USES THEREOF

TECHNICAL FIELD

[0001] The present invention relates to a processing method to produce an immune active milk product and methods of using the immune active milk product to enhance immune response in individuals.

BACKGROUND

[0002] Asthma and allergic diseases continue to expand in the developed world and are growing rapidly in the developing world. Prevention has remained elusive despite years of research.

[0003] Immunosenescence describes the gradual degradation of immune activity brought on through aging. An impaired function of both the innate and acquired arms of the immune system is observed, and results in a higher susceptibility to infectious disease. The use of vaccines is recommended in senior citizens, however, a successful immune response to the vaccine is observed only in an age-dependent proportion of cases. Poor response to vaccines is a common manifestation of immunosenescence. A recent study demonstrated that whey proteins improved vaccine response in an elderly population.

[0004] Stress induced immune suppression, or "Olympic Village Fever" as it is commonly known, has demonstrated a link between high levels of exercise induced stress and greater susceptibility to communicable diseases. Additionally, it is desirable to promote muscle recovery after exercise, particularly intense exercise.

[0005] A number of studies have showed a correlation between decreased allergy and raw milk consumption. Loss et al. in "The protective effect of farm milk consumption on childhood asthma and atopy: The GABRIELA study" in Allergy and Clinical Immunology Vol. 128, Num. 4 (2014) reported that questionnaire-reported consumption of unboiled farm milk was inversely associated with asthma, hay fever, and atopy. Higher levels of the whey proteins BSA, a- lactalbumin, and β-lactoglobulin in milk samples were associated with a reduced risk of asthma. Neither total viable bacterial counts nor the total fat content of the milk were related to asthma or atopy.

[0006] Many clinical studies have demonstrated a link between raw milk consumption and reduced allergy development, in particular, the PASTURE study, Loss, et al. "Consumption of unprocessed cow's milk protects infants from common respiratory infections," Journal of Allergy and Clinical Immunology, 2015 Jan;135(l):56-62,demonstrated raw milk consumption and not farm life exposure were responsible for the observed allergy and respiratory infection reduction. The study's conclusions stated that early life consumption of raw cow's milk reduced the risk of manifest respiratory infections and fever by about 30%.

[0007] Further a study in 2013 (Christen et al., "The Effect of UV-C Pasteurization on

Bacteriostatic and Immunological Proteins of Donor Human Milk", PLOS ONE, 8(12) e85867, showed that human milk' s levels of immunological proteins is significantly higher under UV-C irradiation versus Holder Pasteurization.

[0008] However, raw cow's milk is capable of transmitting serious infections, such as tuberculosis, brucellosis, listeriosis, or enterohemorrhagic E. coli. Introduction of pasteurization and other processing techniques minimized the chances of milk-borne infections. The PASTURE study noted that if the health hazards of raw milk could be overcome, the public health impact of minimally processed but pathogen- free milk might be enormous, given the high prevalence of respiratory infections in the first year of life and the associated direct and indirect costs.

[0009] Bovine milk is a complicated biofluid containing a multitude of carbohydrates, proteins and lipids. Various compounds have been proposed as being responsible for raw milk's protective effects, but no study so far conducted has demonstrated effects as strong as those observed through raw milk consumption. Milk fat globule membrane (MFGM) describes a range of proteins and phospholipids that surround milk's fat globules. These compounds contain a great variety of proteins, many of which have demonstrated immune activity. MFGM components (which includes some or all of the milkfat globule and attendant proteins, phospholipids, and triglycerides) has been successfully extracted and isolated from milk and milk derivatives such as buttermilk.

[0010] Research has demonstrated that many of bovine raw milk' s compounds stimulate human immune systems, however, thermal pasteurization degrades many of these immune active compounds. New methods are required to enable many of these immune active compounds to survive a pasteurization process that continues to meet the required 5 log reduction in pathogens.

[0011] Lactose intolerance affects a large percentage of the world's population.

Additionally, many elderly people, despite no previous intolerance, develop this condition as they age. Enzymatically hydrolyzing lactose into glucose and galactose is a well-known process and reduces lactose levels by at least 95%. Other methods for removing lactose, glucose and/or galactose are known in the art, including ultrafiltration.

[0012] The present invention is directed toward overcoming one or more of the problems discussed above. SUMMARY OF THE EMBODIMENTS

[0013] The present invention includes a method to produce an immune-active milk product. The method includes a step of obtaining a nonfat milk fraction from raw

(unpasteurized) milk; and pasteurizing the nonfat milk fraction by application of a non-thermal pasteurization step, such as by UV-C light, to prepare the immune-active milk product. The method may further include obtaining a composition comprising milk fat globule membrane components (MFGM) and combining the composition comprising MFGM components with the nonfat milk fraction either prior to or following the UV-C pasteurization step.

[0014] The composition comprising MFGM components may be obtained from a commercial supplier and is optionally obtained dried. Alternatively, MFGM may be obtained from a milkfat source or a derivative thereof, such as, for example, from buttermilk, by methods known in the art, and added to the nonfat milk fraction in liquid form.

[0015] In one embodiment, the nonfat milk fraction is treated to lower the levels of sugar, e.g., lactose, galactose, and/or glucose content. In one embodiment, the sugar is removed via hydrolysis of the lactose before or after the UV-C pasteurization step. In another embodiment, the sugar is removed via a mechanical process such as, for example, ultrafiltration.

[0016] In another embodiment, the immune-active milk product is dried to a powder using a low-heat drying process. In one embodiment, the drying step is a freeze-drying step. In another embodiment, the drying step occurs below 45 °C. Alternatively, a combination of ultrafiltration and diafiltration may be used to create a milk protein concentrate.

[0017] In one embodiment, the UV-C light is applied at between 1000-2000 J/liter, or as much as necessary to achieve a 5-log reduction of bacterial content in the composition.

[0018] The present invention includes a method for reducing childhood asthma and allergic diseases, which includes administering an immune-active milk product obtained by the processes of the instant invention to a patient in need thereof.

[0019] The present invention also includes a method for reducing elderly

immunosenescence, which includes administering an immune-active milk product obtained by the processes of the instant invention to a patient in need thereof.

[0020] The present invention also includes a method for reducing stress-induced immune suppression, which includes administering an immune-active milk product obtained by the process processes of the instant invention to a patient in need thereof. Stress-induced immune suppression may be brought on by, for example, exercise. The compositions of the present invention may also be used to enhance muscle recovery after, for example, exercise. [0021] The present invention also includes a method to produce an immune-active milk product which includes the steps of obtaining a nonfat milk fraction from raw (unpasteurized) milk; adding a composition comprising MFGM components, pasteurizing the product by application of UV-C light, drying the pasteurized nonfat milk fraction or alternatively, creating a milk protein concentrate by ultrafiltration/diafiltration to prepare the immune-active milk product.

[0022] Various modifications and additions can be made to the embodiments discussed without departing from the scope of the invention. For example, while the embodiments described above refer to particular features, the scope of this invention also included

embodiments having different combination of features and embodiments that do not include all of the above described features.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] A further understanding of the nature and advantages of particular embodiments may be realized by reference to the remaining portions of the specification and the drawings, in which like reference numerals are used to refer to similar components. In some instances, a sub- label is associated with a reference numeral to denote one of multiple similar components. When reference is made to a reference numeral without specification to an existing sub-label, it is intended to refer to all such multiple similar components.

