadis Limited "Gastran" Batch G01) was sourced from non-irnmunised cows and a stock
preparation was reconstituted at. 300mg/mL in MiliiQ water.
Ios.§tit}snU?l.S.iiQ!M^^lQ,3^iib_.SGF
Incubation mixtures were prepared by combining aiiquots of the 3.12 ug/rnL cholera toxin stock with an equal voulme of either the reconstituted colostrum or MilliQ water. A control mixture of the colostrum preparation and water was also
prepared.
Incubation mixtures 1-2: 100μL of cholera toxin + 100μL of MilliQ water incubation mixtures 3-4: 100,μL of cholera toxin + 100μL of colostrum Incubation mixtures 5-6: 100μl of coloslrusn +100μL of MilliQ water

Mixtures were preheated in a 37°C water bath for 5 minutes before starting treatment by the addition of 50μL of SGF to tubes 1, 3 and 5. Mock treatments were initiated by the addition of 50μL of 0.03M NaCI to tubes 2,4 and 6. All samples were incubated for 15 minutes at 37°C before reactions were stopped by the addition of 0.25 volumes of chilled 160mM Na2CO3 to each tube. Tubes were then centrifuged for 3 minutes at 16,100g in an Eppendorf centrifuge 5415D and stored on ice. Supernotants were filter sterilised and then assayed for toxin activity.
Y1 .,Adrenal Cell Bioassay
Cholera toxin activity was assayed using the Y1 adrenal ceil bioassay. Y1 mouse adrenal ceils were seeded in 96-well microtitre plates at a concentration of 2 X 104 ceils per we!!. After 48 hours, cells were washed 3 times with PBS. The growth medium was replaced with DMEM supplemented with 1% PCS and gentamiein (100 μL), containing duplicate samples of the treatment tubes, serially diluted 2-fold after an initial dilution of 1:10. Cholera toxin causes Y1 adrenal cells to change from their usual elongated morphology to a more rounded shape. These changes are concentration dependent. The ceils were incubated for 18 hours and then examined for typical rounding by using a phase contrast microscope. The end point of the assay was defined as the highest dilution that showed more than 50% cell rounding. The assay was repeated twice in duplicate. Two-fold dilutions of cholera toxin at a starting concentration of 10 ng/mL were assayed in the same plates and the results used to determine the sensitivity of the assay.

RESULTS
(Table Removed)

As shown in the table of results and Figure 6, the treatment of cholera toxin with simulated gastric fluid resulted in the complete abrogation of the biological activity of cholera toxin.
in contrast, the addition of colostrum to the cholera toxin preparation provided protection of the toxin from the effect of the pepsin and acid. In the presence of colostrum, the protected cholera toxin showed no reduction of activity. Using this cholera toxin assay, bovine colostrum has been shown to provide protection of mucosai adjuvant in a simulated gastric environment.
REFERENCES
Hilger C, Grigioni F, De Beaufort C, Michel G, Freilinger J and Hentges F (2001). Differentia! binding of !gG and hgA antibodies to antlgenic determinants of bovine serum albumin. Cltn Exp Immune!, 123:387-394.
Tauschek M, Gorrell R, Strugnell R, Robins-Browne R (2002). Identification of a protein secretory pathway for the secretion of net-labile enterotoxin by an enterotoxigenic strain of Escherichia coli. Proc Natl Acad Sci USA, 99:7066-
7071.
Example 9:
PROTECTION OF LACTOBACILLUS PLANTARUM IN AN IN VIVO MOUSE MODEL
INTRODUCTION:
The role of bovine colostrum as a protectant of probiotic viability in a gastric environment was investigated using an in vivo mouse model. To investigate the protective properties of colostrum mice were fed Lactobacillus plantarum with and without colostrum, or as a positive"control, sodium bicarbonate.
MATERlALS AND METHODS:
Reagents
Lactobacillus plantarum - The Lactobacillus plantarum strain used in this study was the strain referred to in Example 4. 'The strain was cultured on horse biood agar (NBA) plates in a 37°C CO2 incubator for 48 to 72 hours. An inoculum of Lactohacillus was prepared by harvesting a confluent lawn of bacteria from at least 2 plates and inoculating 50 ml of saline to reach a turbidity equivalent to a McFariand 4 standard. The bacteria were pelleted and the supernatant discarded. The bacterial pellet was then rosuspended in 2.5 mL of saline. This inoculum was mixed 1:1 with bioshield, sodium bicarbonate or saline to give a final bacteria! concentration of 1x109 cfu/mL.
