阅读说明：本技术 制备混合型过敏原组合物的方法 (Method for preparing mixed allergen composition ) 是由 C·科宁 O·M·威赫 于 2020-01-23 设计创作，主要内容包括：提供了制备混合型过敏原产品的方法,其中所述混合型过敏原产品基本上不含可复制的活微生物。(Methods of making a hybrid allergen product, wherein the hybrid allergen product is substantially free of viable, reproducible microorganisms, are provided.)

1. A method of making a mixed allergen product substantially free of viable replicating organisms, comprising:

providing a loose mixed allergen material comprising 2 to 20 individual complete food allergens; and

sterilizing the loose mixed allergen raw material by dry heat sterilization, thereby obtaining the mixed allergen product.

2. The method of claim 1, further comprising providing 2 to 20 separate complete food allergens and blending together the 2 to 20 separate complete food allergens to obtain the loose mixed allergen material.

3. The method of claim 2, wherein the individual complete food allergen is selected from the group consisting of almonds, cashews, codfish, eggs, hazelnuts, milk, oats, peanuts, pecans, pistachios, salmon, sesame, shrimp, soybeans, walnuts, and wheat.

4. The method of claim 2 or 3, wherein the admixing further comprises admixing the 2 to 20 separate complete food allergens with one or more than one bulking agent.

5. The method of any one of claims 2 to 4, wherein the blending comprises charging the 2 to 20 separate complete food allergens into a ribbon blender set at about 10Hz to about 50Hz for about 10 minutes to about 50 minutes.

6. The method of any one of claims 2 to 5, wherein after blending, the loose mixed allergen material is discharged into a lined vat.

7. The method of any one of claims 1-6, wherein the dry heat sterilization is performed at a temperature of about 65 ℃ to about 175 ℃ for about 18 hours to about 72 hours.

8. The method of claim 7, wherein the dry heat sterilization comprises using a sterilization system having an oven and a heating chamber, wherein the oven provides heat to the heating chamber from about 250,000BTU to about 750,000 BTU.

9. The method of claim 7 or 8, wherein the loose mixed type allergenic material is first packaged in a heat-resistant package prior to sterilization.

10. The method according to any one of claims 7 to 9, wherein a temperature probe is placed with the loose mixed allergen material during sterilization to monitor the temperature of dry heat sterilization.

11. The method of any one of claims 1 to 10, wherein the mixed allergen product has an aerobic bio-plate count of at least about 1log reduction to at least about 4log reduction as compared to a corresponding bulk mixed allergen raw material that has not been sterilized.

12. The method of any one of claims 1 to 11, wherein the mixed allergen product has less than about 100,000CFU/g of aerobic organisms.

13. The method of any one of claims 1 to 12, wherein the mixed allergen product has a coliform population of less than about 10 CFU/g.

14. The method of any one of claims 1 to 13, wherein the mixed allergen product has less than about 10CFU/g e.

15. The method of any one of claims 1 to 14, wherein the mixed allergen product has less than about 10CFU/g enterobacter.

16. The method of any one of claims 1 to 15, wherein the mixed allergen product has less than about 10CFU/g salmonella.

17. The method of any one of claims 1 to 16, wherein the mixed allergen product has a crotonobacterium content of less than about 10 CFU/g.

18. The method of any one of claims 1 to 17, wherein said mixed allergen product is negative for listeria species or crohn's disease species in a PCR-based assay per 25 grams of said mixed allergen product.

19. The method of any one of claims 1 to 18, wherein the mixed allergen product has a mold count of less than about 10 CFU/g.

20. The method of any one of claims 1 to 19, wherein said mixed allergen product is negative for salmonella species in a PCR-based assay per 375 grams of said mixed allergen product.

21. The method of any one of claims 1 to 20, wherein the mixed allergen product has less than about 10CFU/g staphylococcus aureus.

22. The method of any one of claims 1 to 21, wherein the mixed allergen product has less than about 10CFU/g yeast.

23. The method of any one of claims 1 to 22, wherein the mixed allergen product has a fat content of from about 10% to about 20% by weight.

24. The method of any one of claims 1 to 23, wherein the protein integrity of the mixed allergen product is substantially similar to the protein integrity of a corresponding bulk mixed allergen raw material which has not been sterilized.

25. The method of claim 24, wherein the protein integrity of the mixed allergen product is determined by SDS-PAGE.

26. The method of any one of claims 1 to 25, wherein the mixed allergen product has a protein content of from about 40% to about 50% by weight.

27. The method of any one of claims 1 to 26, wherein the mixed allergen product has a moisture content of from about 1% to about 10% by weight.

28. The method of any one of claims 1 to 27, wherein the mixed allergen product has a water activity of from about 0.1 to about 0.6.

29. The method of any one of claims 1 to 28, wherein less than about 10% by weight of the mixed allergen product is in particles having a diameter greater than 500 microns.

30. The method of any one of claims 1 to 29, wherein the mixed allergen product is darker in color than the corresponding bulk mixed allergen raw material which has not been sterilized.

31. The method of any one of claims 1 to 30, wherein the hybrid allergen product has substantially similar taste characteristics as compared to a corresponding bulk hybrid allergen material without sterilization.

32. The method of any one of claims 1 to 31, wherein sterilizing further comprises one or more additional applications of dry heat sterilization.

33. A method of making a mixed allergen product substantially free of viable replicating organisms, comprising:

providing 2 to 20 individual complete food allergens;

sterilizing each of the 2 to 20 separate complete food allergens by dry heat sterilization, thereby obtaining 2 to 20 sterilized separate complete food allergens; and

blending together said 2 to 20 sterilized individual complete food allergens, thereby obtaining said mixed allergen product.

34. The method of any one of claims 1 to 33, further comprising blending the mixed allergen product with one or more probiotics.

35. The method of claim 34, wherein the one or more than one probiotic is selected from the group consisting of lactobacillus rhamnosus, corynebacterium faecalis, and bifidobacterium longum.

36. The method of any one of claims 1 to 35, further comprising admixing the mixed allergen product with one or more prebiotics.

37. The method of claim 36, wherein the prebiotic is a fructooligosaccharide.

38. The method of any one of claims 1 to 37, further comprising filtering the mixed allergen product.

39. The method of claim 38, wherein the mixed allergen product is filtered through a screen of a #5 mesh (US) screen to a #10 mesh (US) screen on a shaker for about 1 minute to about 10 minutes.

40. The method of any one of claims 1 to 39, further comprising passing the mixed allergen product through a rare earth magnet, a metal detector and a metal separator.

41. A mixed allergen product substantially free of viable replicating organisms produced by the method of any one of claims 1 to 40.

Background

Allergy is a condition of the immune system characterized by an allergic reaction to normally harmless environmental substances. Allergies are caused by allergens that may be present in a wide variety of sources including, but not limited to, pollen or other plant components, dust, mold or fungus, food, additives, latex, blood transfusions, animal or poultry dander, insect venom, radiocontrast agents, drugs or chemicals. Common allergic reactions include eczema, urticaria, hay fever, asthma, and reactions to venom. Mild allergies, such as hay fever, are very common in people and cause symptoms such as allergic conjunctivitis, itching, and runny nose. In some people, severe allergies to environmental allergens, dietary allergens or drugs can lead to life-threatening allergic reactions if left untreated.