[0024] Fig. 1 shows LC/MS TIC chromatograms of the raw milk samples (first and second chromatograms) and 1000 J-treated samples (third to sixth chromatograms.)

[0025] Fig. 2 shows relative levels of a number of protease inhibitors in the raw milk samples, conventionally spray dried samples, pasteurized samples and 1000 J samples (lx or 2x).

[0026] Fig. 3 shows relative levels of several complement proteins in the raw milk samples, conventionally spray dried samples, pasteurized samples and 1000 J samples (lx or 2x).

[0027] Fig. 4 shows relative levels of other immune-active proteins in the raw milk samples, conventionally spray dried samples, pasteurized samples and 1000 J samples (lx or 2x).

[0028] Figure 5 shows a flow chart of the process to prepare an immune-active milk product of the present invention.

[0029] Figure 6 shows a flow chart of the process to prepare an immune-active milk product of the present invention.

[0030] Figure 7 shows a flow chart of the process to prepare an immune-active milk product of the present invention. DETAILED DESCRIPTION

[0031] The present invention allows for the production and use of a product that shares bovine raw milk's ability to stimulate human immune systems, without the infectious disease risk of bovine raw milk. The present invention has developed new methods to treat raw milk to allow for immunologically active proteins and other components such as enzymes, antibodies, and the like to undergo a non-thermal pasteurization process that continues to meet the required 5-log reduction in pathogens of marketed milk. The methods of the invention avoid the use of thermal pasteurization and conventional spray drying, which have been found to reduce the amount and/or quality of immunologically active proteins in milk. For example, the methods can include a low temperature step which exposes milk to a maximum temperature of 42°C for less than 2 minutes, which preserves immunologically active proteins.

[0032] Therefore, in one embodiment, the present invention includes a method to produce immune-active milk products. The invention includes obtaining a raw milk composition, separating the raw milk composition into a nonfat milk fraction and a fat fraction, and pasteurizing the nonfat milk fraction by application of UV-C light (non-thermal pasteurization) to prepare the immune-active milk product. In one embodiment, an MFGM component is added to the nonfat milk fraction prior to non-thermal pasteurization. Figure 5 shows an embodiment of the process described by the instant invention.

[0033] Step 1 of Figure 5 involves obtaining a nonfat raw milk fraction from a raw milk composition. Raw milk is generally obtained whole from the dairy animal, and raw whole milk suitable for the invention includes milk which has not been thermally processed prior to use according to the methods disclosed herein. Generally, raw milk is milk that has not been pasteurized or homogenized. Raw milk can be obtained from, for example, dairy animals, such as cattle, buffaloes, goats, sheep and camels, or other milk animals such as yaks, horses, reindeer and donkeys. In one embodiment, the dairy animal is a cow. Alternatively, a nonfat milk fraction from raw milk may also be obtained.

[0034] In one embodiment, if the starting material is raw whole milk, a nonfat milk fraction is separated from the fat fraction of the milk. Such separation is routine in the art and can be accomplished by any method known in the art. For example, centrifugation is a standard operation used in the food industry for the treatment of milk. Centrifugation uses centrifugal force to separate two mixed liquids or insoluble solids from liquids (suspension). For milk, the fat globules separate from the rest of the milk suspension. The original milk feed at about 3.7% fat is separated into a cream portion (higher than 30% fat) and a skim milk portion (around 0.05% fat). The milk is commonly at about 40°C before entering the centrifuge for optimized liquidity of the fat. Commonly a lower-speed centrifugation step at about 5,000-10,000 x g is used.

[0035] Optionally, the nonfat milk fraction may be treated to lower the amount of sugar present in the milk fraction. Sugars include lactose, glucose and/or galactose. In one embodiment, removal of sugar is accomplished via hydrolysis of lactose, according to Step 2 of Figure 5, although the step comprising hydrolysis of lactose may occur at any point along the process. Optionally, this step occurs after the non-thermal pasteurization e.g. UV-C

pasteurization step described below, or may be performed prior to the non-thermal pasteurization step. If lactose hydrolysis is performed after the non-thermal pasteurization step, a sterile lactase preparation should be used. Lactose intolerance is the inability of many people to digest lactose, which leads to symptoms such as abdominal bloating, cramps, flatulence, and diarrhea in individuals lacking sufficient amounts of the enzyme lactase. It is estimated that approximately 70% of the world's population is intolerant to some extent to lactose. Cow's milk has about 5% lactose. Lactase, or β-galactosidase, can be used to break lactose down into galactose and glucose monomers. Direct treatment of milk using lactase is known in the art and can be accomplished by any method as known in the art. For example, lactase from a source such as fungi of the genus Aspergillus or yeast of the genus Kluyveromyces may be mixed in with the product to break down the lactose in the milk. An example of a suitable lactase is LACTOZYM PURE (Novozymes), and amounts to use, temperatures and procedures to accomplish the suitable amount of hydrolysis of lactose are in accordance with the manufacturer's directions.

[0036] In one embodiment of the present invention, an additional component, e.g., a

MFGM-containing composition, is optionally added to the nonfat milk fraction either before or after the UV-C pasteurization step to prepare the immune-active milk product of the invention, according to Step 3 of Figure 5. The MFGM-containing composition can be obtained from any number of sources that comprise milkfat. Generally, the MFGM-containing composition is obtained from milk, cream, whey, milk serum, or from buttermilk. Milk fat is primarily triglycerides and phospholipids and is secreted from mammary epithelial cells as fat globules, primarily composed of a globule of triglyceride surrounded by a lipid bilayer membrane which helps to stabilize the fat globules in the milk as an emulsion. The milk fat globule includes the milk fat and the associated milk fat globule membrane. The milk fat globule membrane

(MFGM) describes a range of proteins and phospholipids that surround milk fat globules. The term MFGM components defines a composition which is enriched in some or all of the protein components of the milk fat globule membrane. These milk fat globule proteins contain a great variety of proteins, many of which have demonstrated immune activity. The milk fat globule membrane protects the milk fat from naturally occurring lipase enzymes and the development of rancid off-flavors. In conventional heat pasteurization steps, lipase enzymes are inactivated by heat during heat pasteurization which slows down the rancidity process. However, in the processes of the instant invention, enzymes are not inactivated to the same degree.

[0037] In one embodiment, the MFGM-containing composition suitable for use with the present invention may comprise MFGM components. MFGM components may be obtained from sources such as butter serum, whey or buttermilk by methods as known in the art. For example, whole milk is generally 10% cream (4-5% fat). The cream may be churned into butter with a remaining buttermilk that contains less lipids and phospholipids and enhanced MFGM components as compared to the starting cream or whole milk. The buttermilk contains water and is enriched for MFGM as compared to the starting cream. The buttermilk can be used as the source of MFGM and may be added to the nonfat milk portion such that the ratio of MFGM is approximately the same as in raw milk. For example, the buttermilk can be added back to the nonfat milk at a 5% v/v ratio. Generally, preparation processes for MFGM separate most or all of the fat (triglycerides and phospholipids, for example) from the remainder of the components, such that when blended with the nonfat milk fraction or the non-thermally pasteurized e.g., UV-C pasteurized nonfat milk fraction of the present invention, approximates the milkfat level as desired, such as, in some cases to be equivalent to approximately 1-2% fat.

