METHODS FOR LOWERING GLUTEN CONTENT USING FUNGAL CULTURES

Abstract

The present invention provides a method for the preparation of a gluten-containing grain having lowered levels of gluten. The method includes providing a prepared gluten-containing grain which may be optionally sterilized or pasteurized. The prepared gluten-containing grain is then inoculated with a prepared fungal component and incubated. In one embodiment the prepared fungal component myceliates the prepared gluten-containing grain while incubated and during this process hydrolyzes gluten in the prepared gluten-containing grain. The present invention also includes a gluten-containing grain having lowered levels of gluten which has been prepared by the methods of the invention.

Claims

1. A method for preparation of a gluten-containing grain having reduced levels of gluten, comprising: a) providing a prepared gluten-containing grain comprising the steps of: i. providing a gluten-containing grain; ii. sterilizing or pasteurizing the gluten-containing grain to provide prepared gluten-containing grain; b) providing a prepared fungal component; c) inoculating the prepared gluten-containing grain with the prepared fungal component; and d) incubating the prepared gluten-containing grain and prepared fungal component to hydrolyze gluten, resulting in the gluten-containing grain having reduced levels of gluten, wherein the gluten comprises prolamin, glutelin or prolamin/glutelin aggregate.

2. The method of claim 1, wherein the method further comprises drying the gluten-containing grain from step (d).

3. The method of claim 1, wherein the method comprises pasteurizing the gluten-containing grain from step (d).

4. The method of claim 1, wherein the method of step a) further comprises hydrating the gluten-containing grain.

5. The method of claim 1, wherein the gluten-containing grain having lowered levels of gluten has had more than 90% of gluten hydrolyzed compared to the prepared gluten-containing grain.

6. The method of claim 1, wherein the gluten-containing grain having lowered levels of gluten has had more than 95% of gluten hydrolyzed, compared to the prepared gluten-containing grain.

7. The method of claim 1, wherein the gluten-containing grain having lowered levels of gluten has had more than 99.99% of gluten hydrolyzed, compared to the prepared gluten-containing grain.

8. The method of claim 1, wherein the prepared fungal component is a submerged fungal liquid tissue culture.

9. The method of claim 1, wherein the incubating step comprises culturing to induce myceliation.

10. The method of claim 9, wherein the culturing step is performed for between one and forty-two days.

11. The method of claim 1, wherein the submerged fungal liquid tissue culture is grown in a human food-grade media.

12. The method of claim 1, wherein the submerged fungal liquid tissue culture is grown in an animal feed-grade media

13. The method of claim 1, wherein gluten is selected from the group consisting of prolamin, glutelin and an aggregate of prolamin and glutelin.

14. The method of claim 1, wherein gluten content is measured by measuring prolamin, glutenin, or prolamin/glutelin aggregate.

15. The method of claim 1, wherein the prepared fungal component is selected from the group consisting of Hericium erinaceus, Pleurotus ostreatus, Pleurotus eryngii, Pleurotus citrinopileatus, Pleurotus djamor, Trametes versicolor, Lentinula edodes, Armillariella mellea Tricholoma matsutake, Flammulina velutipes, Volvariella volvacea, Agaricus campestris, Agaricus blazei, Grifola frondosa, Pholiota nameko, Agrocybe cylindracea, Boletus ornatipes, Boletus edulis, Ganoderma lucidum, Ganoderma applanatum, Hypsizygus marmoreus, Morchella angusticeps, Morchella esculenta, Morchella hortensis, Phellinus linteus, Auricularia auricula, Tremella fuciformis, Inonotus obliquus, Fomes fomentarius, Laetiporus sulfureus, Bridgeoporus nobillismus, Stropharia rugosoannulata, Clitocybe spp., Cordyceps sinensis, Cordyceps militaris, and Polyporus umbellatus, and combinations thereof.

16. The method of claim 14, wherein the prepared fungal component is M. esculenta.

17. The method of claim 14, wherein the prepared fungal component is T. matsutake.

18. The method of claim 1, wherein the prepared fungal component is prepared by a method comprising screening a number of strains of fungi and selecting a strain having an enhanced ability to tolerate, grow on, metabolize or utilize gluten.