Food allergy is an adverse immune response to food allergens, such as food proteins. Common food allergens are found in shellfish, peanuts, tree nuts, fish, milk, eggs, soybeans, and fresh fruits such as strawberries, mangos, bananas, and apples. Immunoglobulin e (ige) -mediated food allergy is classified as type I immediate hypersensitivity. These allergic reactions have an acute onset (from a few seconds to an hour) and the accompanying symptoms may include: angioedema (soft tissue swelling of the eyelids, face, lips, tongue, throat, and trachea); urticaria; itching of the mouth, throat, eyes and skin; gastrointestinal symptoms such as nausea, vomiting, diarrhea, stomach cramps, and abdominal pain; extravasation or nasal congestion; wheezing; shortness of breath; dysphagia; and anaphylaxis (a severe systemic anaphylaxis that can lead to death). It is estimated that 1 out of 12 children under 21 years of age is diagnosed with food allergies and that the healthcare cost for food allergies is over $ 240 billion per year, most of which is due to about 90,000 food-induced allergies per year in the united states alone. In addition, death events due to fatal food allergies occur each year.

Accordingly, there is a need in the art for allergen compositions that can prevent and/or treat allergy and methods of preparing allergen compositions for preventing and/or treating allergy.

SUMMARY

The present disclosure relates, at least in part, to methods of making hybrid allergen products that are substantially free of viable replicating organisms. For example, in certain embodiments, the method comprises: providing a loose mixed allergen material comprising 2 to 20 individual complete food allergens; sterilizing the loose mixed allergen raw material by dry heat sterilization, thereby obtaining a mixed allergen product.

In certain embodiments, the method further comprises: providing 2 to 20 individual complete food allergens and blending together the 2 to 20 individual complete food allergens resulting in a loose mixed type allergen material.

In other embodiments, the present disclosure provides a method wherein the 2 to 20 separate complete food allergens are selected from the group consisting of almonds, cashews, codfish, eggs, hazelnuts, milk, oats, peanuts, pecans, pistachios, salmon, sesame, shrimp, soybeans, walnuts, and wheat.

In another embodiment, the present disclosure provides a method wherein the admixing further comprises admixing the 2 to 20 separate complete food allergens with one or more bulking agents.

In certain embodiments of the present disclosure, blending comprises charging 2 to 20 individual complete food allergens for about 10 minutes to about 50 minutes in a ribbon blender set at about 10Hz to about 50 Hz. In other embodiments, the loose mixed allergen material is discharged into the liner vat after blending.

In certain embodiments, the methods of the present disclosure further comprise filtering the mixed allergen product. In some embodiments, filtering comprises passing the mixed allergen product through a #5 mesh (US) screen to a #10 mesh (US) screen on a shaker for about 1 minute to about 10 minutes.

In some embodiments, the methods of the present disclosure further comprise passing the mixed allergen product through a rare earth magnet, a metal detector, and a metal separator.

In certain embodiments, the methods of the present disclosure provide for applying dry heat sterilization, wherein the dry heat sterilization is performed at a temperature of about 65 ℃ to about 175 ℃ for about 18 hours to about 72 hours. In some embodiments, dry heat sterilization includes the use of a sterilization system having a furnace and a heating chamber, wherein the furnace provides heat to the heating chamber from about 250,000BTU to about 750,000 BTU. In certain embodiments, the loose mixed allergen material is first packaged in a heat resistant package and then dry heat sterilized. In certain embodiments, a temperature probe is placed with the loose hybrid allergen material during sterilization to monitor the temperature of dry heat sterilization.

In certain embodiments of the present disclosure, the hybrid allergen product has an aerobic bacterial bio-plate count of at least about 1log reduction to at least about 4log reduction as compared to a corresponding unsterilized, loose hybrid allergen material. In some embodiments, the mixed allergen product has less than about 10,000CFU/g aerobic bacterial organisms. In other embodiments, the mixed allergen product has a coliform group, Escherichia coli (Escherichia coli), enterobacter (Enterobacteriaceae), mold, Staphylococcus aureus (Staphylococcus aureus) and yeast of less than about 10 CFU/g. In certain embodiments, the mixed allergen product has less than about 10CFU/g Salmonella (Salmonella). In certain embodiments, the hybrid allergen product has less than about 10CFU/g of Cronobacter sakazakii (Cronobacter). In other embodiments, the mixed allergen product is negative for Listeria (Listeria) species, crohn's disease species, and salmonella species, respectively, in a PCR-based assay per 25 grams of mixed allergen product, and 375 grams of mixed allergen product.

In some embodiments of the present disclosure, the mixed allergen product has a fat content of about 10% to about 20% by weight.

In other embodiments, the protein integrity of the hybrid allergen product is substantially similar to the protein integrity of a corresponding unsterilized loose hybrid allergen raw material. In some embodiments, the protein integrity of the mixed allergen product is determined by SDS-PAGE.

In certain embodiments, the mixed allergen product has a protein content of about 40% to about 50% by weight.

In certain embodiments, the mixed allergen product has from about 1% to about 10% by weight moisture. In other embodiments, the mixed allergen product has a water activity of from about 0.1 to about 0.6.

In certain embodiments of the present disclosure, the mixed allergen product comprises less than about 10% by weight of particles having a diameter greater than 500 microns.

In some embodiments, the hybrid allergen product is darker in color than a corresponding bulk hybrid allergen raw material that has not been sterilized. In other embodiments, the hybrid allergen product has substantially similar taste characteristics as compared to a corresponding bulk hybrid allergen raw material that has not been sterilized.

In certain embodiments, the methods described in the present disclosure comprise one or more further applications of dry heat sterilization.

In an alternative embodiment, the present disclosure describes a method of making a mixed allergen product substantially free of viable replicating organisms comprising: providing 2 to 20 individual complete food allergens; sterilizing 2 to 20 individual complete food allergens individually by dry heat sterilization, thereby obtaining 2 to 20 sterilized individual complete food allergens; and blending together 2 to 20 sterilized separate complete food allergens, thereby obtaining the mixed allergen product.

In another embodiment, the present disclosure describes a method of making a mixed allergen product substantially free of viable and replicating organisms, wherein the method further comprises admixing the mixed allergen product with one or more probiotics selected from the group consisting of Lactobacillus rhamnosus (Lactobacillus rhamnosus), corynebacterium faecalis (anaerobe caccae), and bifidobacterium longum (bifidobacterium longum).

In yet another embodiment, the mixed allergen product is further blended with one or more prebiotics, for example, wherein the prebiotic is a fructooligosaccharide.

Also disclosed is a mixed allergen product substantially free of viable replicating organisms prepared by any of the methods disclosed herein.

Brief description of the drawings

FIG. 1 is a macroscopic image of a sample of unsterilized loose mixed type allergic raw material and a sample of loose mixed type allergic raw material heated in a laboratory oven at 95 deg.C, 105 deg.C, 110 deg.C or 120 deg.C for 5 minutes.