[0038] When MFGM components are not obtained commercially, they may be treated by the non-thermal pasteurization methods as described herein, either prior to or after combination with the nonfat milk portion prepared according to the present invention.

[0039] Compositions comprising MFGM components may also be obtained

commercially, from, for example, Aria Foods Ingredients Group P/S (Denmark), LACPRODAN MFGM- 10 (whey protein concentrate). This material is described as a whey protein concentrate with a high concentration of bioactive proteins and lipid. Typical batches of LACPRODAN MFGM- 10 have a total fat composition of 14-20%, phospholipids of 6-8%, with an IgG level of 4%, lactoferrin of 0.08%, lactose maximum 3%, and sphingomyelin of 1.6%, phosphatidyl ethanolamine of 1.9%, phosphatidyl choline of 1.8%, phosphatidyl inositol of 0.5%, and phosphatidyl serine level of about 0.8%. Other sources of a protein/phospholipid-containing composition include Fonterra, Inc. (New Zealand). MFGM components are also available from commercial suppliers, or may be prepared by methods known in the art, see, e.g., MacGibbon AKH and Taylor MW (2006) Composition and Structure of Bovine Milk Lipids in Advanced Dairy Chemistry Volume 2, Lipids, 3rd Ed, (P.F. Fox and P.L.H. McSweeney, eds.) Springer, New York, pp 1-43. Protein/phospholipid-containing compositions comprising MFGM components may be pasteurized by conventional methods, such as, for example, heat- pasteurization, or optionally, by UV-C pasteurization. For example, MFGM components obtained from commercial sources may have been heat-pasteurized. In these embodiments, the MFGM components may be added to the nonfat milk fraction after the nonfat milk fraction has been treated with the non-thermal pasteurization processes according to the present invention.

[0040] The protein/phospholipid-containing composition optionally comprising MFGM components can be added back to the nonfat fraction of the raw milk composition at any time prior to the optional drying step. If the MFGM composition has been pasteurized, either by conventional heat pasteurization or by UV-C pasteurization, it may be added back to the nonfat milk fraction following the UV-C pasteurization step of the nonfat milk fraction. If the MFGM containing composition has not been pasteurized, it may be added back to the nonfat milk fraction prior to the UV-C pasteurization step of the nonfat milk fraction. In one embodiment, the MFGM may be added back to the nonfat raw milk portion prior to non-thermal

pasteurization.

[0041] The present invention also includes a non-thermal pasteurization of the nonfat milk fraction, according to Step 4 of Figure 5. In one embodiment, the non-thermal

pasteurization includes a UV-C pasteurization. Methods to accomplish a UV-C treatment to accomplish a suitable pasteurization are known in the art. According to the U.S. Food and Drug Administration, milk requires a 5-logio bacterial reduction. Methods to obtain such a reduction in bacterial using UV-C are known in the art. Generally, ultraviolet (UV) irradiation is classified as a non-thermal disinfection method. UV is subdivided by wavelength into UV-A (320-400 nm), UV-B (280-320 nm), UV-C (200-280 nm) and Vacuum-UV (100-200 nm). UV-C has the highest germicidal effect, specifically between 250 and 270 nm, and is capable of destroying bacteria, viruses, protozoa, yeasts, molds and algae. The penetration depth of UV-C depends on the solubility, density and turbidity of a liquid. Milk is difficult to treat with UV-C due to its high absorption coefficient of 300 cm ^-1 at a wavelength of 254 nm. Previous studies showed that UV- C irradiation can be used to successfully reduce the microbial load of opaque liquids such as bovine milk, for example, by creating a turbulent flow around a UV-C source and this therefore exposed the micro-organisms to photons at the interface between the opaque liquid and the photon source. Key factors influencing efficiency of UV-C treatment include reactor design, fluid dynamic parameters and UV-C absorbance of liquid food (Koutchama et al., 2004). Turbulent flow of liquid foods in continuous flow UV reactors increased inactivation of microorganisms in fresh juices, liquid egg whites and milk (Koutchma et al., 2004; Franz et al., 2009). [0042] An example of a UV suitable for the present invention includes that of Choudhary et al. (2011), Performance of coiled tube ultraviolet reactors to inactivate Escherichia coli W1485 and Bacillus cereus endospores in raw cow milk and commercially processed skimmed cow milk. Journal of Food Engineering, 107 (1): 14-20. Choudhary et al. (2011) reported a 4.1 logio CFU/ml reduction of E. coli W 1485 in raw cow milk by UV-C treatment in a coiled tube UV reactor at a flow rate of 100 1pm, corresponding to a R _e (Reynolds number)of 713 at a UV dose of 11.187 mJ/cm ² .

[0043] Another example of UV-C device suitable for the present invention can be found in U.S. 6,916,45, which is incorporated herein by reference in its entirety. A dose rate of 2000 J/1 has been generally found to result in the 5 log pathogen reduction as required by the FDA. An increased UV dose (16.822mJ/cm ² ), can achieve greater than 5-log reductions of E. coli, using a UV-C source was a 8.7 W, 110 V, UV-C germicidal lamp with peak emission at 253.7 nm, having a 505 mm arc length and 15 mm outside diameter (OD) (SBL325, American Ultraviolet Company, Lebanon, IN, USA). The UV lamp was enclosed within a quartz glass sleeve

(American Ultraviolet Company, Lebanon, IN, USA) with a 22 mm OD and an air gap of 2.4 mm between UV lamp and sleeve. Perfluoroalkoxy polymer resin (PFA) tubing with 1.6 mm inside diameter (ID) and 3.2 mm OD was selected to wrap around the glass sleeve based on Geveke (2008) that PFA tubing is highly transparent to UV light and has more chemical and heat resistance than polytetrafluoroethylene and fluorinated ethylene propylene.

[0044] Another example of a UV reactor suitable for the present invention includes the device shown in European Patent No. EP 1255444, title Sterilization of Liquids using Ultraviolet Light, which describes a device and method for UV sterilization of a turbid liquid.

[0045] Optionally, the method further comprises a drying step to form a milk powder according to Step 4 of Figure 5. Conventional drying steps include spray drying, where pasteurized milk is first concentrated in an evaporator to approximately 50% milk solids, then sprayed into a heated chamber where the water almost instantly evaporates. Alternatively, conventional methods for drying milk can be accomplished by drum-drying, where milk is applied as a thin film to the surface of a heated drum, and the dried milk solids removed.

However, as will be appreciated, conventional drying steps utilize heat, which has been found by the present inventors to reduce the immune- stimulating activity of milk. Therefore, in the present invention, these high-heat processes have been avoided. Thus, in this embodiment, the drying step is carried out at a temperature of less than about 45 °C, less than about 42 °C, less than about 40 °C, less than about 32 °C, or less than about 0 °C. Methods by which to dry solids at low temperatures are known in the art. These include freeze-drying, low-temperature drying methods. One method includes using a large volume of air compared to the feed of milk, and the drying process is accomplished fairly slowly over an extended period of time. The temperature of the milk during this process may be increased slowly. For example, during this process, the milk may be exposed to a temperature of 42 °C for approximately 2 minutesor less. One example of an appropriate drying process is the one carried out by LiquaDry, Inc. (Hinckley, UT).