Colostrum Extract (also designated as bioshield) Defatted, freeze dried Bovine
Colostrum Extract (Anadis Limited "Gastran" Batch G01) was sourced from non-immunised cows and irradiated. Sample preparations were reconstituted at 20, 40, and 100 mg/mL in MilliQ water.
Sodium bicarbonate Sodium bicarbonate was also reconstituted at concentrations of 20, 40 and 100 mg/mL in MiliiQ water.
Infection of mice with Lactobacillus
Mice were deprived of food for 3 hours prior to inoculation to ensure that their stomachs were empty. The mice were then inoculated by gavage with 100 μL of each preparation, an inoculum of -1x108 cfu of Lactohaciltus (the actual numbers of Lactobacilli in each preparation was determined by viable plate counts). There were 7 groups of mice, each with 3 mice per group, the mice were given the following preparations:
Lactohacillus + saline Lactobacillus + 20mg/rnL bioshield Lactobacillus + 40mg/mL bioshield Lactobacillus + l00rng/rnL bioshield
Lactobacillus +20mg/mL sodium bicarbonate Lactobacillus + 40rng/mL sodium bicarbonate Lactobacillus + I00mg/mL sodium bicarbonate
Mice were deprived of food for the course of the experiment but were allowed autoctaved water ad libitum. Protection of Lactobacillus in vivo was assayed by counting the number of L pfantarum colonising the large intestine after 3 hours. The large intestine (caecum and colon) was harvested asepticatiy from each mouse into 2 rnL of sterile saline. Samples were weighed and homogenised and ten-fold dilutions prepared in saline. 100 μl, of each dilution was spread onto a LaciohaciHus selective media (horse blood agar containing 10 μg/mL vancomycin and 12.5 μg/mL polymyxin B). Plates were incubated for 48 hours in 5% C02 and the number of colonies per plate counted. Lactobacillus plantarum were readily distinguishable from other Lactobacilli normally present in the normal flora of the mouse due to their opaque white appearance. The number of colony forming units (cfu) per gram of intestine was calculated.
RESULTS
As shown in Figure 7, survival of Lactobacillus plantarum increased in the presence of both bioshield and sodium bicarbonate. Higher concentrations of either bioshieSd or sodium bicarbonate resulted in increased survival and therefore better protection.
Example 10:
COLOSTRUM PROTECTION OF LACTOBACILLUS PLANTARUM VIABILITY UNDER HUMID CONDITIONS
INTRODUCTION;
The role of an extract of bovine colostrum, as a protectant of probiotic viability in different humidity conditions, was investigated using a Lactobacillus viable plate count assay. To investigate the protective properties of colostrum extract, the viability of Lactobacillus plantarum was compared after storage for 14 days in three constant humidity environments.
MATERIALS AND METHODS Reagents
Lactobacillus plantarum The LaciohacH'us plantarum (L.p.) strain used in this study was the strain referred to in Example 4. The strain was cultured on horse blood agar (HBA) plates in a 37°C CO2 incubator for 48 to 72 hours. An inoculum of L.p. was prepared by picking at least 2 colonies from a HBA plate and inoculating 2 ml of saline to reach a turbidity equivalent to a 0.5 McFarland standard. This inoculum was then used for all treatments.
Freeze Drying ...Medium: The freeze-drying medium (FDM) used in these experiments was derived from Conrad et a! (2000), A 2x-concentrated FDM was prepared using 40% w/v α.α-trehalose dihydrate (Sigma) and 5.7% w/v sodium tetraborate decahydrate (Sigma) in sterile 0.6 mM potassium phosphate pH 7.2. The pH of this FDM was initially adjusted to pH 8.5 with solid citric acid (Sigma) and then changed to pH 8.5 with ammonium hydroxide (Sigma 29.5%). The 2x concentrated FDM was then sterile filtered to 0.2μm.
Colostrum: Defatted, freeze-driecd Bovine Colostrum Extract (Anadis Limited "Gastran" Batch G01) was sourced from non-immunised cows and a stock preparation was reconstituted at 40 mg/ml in 2 x concentrated FDM.