Fig. 2A is a schematic diagram showing a validation test 1 in which a tray and a sample cartridge containing a loose mixed type allergen material heat-resistant bag are placed in a dry heat sterilization chamber. B3 of trays 3, 4 and 12 has a compartment probe placed inside the box for temperature monitoring. B4 of trays 7 and 8 has a cell probe placed outside the cassette and B4 of tray 11 has a cell probe placed inside the cassette. Fig. 2B is a schematic diagram showing a validation test 2 in which a tray and a sample cartridge containing a loose mixed type allergen material heat-resistant bag are placed in a dry heat sterilization chamber. B3 of trays 5 and 14 has a compartment probe placed inside the box for temperature monitoring. B4 for trays 4 and 8 has a cell probe placed inside the box and B4 for trays 6 and 9 has a cell probe placed outside the box. The hatched box represents an empty box, without a heat-resistant bag containing loose mixed allergen material. The asterisk (") indicates a box with two heat-resistant pouches containing loose mixed allergen material. "P" denotes a cassette with a MadgeTech probe.

Fig. 3A is a graph showing the temperature profile of the dry heat sterilization chamber during validation test 1. Fig. 3B is a graph showing the temperature profile of the dry heat sterilization chamber during validation test 2. Each line represents a temperature reading of a single compartment probe or a MadgeTech probe.

Detailed Description

A method of making a mixed allergen product substantially free of viable replicating organisms is disclosed.

By "viable replicating organism" it is meant an organism that is capable of propagating, regenerating, proliferating and/or producing Colony Forming Units (CFU) on a plating medium.

By "sterilization" it is meant a process that substantially reduces the number of viable replicating organisms.

As used herein, "complete food allergen alone" refers to a food substance that contains all possible antigenic components of the food substance (e.g., allergenic proteins). The individual complete food allergens may include, but are not limited to, raw or processed food material, concentrated food material, and isolated food material.

As used herein, an "allergenic protein" is an antigenic component of a food allergen, which is directly or indirectly responsible for eliciting a biological allergic reaction. Allergenic proteins can include, but are not limited to, nut proteins such as hazelnut proteins (e.g., Cor a 1, Cor a 2, Cor a 6, Cor a 8, Cor a 9, Cor a 10, Cor a 11, Cor a 12, Cor a 13, and Cor a 14), cashew proteins (e.g., Ana o 1, Ana o 2, and Ana o 3), pistachio proteins (e.g., Pis v 1, Pis v 2, Pis v 3, Pis v 4, and Pis v 5), walnut proteins (e.g., Jug r 1, Jug r 2, Jug r 3, Jug r 4, Jug r 5, Jug r 6, Jug r 7, and Jug 8, Jug n1, Jug n 2, and Jug n 4), pecan american walnut proteins (e.g., Cari 1, Cari 2, and Cari 4), Almond proteins (e.g., Pru du 3, Pru du 4, Pru du 5, Pru du 6, and Pru du 8), peanut proteins (e.g., Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13, Ara h 14, Ara h 15, Ara h 16, and Ara h 17), and brazil nut proteins (e.g., Ber e 1 and Ber e 2). Sensitizing proteins can also include, but are not limited to, animal proteins such as egg proteins (e.g., Gal d 1, Gal d 2, Gal d 3, Gal d 4, Gal d5, Gal d 6, Gal d 7, Gal d 8, Gal d 9, Gal d 10), milk proteins (e.g., Bos d 2, Bos d 3, Bos d 4, Bos d5, Bos d,6, Bos d 7, Bos d 8, Bos d 9, Bos d 10, Bos d 11, and Bos d 12), salmon proteins (e.g., Onc k 5, Sal s 1, Sal s 2, and Sal s 3), cod proteins (e.g., ad pGc 1, Gad m 2, and Gad m 3), shrimp proteins (e.g., Cra c 1, Cra c 2, Cra 4, Cra 5 c5, Cra c 6, Cra c 8, Lit v 1, Lit v 2, Lig v 3, Lit v 4, Met e 1, Pan b 1, Pen a 1, Pen i 1, Pen m 2, Pen m 3, Pen m 4, and Pen m 6) and crab proteins (e.g., Cha f 1, Por p1, Scy p 2, Scy p4, and Scy p 8). Allergenic proteins may also include, but are not limited to, non-nut plant proteins such as wheat proteins (e.g., Tri a 12, Tri a 14, Tri a 15, Tri a 17, Tri a 18, Tri a 19, Tri a 20, Tri a 21, Tri a 25, Tri a 26, Tri a 27, Tri a 28, Tri a 29, Tri a 30, Tri a 31, Tri a 32, Tri a 33, Tri a 34, Tri a 35, Tri a 36, Tri a 37, Tri a 39, Tri a 40, Tri a 41, Tri a 42, Tri a 43, Tri a 44, and Tri a 45), soy proteins (e.g., Gly m 1, Gly m 1.0101, Gly m 2, Gly m 3, Gly m 4, Gly m 5, Gly m 6, Gly m 7, and Gly m 8), Chickpea proteins, sesamin proteins (e.g., Ses i 1, Ses i 2, Ses i 3, Ses i 4, Ses i 5, Ses i 6, and Ses i 7), kiwi proteins (e.g., Act c 1, Act c5, Act c 8, Act c 10, Act d 1, Act d 2, Act d 3, Act d 4, Act d5, Act d 6, Act d 7, Act d 8, Act d 9, Act d 10, Act d 11, Act d 12, and Act d 13), carrot proteins (e.g., Dau c 1, Dau 4, and Dau c5), celery proteins (e.g., Api q 1, Api q 2, Api q 3, Api q 4, Api q 5, and Api q 6), stone fruit proteins (e.g., Pru 1, Pru 3, Pru av 1, Pru av 2, Pru av 3, Pru av 4, Pru p1, Pru p 2, Pru p 3, Pru p4, Pru p 7, and Pru d 3), and oat proteins.

Disclosed, for example, is a method of making a mixed allergen product substantially free of viable replicating organisms, comprising: providing a loose mixed allergen material comprising 2 to 20 individual complete food allergens; and sterilizing the loose mixed type allergen raw material by dry heat sterilization, thereby obtaining a mixed type allergen product.

In certain embodiments, the loose mixed allergen material comprises 2 to 20 individual complete food allergens, e.g., 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 2 to 18, 4 to 18, 6 to 18, 8 to 18, 10 to 18, 12 to 18, 14 to 18, 16 to 18, 2 to 16, 4 to 16, 6 to 16, 8 to 16, 10 to 16, 12 to 16 or 14 to 16 individual complete food allergens. For example, the loose mixed allergen material may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 individual complete food allergens. In a particular embodiment, the loose mixed allergen material comprises 15 or 16 individual complete food allergens.