[0046] Milk contains a number of proteins. Some are known to be immunologically active, although the exact compounds responsible for the full scope of the immunologically active properties of milk remains unknown. Milk proteins include the primary group, the caseins. All other proteins found in milk are called whey proteins. Whey proteins are a mixture of globular proteins, and include all proteins soluble in milk after the pH is dropped to 4.6. The major whey proteins in milk are β-lactoglobulin and a-lactalbumin. Whey proteins denature rapidly at temperatures above 70°C, with thermostability increases from IgG < serum albumin < β-lactoglobulin < a-lactalbumin. The functionality of whey proteins is sensitive to the extent of denaturation.

[0047] Caseins have excellent solubility and heat stability above pH 6. The caseins precipitate out of milk at pH 4.6. Casein proteins include a family of related phosphoproteins (aSl, aS2, β, κ) and are a major component of cheese.

[0048] Milk proteins according to the invention include, for example, protease inhibitors in the whey portion of the milk (milk serum) proteins such as serpin peptidase inhibitor, clade B (ovalbumin), member 4 (bovine)( SERPINB4); serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2 (bovine) (SERPINF2); serpin peptidase inhibitor, clade D (heparin cofactor), member 1 (bovine), (SERPIND1); serpin peptidase inhibitor, clade B (ovalbumin), member linter-alpha-trypsin inhibitor heavy chain 2 (bovine)( SERPINB1); serpin peptidase inhibitor, clade G (CI inhibitor), member 1 (bovine) (ITIH2), serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1 (bovine)(SERPINFl); inter-alpha-trypsin inhibitor heavy chain 1 (bovine)( ITIH1); serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 (bovine)( SERPINA3); serpin peptidase inhibitor, clade C (antithrombin), member 1 (bovine)( SERPINC1); inter-alpha-trypsin inhibitor heavy chain family, member 4 (bovine)(ITIH4); serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 (bovine)(

SERPINA1); serpin peptidase inhibitor, clade A (bovine) (SERPINA5); SERPINX; cystatin C (bovine)(CST-3); cystatin E/M (bovine)(CST6); this list includes orthologs of the above from another dairy animal. [0049] Milk proteins according to the invention also include complement proteins, which is linked to the protease inhibitor system, as protease inhibitors can influence the activation of the complement system. Complement proteins of interest include CD59 molecule, complement regulatory protein (bovine)( CD59); complement factor D (adipsin)(bovine)(CFD); complement component 6(bovine)(C6); complement component 9(bovine)(C9); complement factor H

(bovine)(CFH); complement component 7 (bovine)(C7); complement factor B (bovine) (CFB); complement component 3 (bovine) C3 ; complement component 4 binding protein, alpha (bovine)(C4BPA); CD5 molecule-like (bovine) (CD5L); complement component 4A

(bovine)(C4a); complement factor I (bovine) (CFI); this list includes orthologs of the above from another dairy animal.

[0050] Milk proteins according to the invention also include other known immune active proteins, such as antibacterial proteins, for example, mucin 15, cell surface associated (bovine) (MUC15); mucin 16, cell surface associated (bovine) (MUC16); cathelicidin 2

(bovine)(CATHL2); cathelicidin 4 (bo vine)(CATHL4); cathelicidin 1 (bovine)(CATHLl);

immunoglobulin J polypeptide, linker protein for immunoglobulin alpha and mu polypeptides (bovine)(IGJ); secreted phosphoprotein 1 (bovine)(SPPl); ribonuclease, RNase A family, 1 (bovine)(RNASEl); butyrophilin, subfamily 1, member Al (bovine)(BTNlAl); glycosylation- dependent cell adhesion molecule 1 (bovine) (GLYCAM1); clusterin (bovine) (CLU);

lactoperoxidase (bovine)(LPO); lactotransferrin (bovine)(LTF); xanthine dehydrogenase (bovine) (XDH); CD14 molecule (bovine) (CD14); this list includes orthologs of the above from another dairy animal.

[0051] Other milk proteins according to the present invention include immunoglobulins, such as polymeric immunoglobulin receptor (bovine) (PIGR); joining chain of multimeric IgA and IgM (bovine) (IGJ); Ig kappa chain (bovine)(IGK); immunoglobulin lambda locus (IGL); immunoglobulin M (bovine) (IGM); this list includes orthologs of the above from another dairy animal.

[0052] The nonfat milk fraction, either prior to, or together with the optional returned, composition comprising MFGM components, is then subjected to UV-C treatment to accomplish pasteurization as described elsewhere herein. In one embodiment, the nonfat milk fraction is treated after collection and before addition of a composition comprising MFGM components. The composition comprising MFGM components, or source of MFGM components such as whey or buttermilk, may be UV-C treated prior to additional processing, as UV-C treatment efficacy is enhanced when the liquid has a low viscosity and is more transparent. [0053] Figure 6 shows a flow chart of an embodiment of the present invention for the production of MPC 85 milk or other concentrated milk product of the present invention (also called SUPPLAMILK in the flowcharts). Milk protein concentrates are concentrated preparations of milk proteins that contain milk proteins in the same or a similar ratio to each other as milk or nonfat milk. The processes to prepare milk protein concentrates allow the proteins to stay in their native state and largely un-denatured. Milk protein concentrates may be prepared by filtration processes, such as microfiltration, ultrafiltration and diafiltration. In one embodiment, both ultrafiltration and diafiltration are used. These processes use membrane separation technology to remove the majority of lactose and soluble minerals while retaining the milk protein. MPCs are manufactured ranging in protein content from 42 to 90 percent.

Common MPC products are MPC 42, MPC 70, MPC 80, MPC 85 and MPI (which typically contains 90 percent or more protein by weight). MPC is typically made from skim milk, resulting in fat levels of less than 3 percent. As a rule of thumb, as the protein content of MPCs increases, the lactose levels decrease. For example, NFDM contains about 34 to 36 percent protein and 52 percent lactose, while MPC 42 contains 42 percent protein and 46 percent lactose, and MPC 80 contains 80 percent protein and 5 to 6 percent lactose, respectively. MPCs with even higher protein content also are available. They are generally known as MPIs with a minimum of 90 percent protein (generally in the range of 90 to 91 percent protein by weight) and micellar casein concentrates with 93 to 94 percent protein. Therefore, in Figure 6, the flowchart shows one embodiment of the production of a milk prepared by the processes of the present invention. Raw milk is used to prepare a nonfat raw milk as discussed elsewhere herein, and optionally, a composition comprising MFGM as described herein may be added. A non-thermal

pasteurization step may be performed, followed by the filtration step, e.g., ultrafiltration, to produce the MPC of the desired concentration and lactose level. Optionally, remaining lactose may be removed via enzymatic hydrolysis, as described elsewhere herein, followed by optional low-temperature drying as described herein to produce the milk product of the present invention. Figure 7 shows tan embodiment of the processes of the present invention incorporating a composition comprising MFGM to make an MPC 85 preparation.