19. The method of claim 1, wherein the prepared fungal component is prepared by a method comprising maintaining a strain of fungi on an undefined media comprising organic gluten and an energy source.

20. The method of claim 1, wherein the gluten-containing grain is selected from the group consisting of Triticum spp., Hordeum spp., Secale spp., Zea spp., Sorghum spp., and Avena spp.

21. A gluten-containing grain having lowered levels of gluten prepared by the method of claim 1.

Description

EXAMPLES

Example 1

[0075] Specific and pure strains of fungi obtained from referenced collections were manipulated in sterile environments in 1-10 gal polyproplyene bags, 1 qt-1 gal glass jars or on 9-15 cm Petri plates using undefined, organic fruit and vegetable-based media with 1.5% agar (w/v), in order to monitor and ensure the general vigor and health of strains.

[0076] Mycelial samples were grown in a gentle, ambient sterile airflow for 0.5-4 weeks, then excised from Petri plates and subsequently used for inoculation into liquid-state myceliation employing a similar undefined fruit and vegetable-based media (but with no agar) using ambient air in 1 qt-1 gal glass jars. Some samples were grown in agitated and some were grown in unagitated cultures in ambient air in stainless steel fermenters.

[0077] The unagitated liquid state fermentation formed a floating mass of hyphae which exhibited continuous growth at interface of liquid and air. The mycelium of agitated and/or swirling cultures grew very quickly as hyphal spheres which, being hydrated, remained submerged and had the appearance of small diameter gelatinous beads. Hydrated hyphal spheres collapsed upon desiccation, wherein they were used for inoculating petri-plates for strain propagation and quality control.

[0078] Sphere diameter in liquid-state myceliation was found to be inversely proportional to agitation intensity and volume. Hyphal shear became more efficient at higher agitation and swirling intensity and once sheared hyphae formed new spheres of smallest possible diameter, growing in size until they sheared again. When employed in continuous liquid-state myceliation there existed a constant ratio of sphere diameters, and therefore a constant supply of spheres and hyphal strands adopting spherical morphologies on the order of microns were produced.

[0079] This example thus demonstrated that mycelia sphere diameter was manipulated for more efficient inoculation with inoculation efficiency being inversely proportional to sphere diameter.

Example 2

[0080] Stationary liquid cultures (growth period of 0.1-4 weeks) formed a floating mass of hyphae, which were gently blended with a sharp, sterile cutting device prior to being used for inoculation. Gentle blending was achieved by mixing or low homogenization in a commercial blender in short bursts at slow speeds. Aliquots of blended liquid-state culture were used to inoculate substrate.

Example 3

[0081] For a large batch liquid-state and solid-state operation pure cultures were grown aerobically and inoculated into large industrial liquid-state and large solid-state commercial processors operated continuously for large-scale myceliation of food products. After cultures of media turned completely white or a representative color thereof for a particular species, and had completely overgrown and commanded the medium and were resistant to gentle mixing, the contents were harvested, removed to plastic bags and refrigerated for quick use at either 4° F. or frozen for long-term storage and subsequent utilization at −20° F.

Example 4

[0082] Agar media was prepared with 15-60 g/L agar and 3-22 g/L organic potato starch powder, 0.3-12 g/L organic carrot powder, and 40 mL/L organic mango puree in RO water. The 1.5 L media was then subjected to a 1.5 hour liquid sterilization cycle and cooled in a clean space for 1-1.5 hours and then poured into 120 Petri plates which cooled for 12 hours.

[0083] In sterile operation, the Petri plates were inoculated and cultured for 7-14 days. Upon which time they were sterilely propagated into 4 L Erlenmeyer flasks containing 1.5 L of sterilized and cooled media prepared as above but without the mango puree and agar. This culture was shaken at 60 RPM with a 1.5″ swing radius at room temperature for 1-14 days. This culture was used to inoculate organic hard red winter wheat which was prepared as described below.