For example, in certain embodiments, the individual complete food allergen is selected from the group consisting of nuts, seeds, legumes, eggs, dairy products, fish, and crustaceans. In a specific embodiment, the individual complete food allergens are selected from the group consisting of almonds, cashews, hazelnuts, peanuts, pecans, pistachios, walnuts, sesame, soybeans, eggs, milk, oats, wheat, cod, salmon, and shrimp. In certain embodiments, the separate complete food allergens are almonds, cashews, hazelnuts, peanuts, pecans, pistachios, walnuts, sesame, soybeans, eggs, milk, oats, wheat, cod, salmon, and shrimp. It is contemplated that the complete food allergen alone may be provided as a meal, food powder, powder and/or protein concentrate.

In certain embodiments, the method further comprises providing 2 to 20 separate complete food allergens and blending the 2 to 20 separate complete food allergens together to give a loose mixed allergen material. It will be appreciated that two or more separate complete food allergens may be provided in combination prior to blending. For example, 2 to 20, 4 to 20, 6 to 20, 8 to 20, 10 to 20, 12 to 20, 14 to 20, 16 to 20, 18 to 20, 2 to 18, 4 to 18, 6 to 18, 8 to 18, 10 to 18, 12 to 18, 14 to 18, 16 to 18, 2 to 16, 4 to 16, 6 to 16, 8 to 16, 10 to 16, 12 to 16, or 14 to 16 individual complete food allergens may be provided in combination prior to blending. In another example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 individual complete food allergens may be provided in combination prior to blending.

It will be appreciated that blending may include one or more than one blending step resulting in a loose mixed type allergen material. For example, the individual complete food allergens may be blended in 1 to 19, 1 to 17, 1 to 15, 1 to 13, 1 to 11, 1 to 9, 1 to 7, 1 to 5, or 1 to 3 blending steps. In a particular embodiment, the admixing comprises mixing the individual complete food allergens in a single admixing step.

As contemplated in this disclosure, loose mixed allergen materials are sterilized by dry heat sterilization. It is understood that sterilization may further include one or more additional applications of dry heat sterilization to inactivate heat-resistant spores.

In certain embodiments, dry heat may be applied to a processing chamber of a sterilization system in which loose hybrid allergenic material is contained or transferred, wherein the processing chamber may be heated to a temperature of about 65 ℃ to about 175 ℃. For example, in certain embodiments, the process chamber may be heated to about 65 ℃ to about 80 ℃, about 65 ℃ to about 120 ℃, about 100 ℃ to about 150 ℃, or about 125 ℃ to about 175 ℃. In particular embodiments, the process chamber can be heated to a temperature of about 65 ℃, about 66 ℃, about 67 ℃, about 68 ℃, about 69 ℃, about 70 ℃, about 71 ℃, about 72 ℃, about 73 ℃, about 74 ℃, about 75 ℃, about 85 ℃, about 95 ℃, about 105 ℃, about 115 ℃, about 125 ℃, about 135 ℃, about 145 ℃, about 155 ℃, about 165 ℃, or about 175 ℃. In other embodiments, dry heat may be applied to the loose mixed allergen material for about 3 minutes to about 72 hours. In certain embodiments, for example, the dry heat may be applied for about 3 minutes to about 72 hours, about 30 minutes to about 72 hours, about 12 hours to about 72 hours, about 18 hours to about 72 hours, about 24 hours to about 72 hours, about 30 hours to about 72 hours, about 36 hours to about 72 hours, about 42 hours to about 72 hours, about 48 hours to about 72 hours, about 54 hours to about 72 hours, about 60 hours to about 72 hours, or about 66 hours to about 72 hours. For example, in particular examples, dry heat may be applied to the loose mixed allergen material for about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, or about 72 hours.

In certain embodiments, for example, dry heat may be provided to the process chamber of the sterilization system by a plurality of natural gas fired furnaces that provide heat to the process chamber from about 250,000BTUs to about 750,000 BTUs. For example, heat of about 250,000BTU, about 300,000BTU, about 350,000BTU, about 400,000BTU, about 450,000BTU, about 500,000BTU, about 550,000BTU, about 600,000BTU, about 650,000BTU, about 700,000BTU, or about 750,000BTU may be provided to the process chamber.

In another embodiment, dry heat sterilization may be applied to loose mixed allergen materials packaged in heat resistant packaging. As used herein, "heat resistant" is understood to mean that the application of heat to the packaging material at the same temperature and for the same duration as the loose mixed type allergen material simultaneously applied to the package therein does not result in a change of the integrity and functionality of the packaging material affecting its use as a barrier to chemical or microbial contamination. Furthermore, "heat resistant" is understood to mean that the application of heat to the packaging material at the same temperature and for the same duration as the loose mixed type allergen material simultaneously applied to the package therein does not alter the package to cause the chemicals in the package to be added to the loose mixed type allergen material packaged therein. For example, heat resistant packaging can include, but is not limited to, bulk bags, pouches, or stick-shaped packaging. In another example, heat resistant packaging may be placed in a box and/or on a tray or rack within a heating chamber for dry heat sterilization.

As contemplated in the methods of the present disclosure, blending may include loading 2 to 20 individual substance food allergens (e.g., 4-20, 6-20, 8-20, 10-20, 12-20, 14-20, 16-20, 18-20, 2-18, 4-18, 6-18, 8-18, 10-18, 12-18, 14-18, 16-18, 2-16, 4-16, 6-16, 8-16, 10-16, 12-16, or 14-16 individual complete food allergens) into a mechanical mixer or blender. For example, the mechanical stirrer or mixer may be selected from, but is not limited to: a ribbon blender; a paddle-type masher; a plow mixer; a fluidization agitator; an intensive mixer; a vacuum mixer; a conical mixer; rotating an intermittent agitator; and a two-axis mixer. In a specific embodiment, the individual complete food allergens are loaded into a ribbon blender. Without wishing to be bound by theory, it is believed that blending by loading the complete food allergen alone into a mechanical mixer or blender reduces gritty feel, reduces the number of large particles and increases homogeneity.

It will be understood that each individual complete food allergen may be loaded in different amounts into a mechanical blender or mixer, such as, but not limited to: about 5 kg to about 100 kg; about 10 kg to about 90 kg; about 15 kg to about 80 kg; about 20 kilograms to about 70 kilograms; about 25 kg to about 60 kg; and about 30 kilograms to about 50 kilograms. It will be further appreciated that a mechanical stirrer or mixer may accommodate different combined total amounts of complete food allergen. In certain embodiments, for example, the mechanical stirrer or mixer may hold a combined total amount of complete food allergen of about 100 kg to about 2200 kg or about 500 kg to about 2000 kg. For example, in some embodiments, the mechanical stirrer or mixer can hold a combined total amount of about 500 kilograms, about 600 kilograms, about 700 kilograms, about 800 kilograms, about 900 kilograms, about 1000 kilograms, about 1100 kilograms, about 1200 kilograms, about 1500 kilograms, about 1600 kilograms, about 1700 kilograms, about 1800 kilograms, about 1900 kilograms, or about 2000 kilograms of complete food allergen.