[0054] The suitability of batches of milk prepared by the processes of the invention may be determined by measurement of the level of and/or activity of one or more of the proteins identified hereinabove, compared to raw milk. Optionally, the milk which has been treated according to the methods of the instant invention to result in an immune-active milk composition has at least 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80%, at least 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 74%, at least 73%, at least 72%, at least 71%, at least 70%, at least 69%, at least 68%, at least 67%, at least 66%,at least 65%, at least 64%, at least 63%, at least 62%, at least 61%,at least 60%, at least 59%, at least 58%, at least 57%, at least 56%,at least 55%, at least 54%, at least 53%, at least 52%, at least 51%,at least 50%, at least 49%, at least 48%, at least 47%, at least 46%, at least 45%, at least 40%, at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, at least 26%, at least 25%, or at least 20% of the amount of, and/or activity of, a milk protein of the invention, as compared to raw milk, either as a standardized raw milk or to a specific sample of raw milk. In one embodiment, the immune-active milk composition has at least 90% of the level of, or activity of, at least one protein described herein.

[0055] Resultant immune active milk compositions may be stored prior to consumer use by methods known in the art, i.e., methods known for storage of liquid milk or powdered milk. In one embodiment, the liquid milk and powdered milk formulations are stored under refrigeration.

[0056] The present invention also includes an immune active milk composition of the present invention according to Figures 5, 6, and 7 (called SUPPLAMILK in the figure). This composition, in one embodiment, comprises a UV-C treated nonfat milk. In another embodiment, the immune active milk composition of the present invention includes a UV-C treated nonfat milk and also a composition comprising MFGM components, pasteurized either by UV-C pasteurization or by other non- thermal pasteurization methods, in either liquid or dried form. The compositions of the present invention may also be milk protein concentrates, as discussed herein. The dried composition, in one embodiment, is dried using low-temperature drying methods.

[0057] Immune-active milk compositions of the present invention may be used to treat, reduce and/or prevent various conditions brought about by a dysfunctional immune response. For example, one example of a dysfunctional immune response is asthma and allergic diseases. The method therefore includes a method to reduce or prevent incidence of allergic disease and asthma by administering an immune-active milk composition prepared by the methods of the present invention. Amounts to treat to reduce and/or prevent incidence of allergic disease and asthma include a therapeutic amount. Dosage would be similar to normal intake of milk at each age, e.g., for children, about 200-300 mL per day is a normal intake of milk, which is equivalent to about 24 to 35 g of the powdered milk composition of the invention, for example. Suitable dosages include between about 0.1 g and about 1000 g, about 1 g to about 500 g, from about 5 g to about 100 g, from about 10 g to about 80 g, from about 20 g to about 60 g, or about 24 g to about 35 g; or at least about 2 g, at least about 2 g, at least about 6 g, at least about 8 g, at least about 10 g, at least about 12 g, at least about 14 g, at least about 16 g, at least about 18 g, at least about 20 g, at least about 22 g, at least about 24 g, at least about 26 g, at least about 28 g, at least about 30 g, at least about 32 g, at least about 34 g, at least about 36 g, at least about 40 g or higher amounts. Suitable individuals for treatment using the immune-active milk compositions are children, such as those under age 1, those under age 2, or older children, as well as adults of any age.

[0058] Another example of a dysfunctional immune response is immunosenescence which describes the gradual degradation of immune activity brought on through aging. An impaired function of both the innate and acquired arms of the immune system is observed, and results in a higher susceptibility to infectious disease, which can be observed by the reduction of successful immune responses to a vaccine in an age-dependent proportion of cases. Poor response to vaccines is a common manifestation of immunosenescence. The method therefore includes a method to reduce or prevent immunosenescence by administering an immune-active milk composition prepared by the methods of the present invention. Amounts to treat to reduce and/or prevent immunosenescence include a therapeutic amount. Dosage would be similar to normal intake of milk for the elderly, about 15 g of the powdered milk composition of the invention, for example. Suitable dosages include between about 0.1 g and about 1000 g, about 1 g to about 500 g, from about 2 g to about 100 g, from about 3 g to about 80 g, from about 5 g to about 60 g, or about 10 g to about 35 g; or at least about 2 g, at least about 2 g, at least about 6 g, at least about 8 g, at least about 10 g, at least about 12 g, at least about 14 g, at least about 16 g, at least about 18 g, at least about 20 g, or higher amounts. Optionally, the composition of the invention (as a powder) is given in divided doses, such as 3 times per day. Suitable individuals for treatment using the immune-active milk compositions are older adults, such as those in their 5 ^th , 6 ^th , 7 ^th , 8 ^th , 9 ^th or 10 ^th decades of life, particularly those adults who have demonstrated immune senescence previously.

[0059] Another example of a dysfunctional immune response is stress induced immune suppression. Stress induced immune response is also known as "Olympic Village Fever", has demonstrated a link between high levels of exercise induced stress and greater susceptibility to communicable diseases. Another example of a dysfunctional immune response is immune- compromised individuals, such as those with transplants or diseases causing immune

suppression, such as AIDS. Alternatively, the compositions of the invention may be used to enhance muscle recovery after, for example, intense exercise. The method therefore includes a method to reduce or prevent stress induced immune suppression and/or augment immunity in immune-compromised individuals and/or enhance muscle recovery by administering an immune- active milk composition prepared by the methods of the present invention. Amounts to treat to reduce and/or prevent stress induced immune suppression include a therapeutic amount. Dosage would be similar to normal intake of milk for an adult, e.g., about 200-300 mL per day is a normal intake of milk, which is equivalent to about 24 to 35 g of the powdered milk composition of the invention, for example. Suitable dosages include between about 0.1 g and about 1000 g, about 1 g to about 500 g, from about 5 g to about 100 g, from about 10 g to about 80 g, from about 20 g to about 60 g, or about 24 g to about 35 g; or at least about 2 g, at least about 2 g, at least about 6 g, at least about 8 g, at least about 10 g, at least about 12 g, at least about 14 g, at least about 16 g, at least about 18 g, at least about 20 g, at least about 22 g, at least about 24 g, at least about 26 g, at least about 28 g, at least about 30 g, at least about 32 g, at least about 34 g, at least about 36 g, at least about 40 g or higher amounts. Suitable individuals for treatment using the immune-active milk compositions are children, as well as adults of any age.

[0060] Therapeutically effective amounts of the immune-active milk compositions of the invention can be any amount or dose sufficient to bring about the desired effect and depend, in part, on the severity and stage of the condition, the size and condition of the patient, as well as other factors readily known to those skilled in the art. The dosages can be given as a single dose, or as several doses, for example, divided over the course of several weeks, as discussed elsewhere herein.

[0061] In one embodiment, the immune-active milk compositions are administered orally. Oral administration of immune-active milk compositions are well known to those skilled in the art. Immune-active milk compositions may be used to replace milk in any of or all of an individual's dairy consumption to gain the benefits described herein and/or treat the conditions as described herein.

[0062] Administration can be on an as-needed or as-desired basis, for example, once- monthly, once-weekly, daily, or more than once daily. Similarly, administration can be every other day, week, or month, every third day, week, or month, every fourth day, week, or month, and the like. Administration can be multiple times per day. When utilized as a supplement to ordinary dietetic requirements, the composition may be administered directly to the individual or otherwise contacted with or admixed with daily feed or food. When utilized as a daily feed or food, administration will be well known to those of ordinary skill. [0063] Administration can also be carried out on a regular basis, for example, as part of a treatment regimen in the individual. A treatment regimen may comprise causing the regular ingestion by the individual of an inventive composition in an amount effective to enhance immune function or reduce dysfunctional immune function in the individual. Regular ingestion can be once a day, or two, three, four, or more times per day, on a daily or weekly basis.

