Enriched food product and production method using a bacterium associated with a host from a nutrient-deficient environment

Abstract

Nutrient-enriched food products and methods of producing nutrient-enriched food products using a bacterium associated with a host from a nutrient-deficient environment.

Description

ENRICHED FOOD PRODUCT AND PRODUCTION METHOD USING A BACTERIUM ASSOCIATED WITH A HOST FROM A NUTRIENT-DEFICIENT ENVIRONMENTCross-Reference to Related Applications

[0001] The present application claims the benefit of U.S. Provisional Application Ser. No. 63 / 580,155, filed September 1, 2023, the contents of which are hereby incorporated by reference herein in their entirety.Technical Field

[0002] The present invention relates to nutrient-enriched food products and methods making nutrient enriched food products, and more particularly to food products enriched in vitamins B2, B5, and B6 through culturing with Dyella terrae.Background Art

[0003] Provision of adequate nutrients to the growing population is a major challenge given its environmental impact and increasing economic costs (Nemecek IntJ Life Cycle Assess. 2016 May; 21: 608-620). At least !4 of the global population suffers from nutrient deficiencies (Miller Food Policy Oct 2013; 42:115-128). Biotechnological interventions in the food sector have aimed to improve the nutritional value of food products using enzymology, genetically-modified microorganisms, or whole microbes to meet the nutritional needs of the global population (Arun, Enzymes in probiotics and genetically modified foods. In Value-addition in food products and processing through enzyme technology. 2022; 13-23, Academic Press; LeBlanc Curr Opin Biotechnol. 2013 Apr;24(2): 160-8).

[0004] Whole cell bacteria have been used to preserve food and produce fermented food products and probiotics (Gmoser Bioengineered. 2020 Dec;l 1 ( 1):582-598; Kumar Recent Trends in Food Biotechnology Contributing in Food Production and Processing. In; Kumar, A., Patruni, K., Singh, V. (eds) Recent Advances in Food Biotechnology. 2022. Springer: Singapore). Bacteria, such as Lactobacillus, Lactococcus, Streptococcus , andBifidobacteria, are widely used in fermented foods (Lukjancenjo Microb Ecol. 2012 Apr;63(3):651-73). Because these probiotics are slow growing at acidic pH and unviable during post-fermentation acidification (Shah J Dairy Sci. 2000 Apr;83(4): 894-907), they have been extensively genetically engineered to grow optimally and produce high levels of nutrients (Ahmed Trends Biotechnol. 2003 Nov;21(l 1 ):491 -7; Rossi Nutrients. 2011 Jan;3(l):l 18-34). Given the concern about the mechanisms by which these products are generated (Plavec Microorganisms. 2020 Feb 21 ;8(2):297), having non-genetically modified bacteria that can naturally enhance the nutrient content of foods may be beneficial for consumers.

[0005] Bacteria from hosts dwelling in nutrient-deficient environments represent a promising prospect of improving the nutritional value, digestibility, taste, and texture of food without genetic modification (Sharma Extremophiles as potential resource for food processing enzymes. Microbial Enzyme Technology’ in Food Applications . 2017; 426). Furthermore, a subset of these nutrient-deficient environments are extreme environments containing bacterial components that can withstand the stresses of extreme pH and cold temperatures during industrial processes (i.e., fermentation) given their native environments (Niehaus Appl Microbiol Biotechnol. 1999 June; 51(6): 711-729). For example, probiotic strains (Lactobacillus, Lactococcus, and Bifidobacteria) are slow growing at acidic pH (< 6). Shah Journal of Dairy Science, 2000 April; 83(2):894-907; Shah Milchwissenschaft, 1997;52:71-76; and Rallu Mol Microbiol, 2000 Feb;35(3):517-28. Extreme environments are characterized as locales with extreme chemical and physical factors and organisms that live and reproduce within extreme ranges of environmental variables (Giddings Bioactive compounds from terrestrial extremophiles . 2015; 1-75. Springer International Publishing). These environments are sources of microbial components that are amenable to industrial applications that utilize extreme conditions, such as temperature, pH, and pressure (Coker FlOOORes. 2016; 5: F1000 Faculty Rev-396).Summary of the Embodiments

[0006] In accordance with some embodiments, the invention provides a cultured food product enriched in a nutrient, the cultured food product comprising a host-associated bacterium and the nutrient, the nutrient being produced by the host-associated bacteriumwhen cultured with a food, thereby forming the cultured food product enriched in the nutrient.