It will also be appreciated that the setting of the mechanical stirrer or mixer may be adjusted in order to achieve optimal blending and/or mixing of the individual complete food allergens. For example, the mechanical stirrer can be set at about 10Hz to about 50Hz (e.g., about 15Hz to about 45Hz, about 20Hz to about 40Hz, or about 25Hz to about 35 Hz). The mechanical stirrer may also be set to run for different lengths of time. For example, the mechanical mixer may blend the individual food allergens for about 5 minutes to about 30 minutes, about 7.5 minutes to about 25 minutes, or about 10 minutes to about 20 minutes. In a specific embodiment, the mechanical stirrer or mixer is a ribbon blender with a capacity of 1000 kg and blends the individual complete food allergens for 15 minutes at 30 Hz.

In certain embodiments, the methods of the present disclosure further comprise admixing 2 to 20 separate complete food allergens with one or more than one bulking agent. Contemplated fillers may include any of the fillers described herein. In certain embodiments, the bulking agent comprises a sugar, for example, sucrose, maltodextrin, trehalose dihydrate, isomalt, mannitol, lactose, glucose, fructose, raffinose, or any combination thereof. In certain embodiments, the bulking agent comprises maltodextrin or sucrose, or a combination thereof.

In certain embodiments, the loose mixed allergen material may be discharged from a mechanical blender or mixer into a liner vat and then recharged for sterilization. In other embodiments, the bulk mixed allergen material discharged into the lined vat may be packaged into a bag or pouch.

Also contemplated is a method of making a mixed allergen product, which further comprises filtering the mixed allergen product to remove agglomerated particles. For example, the mixed allergen product may be passed through a screen ranging from a #5 mesh (US) screen to a #10 mesh (US) screen on a shaker for about 1 minute to about 10 minutes. For example, the screen mesh may be a #5 mesh (US) screen, a #6 mesh (US) screen, a #7 mesh (US) screen, a #8 mesh (US) screen, a #9 mesh (US) screen, or a #10 mesh (US) screen. In a specific embodiment, the mixed allergen product is filtered through a screen ranging from a #5 mesh (US) screen to a #10 mesh (US) screen on a shaker for about 3 minutes.

In certain embodiments, the method of making a hybrid allergen product may further comprise magnetically sieving the hybrid allergen product to remove metal particles. For example, magnetic sieving may include passing the mixed allergen product through a rare earth magnet, a metal detector, and/or a metal separator. In another example, the metal detector can detect stainless steel particles having a size of at least about 2 millimeters to about 2.5 millimeters, ferrous particles having a size of at least about 1.5 millimeters to about 2 millimeters, and non-ferrous particles having a sensitivity of about 1.5 to 2 parts per million.

In other embodiments, the method of preparing a mixed allergen product may be a continuous process, wherein the total completion time of the method is from about 3 minutes to about 72 hours, from about 30 minutes to about 72 hours, from about 12 hours to about 72 hours, from about 18 hours to about 72 hours, from about 24 hours to about 72 hours, from about 30 hours to about 72 hours, from about 36 hours to about 72 hours, from about 42 hours to about 72 hours, from about 48 hours to about 72 hours, from about 54 hours to about 72 hours, from about 60 hours to about 72 hours, or from about 66 hours to about 72 hours. For example, in particular examples, the total completion time for the method is about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, or about 72 hours.

In the disclosed method, sterilizing a loose mixed allergen material by dry heat sterilization results in a mixed allergen product that is substantially free of viable replicating organisms.

In certain embodiments, the mixed allergen products of the present disclosure are substantially free of viable replicating bacteria, yeasts, and/or molds. In some embodiments, the hybrid allergen product may have: at least about a 1log reduction to at least about a 9log reduction in aerobic plate count; at least about a 1log reduction to about a 4log reduction; at least about a 1log reduction to about a 5log reduction; at least about a 1log reduction to about a 6log reduction; at least about a 1log reduction to about a 7log reduction; at least about a 1log reduction to about an 8log reduction or at least about a 3log reduction to about a 9log reduction. For example, the hybrid allergen product can have an aerobic bacteria bio-plate count of at least about 1log reduction, at least about 2log reduction, at least about 3log reduction, at least about 4log reduction, at least about 5log reduction, at least about 6log reduction, at least about 7log reduction, at least about 8log reduction, or at least about 9log reduction, as compared to a corresponding bulk hybrid allergen starting material that has not been sterilized. In another example, the mixed allergen product has less than about 100,000CFU/g, 10,000CFU/g, less than about 1,000CFU/g, less than about 100CFU/g, or less than about 10CFU/g of aerobic organisms. In another example, the mixed allergen product has a coliform population of less than about 100CFU/g, less than about 10CFU/g, or less than about 1 CFU/g. In yet another example, the mixed allergen product can have a pathogenic microorganism strain less than about 10 CFU/g. For example, the mixed allergen product has less than about 100CFU/g, less than about 10CFU/g, or less than about 1CFU/g of E.coli. In another example, the mixed allergen product has enterobacter bacteria less than about 100CFU/g, less than about 10CFU/g, or less than about 1 CFU/g. In another example, the mixed allergen product has less than about 100CFU/g or less than about 10CFU/g of Salmonella. In another example, the mixed allergen product has less than about 100CFU/g or less than about 10CFU/g Cronobacter sakazakii. In yet another example, the mixed allergen product is negative for listeria or crohn's species per 25 gram sample of the mixed allergen product as determined by a PCR-based assay. In another example, the mixed allergen product has less than about 100CFU/g, less than about 10CFU/g, or less than about 1CFU/g mold. In yet another example, the mixed allergen product is negative for Salmonella species per 375 gram sample of the mixed allergen product as determined by a PCR-based assay. In another example, the mixed allergen product has less than about 100CFU/g, less than about 10CFU/g, or less than about 1CFU/g of Staphylococcus aureus. In another example, the mixed allergen product has less than about 100CFU/g, less than about 10CFU/g, or less than about 1CFU/g yeast.

In certain embodiments, the hybrid allergen products of the present disclosure have a fat content of at least about 5% by weight. For example, the mixed allergen product may have a fat content of about 10% to about 20% by weight. In some embodiments, for example, the mixed allergen product may have a fat content of about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% by weight.

It is noted that the application of dry heat sterilization to loose mixed allergen materials does not substantially alter protein integrity. For example, in certain embodiments, the sterilized mixed allergen product has the same protein integrity as a corresponding bulk mixed allergen raw material that has not been sterilized. In another example, the protein integrity is determined by SDS-PAGE, wherein the mixed allergen product has substantially similar protein bands, protein band distribution and protein band intensity as the bulk mixed allergen starting material without sterilisation.

It is further contemplated that the application of dry heat sterilization to the loose mixed allergen material does not substantially affect the ability of the loose mixed allergen material to elicit an allergenic effect when consumed by an individual. As used herein, "allergenic effect" is understood to mean an immune response to one or more than one antigenic component, characterized by, but not limited to, immune cell activation, cytokine production, and IgE production. In certain embodiments, the hybrid allergen product has an allergenic effect when consumed by an individual that is substantially similar to an effect of a corresponding bulk hybrid allergen material that is not sterilized and consumes substantially similar protein qualities. In certain embodiments, the allergenic effect is determined by production of IgE or cytokines or by measuring immune cell activation responses produced by an individual in response to consumption of loose mixed allergen raw material or mixed allergen product. In other embodiments, the allergen effect is determined by an in vitro immune response, e.g., measuring IgE or cytokine production, or measuring activation of immune cell cultures after treatment with loose mixed allergen raw material or mixed allergen product.