Similarly, regular administration can be every other day or week, every third day or week, every fourth day or week, every fifth day or week, or every sixth day or week, and in such a regimen, administration can be multiple times per day. The goal of regular administration is to provide the individual with optimal dose of an inventive composition, as exemplified herein.

[0064] The compositions provided herein are, in one embodiment, intended for "long term" consumption, sometimes referred to herein as for 'extended' periods. "Long term" administration as used herein generally refers to periods in excess of one month. Periods of longer than two, three, or four months comprise one embodiment of the instant invention. Also included are embodiments comprising more extended periods that include longer than 5, 6, 7, 8, 9, or 10 months. Periods in excess of 11 months or 1 year are also included. Longer terms use extending over 1, 2, 3 or more years are also contemplated herein. In some individuals, it is envisioned that the individual would continue consuming the compositions for the remainder of the individual's life on a regular basis. "Regular basis" as used herein refers to at least weekly, dosing with or consumption of the compositions. More frequent dosing or consumption, such as twice or thrice weekly are included. Also included are regimens that comprise at least once daily consumption. The skilled artisan will appreciate that the reduction of or prevention of a dysfunctional immune response may be a valuable measure of dosing frequency. Any frequency, regardless of whether expressly exemplified herein, that allows maintenance of a functional immune response within acceptable ranges can be considered useful herein.

[0065] As used herein, the term "oral administration" or "orally administering" means that the mammal ingests, or a caretaker is directed to feed, or does feed, the individual one or more of the compositions described herein. The immune-active milk compositions of the present invention may be used as a food product, which will include foods and nutrients intended to supply necessary dietary requirements of a human being as well as other human dietary supplements. In a one embodiment, the food products formulated for human consumption are complete and nutritionally balanced, while in others they are intended as nutritional supplements to be used in connection with a well-balanced or formulated diet.

[0066] In another embodiment, the composition is a food supplement, such as drinking water, beverage, liquid concentrate, gel, yogurt, powder, granule, paste, suspension, chew, morsel, treat, snack, pellet, pill, capsule, tablet, or any other delivery form including milk, powdered milk, or incorporated into any foods containing dairy as known in the art. The nutritional supplements can be specially formulated for consumption by a particular species or even an individual mammal, such as companion mammal, or a human. In one embodiment, the nutritional supplement can comprise a relatively concentrated dose of the immune-active milk composition such that the supplement can be administered to the individual in small amounts, or can be diluted before administration to an individual. In some embodiments, the nutritional supplement or other MCT-containing composition may require admixing with water or the like prior to administration to the individual, for example to adjust the dose, to make it more palatable, or to allow for more frequent administration in smaller doses.

[0067] In some embodiments, the individual is specifically a human. Other individuals include mammals such as companion animals, such as a dog or cat.

[0068] While various aspects and features of certain embodiments have been summarized above, the following detailed description illustrates a few embodiments in further detail to enable one of skill in the art to practice such embodiments. The described examples are provided for illustrative purposes and are not intended to limit the scope of the invention.

[0069] In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the described

embodiments. It will be apparent to one skilled in the art, however, that other embodiments of the present invention may be practiced without some of these specific details. Several embodiments are described and claimed herein, and while various features are ascribed to different embodiments, it should be appreciated that the features described with respect to one embodiment may be incorporated with other embodiments as well. By the same token, however, no single feature or features of any described or claimed embodiment should be considered essential to every embodiment of the invention, as other embodiments of the invention may omit such features.

[0070] Unless otherwise indicated, all numbers used herein to express quantities, dimensions, and so forth used should be understood as being modified in all instances by the term "about." In this application, the use of the singular includes the plural unless specifically stated otherwise, and use of the terms "and" and "or" means "and/or" unless otherwise indicated. Moreover, the use of the term "including," as well as other forms, such as "includes" and "included," should be considered non-exclusive. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one unit, unless specifically stated otherwise. EXAMPLES

[0071] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1

[0072] UV-C treatment of raw milk

[0073] Milk was UV-C pasteurized at 1000 J/liter. Samples of raw milk and two separate

UV-C samples were compared to standard pasteurized whole milk and pasteurized and spray dried whole milk powder.

[0074] LC/MS chromatography. Figure 1 shows LC/MS TIC chromatograms of the raw samples (first and second chromatograms) and 1000 J samples (third to sixth chromatograms.) Using an overall statistical analysis of protein change using Perseus software (available from MaxQuant) which is useful to analyze expression proteomics, posttranslational modifications, interaction proteomics, machine learning for biomarker discovery, time series analysis and cross- omics data analysis. No proteins were statistically significantly different between the raw v. 1000 J samples. Following this test, the samples grouped based on gene ontology (biological function) and plotted the difference between the two samples. Ultracentrifugation at pH 4.6, to make milk serum was performed on the raw milk and the 1000 J treated milk, to remove denatured proteins.

[0075] The supernatant was prepared using filter-aided sample preparation (FASP) was employed, resulting in two samples obtained from the raw milk and 4 samples from the 1000 J treated milk. Supernatants were prepared in the presence of high concentrations of detergent. Disulfide bridges were reduced with dithiothreitol (DTT). Detergent micelles and protein detergent complexes were dissociated in the presence of 8 M urea. The detergent, DTT and other low-molecular-weight components were removed by utrafiltration (Microcon units) facilitated by centrifugation. Thiols were carboxyamidomethylated with iodoacetamide (IAA) and excess reagent was removed by ultrafiltration. In repeated washes with 8 M urea any remaining detergent was depleted from the proteins. The protein suspension was digested with

endoproteinase, and the resulting peptides were collected as a filtrate. High-molecular-weight molecules including the endopro tease are retained on the filter. When nuclei or total cell ly sates are processed in the units, DNA is retained on the filter.

[0076] Protease inhibitors have been linked to allergy and asthma. It was found that protease inhibitors, a heat-sensitive group of milk proteins, significant degradation is seen for the pasteurized and spray-dried samples. It was found that proteins (shown by their gene code) were treated, all the data points showed identical intensities for the samples, but significant degradation was observed in the pasteurized and spray dried samples. While spray-dried samples and pasteurized samples show decreases, none of the proteins tested showed a clear decreasing trend after the UV-C treatment. See Figure 2.

[0077] Complement proteins. Complement system is an innate immune system. C3 is one of the central components of this system, and also is the most abundant protein of this system in milk. The complement system is linked to the protease inhibitor system, as protease inhibitors can influence activation of the complement system. Significantly lower levels of complement proteins are shown in the pasteurized and spray-dried samples. While spray-dried samples and pasteurized samples show decreases, none of the proteins tested showed a clear decreasing trend after the UV-C treatment. See Figure 3.

[0078] Other immune-active proteins were also tested. See Figure 4. SPP1 (osteopontin) and LTF (lactoferrin) are the most abundant immunologic ally- active proteins in milk.

Lactoferrin is especially known for its temperature sensitivity, as it easily denatures even under low-pasteurization conditions. While spray-dried samples and pasteurized samples show decreases, none of the proteins tested showed a clear decreasing trend after the UV-C treatment.