[0084] Approximately 0.45 kg samples of organic hard red winter wheat was placed inside 2.2 mm thick polypropylene bags with dimensions of 8″×5″×19″ (width×depth×height) outfitted with a 0.2 μm breather patch. RO filtered water was added at 50% v/m. The bags were subsequently folded around the grain such that the grain was never inverted and the bags were loosely wrapped with EPDM bands.

[0085] The bags were loaded into an autoclave and subjected to a 1.5 hr liquid sterilization cycle. Once sterilized the bags were cooled in a clean space for 12 hours. Each bag was then inoculated with 170 mL of a submerged liquid tissue culture of M. esculenta which had been cultured for 8 days and then sealed with a bag sealer.

[0086] The bags myceliated in a clean space for 21 days, upon which time there was resplendent growth of mycelial hyphae throughout the wheat. The bags' content were dried indoors for three days on parchment paper in a laminar air flow. At this point, duplicate samples of myceliated and autoclaved grain were sent to a third-party laboratory to detect the gluten and glutenin fractions of gluten using an ELISA method (Elisa Technologies, Gainesville, Fla., Aller-Tek® Gluten ELISA test kit). The autoclaved control samples were stored at −20 ° C. for the duration of the myceliation.

[0087] The third-party report concluded an average gluten content of 30 ppm while the control had a level too high to quantitate (the upper limit of quantitation was 80 ppm). The estimated gluten content of the control based on the total protein content and the typical gluten precursor fraction of such wheat is 135,000-160,000 ppm, indicating a greater than 99.99% reduction in gluten content. Once rehydrated, the myceliated gluten-containing grain was essentially aductile and did not knead.

[0088] Tasters found that the wheat had a pleasant flavor and M. esculenta imbued the grain with a honeysuckle taste.

[0089] It was found that there was increased crude nitrogen upon completion of the myceliation. The increased protein content was demonstrated as compared to the identically hydrated and autoclaved non-myceliated grain. This finding was unexpected, because in one embodiment instead of a slightly imperfect mass balance between substrate and mycelial nitrogen, the myceliated gluten-containing grain showed a statistically significant increase in total nitrogen content of the culture by 17%. These strains typically have fruit-bodies with high protein content.

Example 5

[0090] Eight 1.023 kg samples of hard red winter wheat with an initial moisture content of 10.3% was ultimately hydrated to 55% with 767 mL of RO water added pre-sterilization and 250 mL inoculant. The mass occupied 25% of a 12 L polypropylene bag with 0.2 μm breather patch. Four bags were inoculated with M. esculenta and four with T. matsutake grown in a media comprising 20 g/L organic potato starch powder and 8 g/L organic carrot powder and were cultured for 12 days. For each strain two bags were set at room temperature and two at 65° F. Visible myceliation was noticed after 3-5 days for every culture and by day 21 the bags were fully colonized. Samples were sent for gluten ELISAs. The M. esculenta cultures yielded a gluten content of 19.67 ppm while the T. matsutake cultures were at 50 ppm.

Example 6

[0091] The grain is dispensed into a food grade pressure vessel capable of agitation, and is sterilized. This is the most efficient way to sterilize and cool large volumes of grain. The grain is inoculated in situ, with controlled humidity, sterile air supply and temperature. Once myceliation is deemed complete, a process that will take anywhere from 1-22 days if done properly (e.g. the culture is at peak vigor of the log phase, the grain serendipitously provides a desirable carbon/nitrogen ratio, contamination is kept at bay, humidity and temperature are controlled for, etc.), and even shorter with some strains. The grain can then be pasteurized in this pressure vessel by methods known in the art, if so desired, and even dried therein. Both of these steps can be accomplished by other methods known in the art. The grain is considered ‘gluten-free’ by many of the world's regulatory authorities. Methods known in the art can easily produce a ‘gluten-free’ flour according to the FDA, and some embodiments of the invention will produce a grain with gluten content less than 20 ppm.