In certain embodiments, the mixed allergen products of the present disclosure have a protein content of about 40% to about 50% by weight. For example, the mixed allergen product may have a protein content of about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% by weight.

In certain embodiments, the hybrid allergen products of the present disclosure have from about 1% to about 10% by weight moisture. For example, the mixed allergen product may have about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by weight moisture.

In certain embodiments, the mixed allergen products of the present disclosure have a water activity of from about 0.1 to about 0.6. "water activity" is understood to mean the ratio of the vapour pressure of the mixed allergen product to the vapour pressure of distilled water under the same conditions. It will be appreciated that water activity is a measure of the water that does not bind to molecules of the mixed allergen product and is therefore able to support the growth of bacteria, yeast and moulds. Further, it will be understood that water activity may be measured using suitable electronics, such as a hygrometer, a moisture-hygrometer, a humidity meter, and a relative humidity system.

In certain embodiments, the hybrid allergen products of the present disclosure have less than about 10% by weight of particles having a diameter greater than 500 microns.

In yet another embodiment, the hybrid allergen product of the present disclosure has substantially similar taste characteristics as compared to a corresponding bulk hybrid allergen material that has not been sterilized. For example, the mixed allergen product may have one or more than one aroma selected from, but not limited to, salty, nutty, seafood, barbecue, and brown sauce. In another example, the mixed allergen product is free of any off-flavors resulting from processing.

In another embodiment, a method of making a mixed allergen product substantially free of viable replicating organisms is contemplated, wherein the 2 to 20 individual food allergens are sterilized by dry heat sterilization prior to blending to obtain the mixed allergen product.

In certain embodiments, the method of making a hybrid allergen product further comprises admixing the hybrid allergen product with one or more probiotics selected from the group including, but not limited to: lactobacillus (Lactobacillus) species, Bifidobacterium (Bifidobacterium) species, anacetostripes species and Bacillus (Bacillus) species. For example, the one or more probiotics may be selected from the group including, but not limited to: lactobacillus rhamnosus (Lactobacillus rhamnosis), Corynebacterium faecalis, Bifidobacterium longum, Bifidobacterium lactis (Bifidobacterium lactis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus casei (Lactobacillus casei), Lactobacillus brevis (Lactobacillus brevis), Lactobacillus salivarius (Lactobacillus salivarius), Lactobacillus lactis (Lactobacillus lactis), Bacillus coagulans (Bacillus coagulans), Lactobacillus bulgaricus (Lactobacillus bulgaricus), Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus paracasei (Lactobacillus paracasei), Bacillus coagulans and Bifidobacterium bifidum (Bifidobacterium bifidum). In a particular embodiment, the method of preparing a mixed allergen product further comprises blending the mixed allergen product with lactobacillus rhamnosus and/or bifidobacterium longum. As used herein, "probiotic" refers to a culture (pure or mixed) comprising microorganisms that, when ingested, produce a beneficial effect on the host.

In other embodiments, the method of making a hybrid allergen product further comprises admixing the hybrid allergen product with one or more prebiotics selected from the group including, but not limited to: fructooligosaccharides, inulin, isomalto-oligosaccharides, lactitol, lactosucrose (lactosucrose), lactulose, pyrodextrins, soy oligosaccharides, xylooligosaccharides (xylooligosaccharides), trans-galactooligosaccharides, oligosaccharides, soluble corn fiber, beta-glucans, oligofructose (oligofructose) and dimers. In a particular embodiment, the method of making a mixed allergen product further comprises blending the mixed allergen product with fructooligosaccharide. As used herein, the term "prebiotic" refers to a specific dietary food or nutrient that promotes the growth, proliferation, and/or activity of a specific, desired bacterial strain.

The disclosed mixed allergen products may also contain one or more vitamins, as desired. Vitamins that may be present include, for example, vitamin A (e.g., in an amount of about 1 to about 35,000 International units), vitamin C (e.g., in an amount of about 1 to about 1,000 milligrams), vitamin D (e.g., in an amount of about 1 to about 4,000 International units, i.e., in an amount of about 0.025 to about 100 micrograms), vitamin E (e.g., in an amount of about 1 to about 450 International units), vitamin K (e.g., in an amount of about 1 to about 250 micrograms), vitamin B-1 (thiamin an amount of, for example, about 1 to about 15 milligrams), vitamin B-2 (riboflavin; for example, in an amount of about 1 to about 17 milligrams), vitamin B-3 (niacin; for example, in an amount of about 1 to about 200 milligrams), vitamin B-5 (pantothenic acid; for example, in an amount of about 1 to about 100 milligrams), vitamin B-6 (pyridoxine; for example, in an amount of about 1 to about 30 milligrams), vitamin B-9 (folic acid; for example, in an amount of about 1 to about 4,000 micrograms), vitamin B-12 (cobalamin; e.g., in an amount of about 1 to about 250 micrograms), vitamin H (biotin; e.g., in an amount of about 1 to about 1,000 micrograms), and combinations thereof. In certain embodiments, the mixed allergen product comprises vitamin D. In certain embodiments, the mixed allergen product comprises 400 international units, i.e., about 10 micrograms of vitamin D.

Also contemplated are methods of making the mixed allergen product, wherein the mixed allergen product is further mixed with a physiologically acceptable delivery vehicle to produce a physiologically acceptable composition. The mixed allergen product can be further incorporated into various formulations for administration to a subject. More specifically, the mixed allergen product may be formulated into a physiologically acceptable composition by combining with a suitable, physiologically acceptable carrier or diluent (e.g., vegetable oil).

In certain embodiments, the disclosed mixed allergen products are designed for oral immunotherapy of food allergies in children or adults, e.g., as dispersible powders or granules, food products, tablets, lozenges, pastilles, fudge, emulsions, and the like. Compositions intended for oral use may be prepared according to any convenient protocol for the manufacture of oral compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents (e.g., glycerol, propylene glycol, sorbitol or sucrose), flavoring agents, coloring agents and preserving agents in order to provide suitable formulations.

Also contemplated is a method of making a hybrid allergen product, wherein the hybrid allergen product is mixed or blended with a food that is not allergic to children or adults. For example, food products may include, but are not limited to: infant or toddler formula, infant food (e.g., purees suitable for consumption by infants or toddlers), potato chips, cookies, bread, spreads, creams, yoghurts, liquid beverages, chocolate-containing products, candy, ice cream, cereals, coffee, purees, and the like.

Throughout the specification, when an apparatus, device, or system is described as having, including, or containing specific components, or a process or method is described as having, including, or containing specific steps, it is contemplated that an apparatus, device, or system consisting essentially of, or consisting of, the recited components and that a process or method consisting essentially of, or consisting of, the recited processing steps may additionally be present.

All examples provided in this application are for illustrative purposes only and should not be construed as limiting in any way.