[0007] In accordance with some embodiments, the invention provides a method of producing a cultured food product enriched in a nutrient, the method comprising culturing a food with a host-associated bacterium, wherein the host-associated bacterium produces the nutrient during the culturing, thereby forming the cultured food product enriched in the nutrient.

[0008] In some embodiments, the host-associated bacterium is Dyella terrae. The host-associated bacterium may be Dyella terrae Ely Copper Mine. In some embodiments, the nutrient is riboflavin (vitamin B2), pantothenic acid (vitamin B5), and / or pyridoxine (vitamin B6).

[0009] The food may be a dairy product, a milk alternative, a fruit, a meat, a fish, or a vegetable. The dairy product may be milk. The milk substitute may be almond milk, oat milk, or soy milk. The vegetable may be a cabbage, a carrot, an onion, a radish, or a garlic. The fruit may be an apple or a pepper.

[0010] The cultured food product may be a cultured dairy product, a cultured milk alternative, a fermented fruit product, a fermented fish, a fermented meat, kombucha, or a fermented vegetable product. In some embodiments, the cultured dairy product is cultured milk or a yogurt. The cultured milk substitute may be cultured almond milk, cultured oat milk, or cultured soy milk. The fermented vegetable product may be kimchi or sauerkraut. The fermented fruit product may be a pepper sauce or an apple sauce.Brief Description of the Drawings

[0011] The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

[0012] Fig. 1 is a plot showing a Dyella terrae Ely Copper Mine bacteria growth curve, demonstrating the time it takes for cells to reach stationary phase under the specified growth conditions.

[0013] Fig. 2 shows relative absorbance at 450 nm of UHPLC traces of methanol extracted R2A, Dyella grown in R2A broth, and a riboflavin standard. Lumichrome can beobserved at 4.94 minutes in Dyella cultures in R2A broth. A spike in absorbance at 450 nni for Dyella grown in R2A broth indicates increased riboflavin production, in accordance with embodiments of the invention.

[0014] Fig. 3 shows MS / MS spectra of lumichrome and riboflavin in Dyella in R2A broth showing fragmentation patterns of these flavins, in accordance with embodiments of the invention.

[0015] Fig. 4 shows UV-vis traces at 450 nm of media blank, riboflavin standard, and Dyella extracts analyzed after 2 days, 6 days, and 8 days of growth, in accordance with embodiments of the invention.

[0016] Fig. 5 shows images of A) skim milk cultured with (right) and without (left) Dyella, B) almond milk cultured with (right) and without (left) Dyella, C) oat milk cultured with (right) and without (left) Dyella, and D) yogurt made from skim milk cultured with (left) and without (right) Dyella, and demonstrating visible yellow / orange riboflavin produced in products inoculated with Dyella, in accordance with embodiments of the invention.

[0017] Fig. 6 shows extracted ion chromatograms for vitamin B5 (a.k.a. pantothenic acid) (m / z 220.1185, [M+H]) in whole milk, whole milk inoculated with Dyella, and a pantothenic acid standard, in accordance with embodiments of the invention.

[0018] Fig. 7 shows extracted ion chromatograms for vitamin B6 (a.k.a. pyridoxine) (m / z 170.08172, [M+H]) in whole milk, whole milk inoculated with Dyella, and a pyridoxine standard, in accordance with embodiments of the invention.

[0019] Fig. 8 shows a plot of absorbance at 450 nm of riboflavin standards (0-2.5 mg / ml) in water (black) and Dyella containing whey spiked with each riboflavin standard (gray), in accordance with embodiments of the invention.Detailed Description of Specific Embodiments

[0020] Definitions. As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:

[0021] The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0022] “Nutrient” means a substance that is essential for growth and maintenance of life. In some embodiments, a nutrient is essential for growth and maintenance of mammalian life. A nutrient includes, but is not limited to, vitamins and provitamins.

[0023] A “host-associated bacterium” means a bacterium occurring on or inside surfaces of a non-mammalian host, wherein, during the association, the bacterium provides the non-mammalian host with a nutrient that the non-mammalian host is unable to synthesize in quantities sufficient for the sustainment of life and is unable to obtain from its environment. In some embodiments, the non-mammalian host is an amoeba. As used herein, such an environment is referred to as a “nutrient-deficient environment.”

[0024] A “dairy product” means a product made from dairy, for example, milk.

[0025] A “milk substitute” or “milk alternative” means a substance that resembles milk and which can be used in the same ways as milk. Non-limiting examples of a milk substitute include soy milk, oat milk, and almond milk.