Preparation of sample Four aliquots of each of the following mixtures were made prior to freeze drying:
(a) L.p. + 2x FDM (0,5 ml + 0.5 ml)
(b) L.p, * 40 mg/ml colostrum in 2x FDM (0.5 ml + 0.5 mi)
These samples were mixed and then incubated at room temperature for 1 hr and then frozen on dry ice. Samples were then freeze-dried overnight.
Stability of Lactobacillusplantarum at different relative humidities
Three constant humidity chambers were set up using sealed boxes containing different solutions. A saturated solution of LiCI (Sigma) maintained the relative humidity (RH) at 11.3% (water activity aw » 0.113). An 80% w/w solution of glycerol (BDH Analar) in water produced a 50% RH (aw ™ 0,50) and a saturated KCI (BDH Analar) solution maintained an RH of 85% (aw = 0.85) (refer to Forney & Brand! 1992 and Merck index). After freeze drying, each sample was gently ground to a fine powder using a mortar and pestle. Two samples (L.p. + FDM and L.p. + Colostrum in FDM) were incubated in open wide-mouth vials in each chamber, for 14 days at 20°C. A control sample of each mixture was stored at 20°C in closed vials.
Lactobacillus Viable Plate Counts
Lactobacillus survival, after incubation at different relative -humidities, was assayed using a viable plate count technique. Ten-fold dilutions of the incubation mixtures were prepared in saline containing 0.05% of the surfactant Tween 20. 100μl of each dilution was then spread onto duplicate plates. Plates were incubated for 48 hours and the number of colonies per plate counted. The number of colony forming units (cfu) per rnl In the incubation mixture was then calculated by multiplying the number of cfu/ml of diluted suspension by the dilution factor. These counts were then expressed as a percentage of the control sample.
RESULTS
(Table Removed)
An extract of bovine colostrum increased the survival of a fresco-dried probiotic, Lactobacillus plantarum, during exposure to medium and high humidity conditions.
REFERENCES
Conrad PB, Miller DP, Cielenski PR and da Pablo JJ (2000). Stabilization and
Preservation of Lactobacillus acidophilus in Saccharide Matrices. Cryoblology
41:17-24.
Forney CF and Brand! DG (1992). Control of Humidity in Small Controlled-environment Chambers using Glycerol-Water Solutions. HortTechnology Jan
Mar 2(1): 52-54.
Merck index: Misc-98 "Saturated Solutions" and Misc-103 "Constant Humidity Solutions".
Finally, it is understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims:
1. An in vitro method of improving the viability of a labile bioactive
substance on administration to a hostile collecting colostrum from bovine
animals within 4 days post parturition; mixing colostrum with the bioactive
substance in an in vitro mixing process in a ratio of colostrum to bioactive
substance of at least 0.5:1 wherein the labile bioactive substance
selected from probiotic bacteria; antibodies and fragments thereof said
antibodies and fragments thereof directed against pathogenic bacteria;
and vaccines directed against pathogenic Bacteria or toxins produced
thereby.
2. An in vitro method according to claim 1 wherein the labile bioactive
substance is selected from antibodies and vaccines directed against
bacterial pathogens responsible for gastrointestinal disease.
3. An in vitro method according to claim 1 wherein the labile, bioactive
substance is a probiotic Lactobacillus bacteria.
.4. An in vitro method according to claim 1 wherein the labile bioactive substance comprises antibodies or antibody fragments directed against pathtogenic bacteria selected from Helicobacter pylori, Enterotoxigenic E Coli, Yersinia p&stis and anthrax.
5. An in vitro method according to claim 1 wherein the labile bioactive
substance is selected from antibodies directed against Enterotoxigenic E.
Coii,
6. A method as claimed in claim 1, wherein said weight ratio is greater than
2:1.
7. A method as claimed in claim 1 wherein the mixture is for administration
in a composition comprising one or more carriers or axcipients suitable
for administration orally or by inhalation.

8. A method as claimed in any one of claims 1 to 7 wherein the colostrum
comprises bovine colostrum retained from no more than Mo days post
parturition.
9. A method as claimed in claim 8 wherein the bovine colostrum comprises
colostrum retained from the day post parturition.
10. A method as claimed in any one of claims 1 to 7 wherein the colostrum is
in liquid form and after forming the mixture of colostrum and bioactive
substance in vitro the mixture is dried to form a solid.
11. A method as claimed in any one of claims 1 to 7 wherein the mammalian
colostrum is dried.