Examples

Examples1

This example describes the selection of allergenic food ingredients included in an exemplary dry powdered mixed allergen product that contains 16 individual complete food allergens (almonds, cashews, hazelnuts, peanuts, pecans, pistachios, walnuts, sesame, soybeans, eggs, milk, oats, wheat, cod, salmon, and shrimp).

The source of the ingredients is mainly focused on domestic commercial availability, with the exception of the individual complete food allergens which are only available internationally. Successful commercial procurement of multiple selections of each individual complete food allergen has led to the development of selection criteria in order to select the best commercial food allergen to be tested. The attributes screened include: protein content; a content of bulky material; sensory characteristics such as taste, presence of abnormal odor, and gritty feel; and solubility. Ingredients with relatively low protein content or with a large proportion of leavening material are eliminated from competition. Complete food allergens alone were dry tasted to determine the presence of off-flavors and to assess grittiness.

Table 1 shows sample formulations of the mixed allergen product and representative pre-weighed batch amounts of complete food allergen alone provided for blending and sterilization.

TABLE 1

Complete food allergen alone	Formulation (g)	Formulation (%)	Sterilization run batch (kg)	Actual batch (kg)
					50 percent of almond powder	0.6895	6.620	33.10	33.10
Cashew nut powder 35%	0.10720	10.294	51.47	51.47
					Cod powder	0.02291	2.200	11.00	11.00
Whole egg powder	0.06742	6.473	32.37	32.37
					Hazelnut powder	0.08824	8.472	42.36	42.36
Milk protein isolate	0.03534	3.393	16.96	6.35
					Oat protein	0.06266	6.017	30.08	30.08
Peanut powder	0.06122	5.879	29.39	29.39
					American hickory nut powder	0.09816	9.425	47.13	47.13
Pistachio nut powder	0.08867	8.514	42.57	42.57
					Salmon protein powder	0.07560	7.259	36.30	36.30
Sesame seed powder	0.06100	5.857	29.28	29.28
					Shrimp meal	0.04261	4.092	20.46	20.46
Soy protein isolate	0.03736	3.588	17.94	17.94
					Walnut powder	0.08316	7.985	39.93	39.93
Wheat gluten	0.04096	3.933	19.67	19.67
					Total of	1.04146	100.00	500.00	489.39

Example 2

This example describes an exemplary blending method for preparing a mixed allergen product containing 16 separate complete food allergens (almonds, cashews, hazelnuts, peanuts, pecans, pistachios, walnuts, sesame, soybeans, eggs, milk, oats, wheat, cod, salmon, and shrimp).

1,000 kg ribbon blender (Prince Industries, india) was cleaned, sterilized and tested for Adenosine Triphosphate (ATP) and allergens. Actual batches (in kg) of 16 individual complete food allergens shown in table 1 were loaded into a ribbon blender and blended at 30Hz for 15 minutes.

The loose mixed allergen material is discharged from the ribbon blender into lined vats (40 to 50 kg per vat).

Example 3

This example describes an alternative blending and milling process for preparing a mixed allergen product containing 16 separate complete food allergens (almonds, cashews, hazelnuts, peanuts, pecans, pistachios, walnuts, sesame, soy, eggs, milk, oats, wheat, cod, salmon, and shrimp).

All individual complete food allergens were weighed to make an 8 kg batch. The individual complete food allergens were transferred to a D500 Hobart mixer equipped with a D-30 bowl and safety cage. The complete food allergen alone was mixed with a paddle attachment "B" flat plate mixer on setting 1 for 2 minutes. The blended complete food allergen was transferred into large food grade plastic bags, sealed and stored at room temperature until ground. The blended complete food allergen was slowly fed into a Quadro SLS-L1A FitzMill equipped with a #20 mesh screen and set at 9,000rpm to achieve a target particle size of <500 microns in diameter. The ground complete food allergen was passed through a metal detector and a #35 mesh screen prior to packaging.

Example 4

Preliminary tests of heat sensitivity were performed to determine the suitability of loose mixed allergen materials for sterilisation treatment.

Loose mixed allergen materials were placed in pans and subjected to different temperatures in a laboratory oven for 5 minutes.

As shown in fig. 1, the loose mixed type allergen raw material does not show any other physical change that would make the material unsuitable for sterilization treatment when heat-treated at 95 ℃, 105 ℃, 110 ℃ or 120 ℃ for 5 minutes, except that the color becomes slightly darker as the temperature increases.

Example 5

This example describes an exemplary dry heat sterilization process that can be used to prepare a mixed allergen product containing 16 individual food allergens (almond, cashew, hazelnut, peanut, pecan, pistachio, walnut, sesame, soybean, egg, milk, oat, wheat, cod, salmon, and shrimp). A dry heat sterilization process similar to that contemplated by the present disclosure is described in US 2014/0023757, which is incorporated herein by reference.

The loose mixed allergen material blended as described in example 2 or 3 may be packed in a heat-resistant packaging bag. The bagged loose mixed type allergenic material may then be placed into an insulated processing chamber, wherein heat is provided to the processing chamber by a plurality of natural gas burners. Multiple intake and exhaust conduits may be used to circulate heat through the insulated treatment chamber to promote uniform heat absorption by the bagged loose mixed allergen material.

For example, 6 bags of loose mixed type allergenic material (weighing about 25 pounds per 12 kilograms per bag) may be placed as a single layer on a tray, and 14 trays may be loaded into a heating chamber having dimensions of 40 feet long, 10 feet high, and 9 feet wide.

The heating chamber may then be sealed and the chamber may be heated to reach the target temperature of 74 ℃. The pre-heating time for the chamber to reach the target temperature may take about 24 hours.

Six probes within the heating chamber may be used to monitor the temperature of the heating chamber. For example, two probes may be placed at the rear of the heating chamber, two probes may be placed at the middle of the heating chamber, and two probes may be placed at the front of the heating chamber.

Once the target temperature is reached, the heating chamber may be maintained for a 24 hour period.

The temperature of the insulated processing chamber, the plurality of natural gas burners, and the bagged loose mixed type allergenic material can be monitored to ensure that the loose mixed type allergenic material is dry heat sterilized for a period of 24 hours using a temperature of about 65 ℃ to about 175 ℃.

After the dry heat treatment, the sterilized mixed allergen product may be unloaded from the heating chamber. The water activity, moisture level and total dish count of the sterilized mixed allergen product sample can be measured and compared to a loose mixed allergen raw material sample prior to dry heat sterilization.

For example, samples can be diluted and plated on common recovery media dishes to measure colony forming units (CFU/g) per gram of product or material for total aerobic and enterobacteria counts. Samples were diluted and spread on 3M Petrifim, respectively, according to the manufacturer's instructions (3M, St. Paul, Minnesota)^TMColi/coliform and 3M Petrifilm^TMColi/coliform and staphylococcus aureus counts were determined on Staph Express counting plates.

Can be used according to the manufacturer's instructionsThe PCR detection system (Hygiena, Camarillo, California) additionally tested the samples for the presence of Listeria and Salmonella.

It can also be prepared by diluting the sample and spreading it on 3M Petrifilm according to the manufacturer's instructions^TMTotal mold and yeast counts were determined on mold and yeast count plates.