[0079] The conclusions drawn from this study show that overall protein, and specific known immune-active proteins, are not decreased after 1000 J UV-C.

Example 2

[0080] Preparation of UV-C milk product. Raw cow's milk was obtained, and separated into a nonfat milk fraction and a fat fraction through centrifugation according to standard methods. The resultant nonfat milk had between 0.1% and 0.3% fat. The nonfat milk fraction was enzymatically treated to hydrolyze lactose using LACTOZYM PURE (Novozym, Denmark), according to manufacturer instructions.

[0081] A composition comprising milk fat globule membrane components (MFGM) obtained from raw buttermilk by methods known in the art and combined with the raw nonfat milk. The MFGM supplemented nonfat milk was pasteurized with a SurePure SP40 turbulent flow UV-C pasteurizing system (obtainable from SurePure, AG., Capetown, South Africa; see U.S. 6,916,45); the milk is processed in this system two times to ensure achievement of 2000 J/1.

[0082] The solution comprising the UV-C pasteurized nonfat milk was dried into a powder at low temperatures (below 45°C) or through freeze-drying. Procedures used exposed the milk to a maximum temperature of 42°C for less than 2 minutes. Alternatively, an MPC 85 was prepared. The final fat concentration of the resultant product is approximately 1-1.5%. [0083] A milk prepared according to the present invention, according to the process of

Figure 6, without the optional MFGM addition and without the optional enzymatic hydrolysis of lactose, and with the optional low-temperature drying, has the following characteristics:

Example 3

[0084] A composition prepared according to the methods of Example 2 is obtained and analyzed by the methods of Example 1. The identified proteins were compared between the composition of the invention (called SUPPLAMILK NF) and nonfat milk powder, obtained by conventional methods. As can be seen, the amount of the measured proteins remaining (by MS) is significantly higher for all proteins other than SPP1.

[0085] Many clinical trials have proven raw cow's milk consumption sufficiently activates human immune systems to significantly reduce allergy and respiratory infection development in children. Additional studies have shown milk protein consumption enhances elderly vaccine response. Milk products prepared in accordance with the present invention retain most of the immune active proteins found in raw milk, while standard nonfat milk powder does not. Many researchers believe these immune active proteins are responsible for raw milk's ability to stimulate immune responses. The below table shows a summary for each protein class. Overall, the milk product of the present invention contains a much greater percentage of each protein, compared with raw milk, than nonfat milk protein prepared by conventional methods.

Example 4

[0086] MARINA - large weaned infant study comparing intervention with SupplaMilk to pasteurized, homogenized and spray dried whole milk powder for respiratory infection, allergies and asthma. The immune-active milk composition (Intervention) to be tested is prepared by the following steps: USD A Grade A whole milk, non-homogenized, but continuously stirred during storage; pasteurized using turbulent flow ultraviolet light in the "C" bandwidth (200-280 nm) with 1,100 J of exposure per liter of milk; pathogen testing of the UV-C pasteurized milk and dried into a powder containing less than 5% moisture using a low temperature drying system with maximum temperature exposure of 41°C (106°F). This is compared to (comparator) USD A Grade A milk from same batch as Intervention, ultra-high temperature pasteurized, homogenized and spray dried to less than 5% moisture using conventional high temperature spray drying techniques. The immune active milk composition is found to found to enhance infant immunity compared to milk not treated by methods of the invention.

Example 5

[0087] Immunosenescence - compare elderly vaccine response with the immune-active milk of the invention intervention compared to pasteurized, homogenized and spray dried whole milk powder. Vaccination is the most effective strategy against infectious disease; however, immunological function in the elderly often falters due to immunosenescence-a condition in which the immune system mounts a less robust response to pathogens. Likewise,

immunosenescence often decreases the efficacy of vaccines in the elderly, which makes this particular demographic more susceptible to severe infection. Indeed, several reports have demonstrated that older individuals lack robust responses to conventional vaccines, and particularly antibody responses to influenza and tick-borne encephalitis vaccine were impaired in the elderly. These data support the concept that immunosenescence is an important issue to overcome in the development of effective vaccine responses in the aged. Vaccines have proven to be one of the most effective ways to prevent infection; however, 90% of deaths in the elderly are from vaccine -preventable diseases. Taken together with the fact that Western societies are in the midst of a profound demographic change in which there are increased proportions of persons living well beyond 65, these data suggest that alternative strategies may be required to enhance immune responses in aged populations (>60 years old).

[0088] The impact of diet, and particularly inadequate protein intake on immune function has previously been described. Conversely, we have formerly demonstrated that a diet supplemented with whey protein significantly enhanced vaccine response by increasing antibody titers against Streptococcus pneumoniae vaccine compared to soy protein as a control. Given the proven health benefits, including increased vaccine response, in people who consume adequate quantities of high quality protein, we hypothesize:The daily consumption of a UV pasteurized milk supplement (SUPPLAMILK) will significantly increase vaccine efficacy in the elderly. To test this hypothesis, we propose the following specific aims: In Aim 1, we will characterize and quantify tetanus and diptheria- specific antibodies from the peripheral blood of participants. In Aim 2, we will determine how consumption of UV pasteurized milk supplements correlate with tetanus and diptheria- specific systemic IgG. These aims are based on prospectively studying recruited volunteers from one or more community residences in Northern California that wish to participate in our study.

Recruitment and Screening

[0089] Potential participants living in community residences will be approached to assess their willingness to hear about the study. The study will be explained to interested individuals and a consent form provided. Individuals will be encouraged to ask questions and discuss the study with their friends and family before a follow-up appointment is scheduled for a final decision about participation. Given the personal health information (the individual's name and whether they have had either Tdap or Boostrix vaccine in the past five years) is necessary to identify eligible subjects, we are requesting a HIPAA waiver for recruitment (see attached). The names of the eligible individuals will be written on a piece of paper by the clinical research coordinator or Principal Investigator and if the individual has decided to participate this will be noted on the paper. The purpose of this piece of paper is to ensure that we do not approach eligible individuals more than once. The paper will be kept in a locked cabinet and destroyed at completion of enrollment. [0090] Inclusion criteria include participants that are greater than 60 years of age and are willing to participate. Subjects will have to undergo a physical examination and health assessment by a physician to ensure the absence of exclusion criteria which include:

administration of a Tdap or Boostrix vaccine in the past five years, regular consumption of greater than one unit of milk and/or milk products (milk, yogurt, fresh cheese, etc) a day at the time of enrollment, known milk allergy, food faddism, other non-traditional diet, prolonged consumption of dairy supplements (greater than one daily during the previous four (4) weeks), use of tobacco products in the previous 10 years, underlying neoplasia or immunological disease, including hypergammaglobunemia, renal disease or failure, use of steroids or

immunosuppressive drugs in the previous eight (8) weeks, reduced physical activity (New York Heart Association (NYHA), classes III-IV).