Example 6

This example describes the validation of the dry heat process for sterilization of loose mixed allergen materials.

In this dry heat method, a source derived from Enterococcus Faecium (Enterococcus faecalis)8459 strain830 were used as surrogate microorganisms to evaluate lethality of dry heat sterilization to salmonella and crohn's disease pathogens.1830 have a heat tolerance similar to or greater than that of Salmonella and Cronobacterium. Verifying that it is intended to be viable1830 the number of organisms achieves at least a 5-log reduction. Two validation trials were performed to assess variability.

Before dry heat sterilization, use about 1.4X10⁶ 1830 Bioseed 70 thermotolerant bags each containing 25 grams of loose mixed allergen material blended as described in example 2 or 3. Each inoculated heat-resistant bag was placed in a box.

The 6 inoculated loose mixed allergen material bags were not introduced into the dry heat sterilization process and were used as untreated control samples.

For validation tests 1 and 2, 14 trays, each containing four boxes of inoculated heat-resistant bags of loose mixed allergen material, were loaded into a heating chamber as shown in fig. 2A and 2B, respectively. A second inoculated heat-resistant bag of loose mixed allergen material was placed in a number 3 and/or 4 box on number 1, 6, 9 and 14 trays.

The temperature of the heating chamber was monitored throughout the validation run using the heating chamber probe: two at the front of the heating chamber, two in the middle of the heating chamber, and two at the rear of the heating chamber. In addition, a MadgeTech probe is included to monitor the temperature of the heating chamber during dry heat sterilization. The MadgeTech probe is placed inside a heat-resistant bag that has selected the inoculated loose mixed allergen material, or outside the cassette.

As shown in fig. 3A and 3B of validation experiments 1 and 2, respectively, the temperatures recorded by the MadgeTech probe were relatively uniform at different locations within the room. The temperature recorded by the heating chamber probe varies from one room location to another (i.e., rear, middle, or front), but the temperature between two probes at the same location is relatively similar during the dry heat sterilization process.

For validation test 1 (FIG. 3A), the dry heat treatment was initially set at 175 deg.F/80 deg.C. It took 18 hours for all probes to reach the target temperature. Once the target temperature was reached, the temperature was reduced to 165F/74F and maintained at this temperature for an additional 24 hours.

For validation test 2 (FIG. 3B), the dry heat treatment was set at 165 ℃ F./74 ℃. It took 17 hours for all probes to reach the target temperature. Once the target temperature was reached, the dry heat sterilization temperature was maintained at 165 ° F/74 ℃ for an additional 24 hours.

As shown in table 2, it was verified that the water activity and moisture content of the sterilized mixed allergen product after dry heat sterilization of test 1 was lower than those of the loose mixed allergen raw material before dry heat treatment. The loose mixed allergen raw material before dry heat sterilization and the plate total count of the mixed allergen product after dry heat sterilization on selective medium were similar.

TABLE 2

As shown in Table 3, the loose mixed type allergen raw material samples without dry heat sterilization treatment had an average of 8.1. + -. 0.1log cfu/g1830 count.

TABLE 3

As shown in table 4, was mixed with the seeded bulk without dry heat treatmentComparison of the type-Combined allergen material samples, achieved in all samples after dry heat sterilization in validation test 1Greater than 5log reduction of 1830 CFU/g.

TABLE 4

As shown in table 5, the water activity of the sterilized mixed allergen product after dry heat sterilization of verification test 2 was similar to the water activity of the loose mixed allergen raw material before dry heat treatment. The moisture content and the total plate count after the dry heat sterilization treatment in proof test 2 were lower than those of the loose mixed type allergen raw material which was not dry heat sterilized.

TABLE 5

As shown in Table 6, the loose mixed type allergen raw material samples without dry heat sterilization treatment had an average of 8.1. + -. 0.1log cfu/g1830 count.

TABLE 6

As shown in table 7, and verificationThe dry heat sterilization in test 1 is similar, verifying that the dry heat sterilization in test 2 results in all samples compared to inoculated loose mixed allergen material samples without dry heat treatmentGreater than 5log reduction of 1830 CFU/g.

TABLE 7

Example 7

The particle size distribution of a sample of the mixed allergen product treated with dry heat sterilization can be measured and compared to corresponding loose particles of mixed allergen material not subjected to dry heat sterilization. Can useThe samples were filtered for 3 minutes with a vibrating screen and a series of mesh sizes consisting of: #10, #12, #14, #16, #18, #20, #25, #30, #35, #40 and #50 mesh. The filtered particles and the particles retained by each screen can be removed individually, weighed, and the distribution plotted as% weight versus particle size (in microns).

Example 8

The protein integrity of the mixed allergen product sample treated with dry heat sterilization can be compared to a loose mixed allergen material not subjected to dry heat sterilization. Protein integrity can be assessed by SDS-PAGE analysis of all proteins present in unsterilized and dry heat sterilized samples.

1 mg of loose mixed allergen raw material or mixed allergen product sterilized by dry heat may be weighed into a 2 ml microtube. Based on their estimated protein concentration, samples can be diluted to a protein concentration of 1 mg/ml with Laemmli buffer containing 5% 2-mercaptoethanol (BioRad, Hercules, ca). The protein suspension may be mixed by gentle inversion or intermittent vortex mixing for 5 minutes and then heated to 95 ℃ to 100 ℃ for 5 minutes. Samples can be centrifuged at 1,000RPM for 1 minute, and then 10 microliters of each sample per well can be loaded in triplicate on a 4-20% polyacrylamide gradient gel (BioRad, Hercules, ca) immersed in 1X SDS-PAGE running buffer (BioRad, Hercules, ca). The gel may be run at 100V for 80-90 minutes (or 200V for 30-40 minutes). The gel can then be washed 3 times with distilled water on an orbital shaker at room temperature for 5 minutes each. The gel can be fixed in fixative (Sigma-Aldrich, st. louis, missouri) or 50% methanol, 10% acetic acid for 15 minutes. Gels can be stained with EX Blue gel staining reagent (Sigma-Aldrich, st. louis, missouri) for 2 hours or overnight, and then destained by washing in distilled water for 1-2 hours or overnight. When the wash is complete, the gel may be covered with clear plastic and scanned using a gel reader. Protein band density measurements can be analyzed using ImageJ public domain software (NIH, Bethesda, maryland).

Example 9

The visual and sensory characteristics of the mixed allergen product sample treated with dry heat sterilization can be compared to a bulk mixed allergen material not subjected to dry heat sterilization application. A comparison of the taste characteristics of the dry heat sterilized mixed allergen product and the bulk mixed allergen that has not been sterilized can also be made.

Incorporation by reference

All publications and patents mentioned in this specification are herein incorporated in their entirety by reference into the specification, for all purposes. In case of conflict, the present application, including any definitions herein, will control.

Equivalent scheme

While specific embodiments of the present invention have been discussed, the above description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon reading the specification. The full scope of the invention should be determined by reference to the claims and their full scope of equivalents, and this specification, as well as these variations.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.