Human Samples

[0091] Peripheral blood samples will be collected at the time of enrollment (week 0) and serum will be stored for later analysis. Participants will then be randomized into two groups and provided with equal concentration and quantity of either soy or SupplaMilk protein supplement, which is provided in powdered form in color-coded, single-serving bags. Both participants and examiners will be blinded as to what type of protein they are receiving. Participants will be asked to consume two servings of protein powder (6 grams/packet) with 4 ounces of water twice per day, with meals for a total of 8 weeks. At week four participants will be vaccinated with Boostrix vaccine (GlaxoSmithKline, USA), which includes Tetanus Toxoid, Reduced Diptheria Toxoid and Acellular Pertussis Vaccine, Adsorbed. A second blood draw will be obtained 4 weeks after vaccination (week 8) and serum collected for tetanus and diphtheria- specific antibody quantification.

Protein Supplement

[0092] Tamarack Biotics LLC will provide the UV pasteurized milk protein supplement

(SupplaMilk). Importantly, raw milk consumption correlates with health benefits, including significantly decreased incidence of respiratory disease and allergy in children. Conversely;

however, consumption of raw milk does come with notable risks caused by ingestion of milkborne pathogens, which far outweighs the health benefits.. In order to mitigate these risks, while still retaining the immunologically active factors in raw milk, Tamarack will employ a nonthermal, turbulent flow ultraviolet pasteurization technique, which has successfully been commercialized for water

decontamination, pasteurization of fruit juices and most recently pasteurization of milk, as described by Crook et al, 2015. Briefly, raw milk will be exposed to UV-C light in turbulent flow at a rate of 4,000 L/hour and applied dose of 2,000 J/L. [0093] After pasteurization, the milk will be dried using a high volume air dryer with maximum temperature exposure of 42°F for 2 minutes, packaged into 25 kg aluminum storage bags with oxygen scavenger and stored frozen.

[0094] Prior to participant consumption, the batch of UV pasteurized milk powder will be analyzed by third-party National Food Laboratory, LLC in which the following minimum guidelines must be met, as stipulated by the FDA Pasteurized Milk Ordinance (PMO, 2013):

Individual producer milk is milk that is still on the farm.

² Commingled milk is milk that has left the farm and has been mixed with other individual producer milk in a tank, either during shipment or at the processing plant.

[0095] Soy protein will be purchased commercially from ADM, Minneapolis, USA as previously reported.

[0096] Each participant will require a total of 112 servings for the 8-week duration of the study; a 4-week supply will be provided at the first visit (week 0) and at the time of vaccine administration (week 4). Six grams of UV pasteurized milk protein (Tamarack Biotics LLC, Fresno, CA USA) or 6 grams of low isoflavone soy protein (ADM, Minneapolis, MN) and 2 grams of a flavoring ingredient (Nesquick ®, Nestle) will be measured into single serving bags. Bags will be color-coded based on protein type and both participants and examiners will be blind to the underlying code. All subjects will be instructed on how to prepare and consume the supplement— with water or added to a non-hot food, twice a day in addition to their normal diet. It will be advised to the participants that the supplement should not be used as a substitute for any part of their normal diet.

BOOSTRIX Vaccine [0097] BOOSTRIX (Tetanus Toxoid, Reduced Diptheria Toxoid and Acellular Pertussis

Vaccines, Adsorbed) is a noninfectious, sterile, single-shot vaccine given as an intramuscular injection. Importantly, it is the only Tdap vaccine that is approved by the FDA for adults 65 years and older (http://www.fda.gov/downloads/BiologicsBloodVaccines/UCM1528 42.pdf). It contains tetanus toxoid, diphtheria toxoid, and pertussis antigens (inactivated pertussis toxin and fomaldehy de-treated filamentous hemagglutinin and pertactin). The antigens are the same as used in INFANRIX, but BOOSTRIX is formulated with reduced quantities of these antigens. Each antigen is individually adsorbed onto aluminum hydroxide, which acts as an adjuvant to increase immune responses, and in total represents 0.39 mg aluminum per dose. In a typical 0.5 mL dose, there are 5 Lf tetanus toxoid, 2.5 Lf diphtheria toxoid, 8 meg of inactivated PT, 8 meg of FHA, and 2.5 meg of pertactin.

[0098] Adverse reactions were reported in >15% people over 60 and include: pain, redness and swelling at the injection site; headache; fatigue; gastrointestinal problems.

Measurement of Antibody Concentration

[0099] Blood samples will be obtained at week 0 and week 8, serum will be separated and stored at -20° C until being analyzed for concentration of immunoglobulin G (IgG) antibodies specific to tetanus or diphtheria. Toxoid- specific antibodies will be quantified using commercially available enzyme-linked immunosorbant assays (ELISA). Positive and negative controls will also be analyzed. Antibody concentration will be determined from samples taken at week 0 ([Abo]) and week 8 ([Abg]) for both toxins and will be expressed quantitatively ^g/mL) based on standard curves for each ELISA. Raw values will be log-transformed to approximate normality and reduce outliers.

Quantifying Vaccine Responsiveness

[00100] Type-specific antibody concentration post-vaccine ([Ab ₈ ]T _X ) minus the concentration pre- vaccine ([AbohJ will be used to determine the change in antibody concentration for a given serotype (A[Ab]x _x ). The log-change in antibody concentration will be used to define a given serotype response (RT _x ). A log-change in antibody concentration of 1.5 or greater (A[Ab]x _x > 1.5) will be considered a positive serotype response and given a value of 1 (R _Tx = 1). The sum of the 14 serotype responses (∑RT _x ), out of 14 possible serotypes, will be used to define an individual's overall vaccine response, as shown previously.

Statistical analysis

[00101] Non-parametric statistical test will be used to determine raw values due to non- normality of the data. Mean and standard error of the mean (SEM) will be reported for the log- transformed change in antibody concentration for all 14 serotypes. Serotype response to a given serotype will be reported as the proportion of individuals with a positive response. Mean and SEM will be reported for the sum of overall vaccine responses. The average log-change in antibody concentration, proportion of positive responders and average overall vaccine response will be compared in the soy and pasteurized raw milk treatment groups. Data between different groups will be compared using Student's i-test for continuous variable and chi square test for categorical variables. All analyses will be two-tailed and p-values <0.05 will be considered statistically significant. The statistical power for each test was determined and a theoretical n was predicted from the population variance and difference in mean. All analyses will be performed using Prism 5.0 for Mac OSX (GraphPad Software Inc, LaJolla, USA).

Diet Record

[00102] Participants will be instructed to record all food consumed for 3 days prior to commencement of week 0 and again at week 4. Detailed instructions will be given on how to accurately record food intake and participants will be provided a food diary for record keeping. Completed records will be returned to the study coordinator at week 4 and week 8. Written records will be analyzed using diet analysis software Nutrition Data System for Research (NDSR, University of Minnesota, USA). Briefly, daily kilocalorie and protein consumption will be calculated by the system and theoretical values for inadequate protein (IP), RDA and optimal protein (OP) will be calculated based on body weight (g/kg body weight).

[00103] The study will conclude that daily consumption of a UV pasteurized milk supplement (SUPPLAMILK) significantly increases vaccine efficacy in the elderly.

[00104] The description of the various embodiments has been presented for purposes of illustration and description, but is not intended to be exhaustive or limiting of the invention to the form disclosed. The scope of the present invention is limited only by the scope of the following claims. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments described and shown in the figures were chosen and described in order to explain the principles of the invention, the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated. All references cited herein are incorporated in their entirety by reference.