[0026] A “fermented vegetable product” means a vegetable-based food product produced through controlled bacterial growth of a bacterium with a vegetable, resulting in conversion of a vegetable component through enzymatic action of the bacterium. The controlled bacterial growth may be aerobic or anaerobic. Non limiting examples of fermented vegetable products include kimchi and sauerkraut.

[0027] A “fermented fruit product” means a fruit-based food product produced through controlled bacterial growth of a bacterium with a fruit, resulting in conversion of a fruit component through enzymatic action of the bacterium. The controlled bacterial growth may be aerobic or anaerobic. Non-limiting examples of fermented fruit products include pepper sauce, such as hot sauce, and fermented apple products, such as apple sauce.

[0028] Here, we disclose use of a host-associated bacterium obtained from a nutrientdeficient environment to produce cultured food products enriched in a nutrient, the nutrient being produced by the host-associated bacterium when cultured with a food. In someembodiments, the host-associated bacterium provides a host with the nutrient in the nutrientdeficient environment.

[0029] We have, surprisingly, found that a host-associated bacterium, Dyella terrae Ely Copper Mine, isolated and identified from acid rock drainage from Ely Copper Mine, Vermont, (Giddings Front Microbiol. 2022 May 23; 13 : 856908) produces a significant amount of nutrients including riboflavin (vitamin B2), pantothenic acid (vitamin B5), and pyridoxine (vitamin B6) when cultured. It is likely that D. terrae supplies its amoeba host with these nutrients, which are deficient in the acid rock drainage. We further show that when cultured with food, Dyella terrae produces riboflavin (vitamin B2), pantothenic acid (vitamin B5), and pyridoxine (vitamin B6), thereby forming a food product enriched in these nutrients.

[0030] In accordance with some embodiments, the invention provides a cultured food product enriched in a nutrient, the cultured food product comprising a host-associated bacterium and the nutrient, the nutrient being produced by the host-associated bacterium when cultured with a food, thereby forming the cultured food product enriched in the nutrient.

[0031] In accordance with some embodiments, the invention provides a method of producing a cultured food product enriched in a nutrient, the method comprising culturing a food with a host-associated bacterium, wherein the host-associated bacterium produces the nutrient during the culturing, thereby forming the cultured food product enriched in the nutrient.

[0032] In some embodiments, the host-associated bacterium is Dyella terrae. The host-associated bacterium may be Dyella terrae Ely Copper Mine. In some embodiments, the nutrient is riboflavin (vitamin B2), pantothenic acid (vitamin B5), and / or pyridoxine (vitamin B6).

[0033] The food may be a dairy product, a milk alternative, a fruit, a meat, a fish, or a vegetable. The dairy product may be milk. The milk substitute may be almond milk, oat milk, or soy milk. The vegetable may be a cabbage, a carrot, an onion, a radish, or a garlic. The fruit may be a pepper or an apple.

[0034] The cultured food product may be a cultured dairy product, a cultured milk alternative, a fermented fruit product, a fermented fish, a fermented meat, kombucha, or afermented vegetable product. In some embodiments, the cultured dairy product is cultured milk or a yogurt. The cultured milk substitute may be cultured almond milk, cultured oat milk, or cultured soy milk. The fermented vegetable product may be kimchi or sauerkraut. The fermented fruit product may be a pepper sauce or fermented apple product.

[0035] Example 1: Isolation of Amoeba Host from a Nutrient-Deficient Environment

[0036] Acid mine drainage is an acidic metal-rich effluent that has low carbon levels and an acidic pH, making it difficult for microorganisms to thrive (Giddings PLoS One. 2020 Aug 12; 15(8):e0237599). This extreme environment is an example of a nutrient-deficient aqueous environment that maybe sampled for a host, e.g., an amoeba, which harbors a host- associated microbe required for the host’s survival (Giddings Front Microbiol. 2022 May 23; 13 :856908).

[0037] Acid mine drainage was collected in autoclaved glass containers and 100 mL were filtered through 3 -pm nitrocellulose filter paper by vacuum filtration as described in Giddings Front Microbiol. 2022 May 23; 13 :856908. The resulting filter paper was placed upside down on non-nutrient agar (sodium chloride, 60 g / L; sodium citrate tribasic, 0.8 g / L; magnesium sulfate, 4 mM; monobasic sodium phosphate, 2.5 mM; monobasic potassium phosphate, 2.5 mM; calcium chloride, 0.5 mM; and agar, 15 g / L) and plates were seeded with 104Escherichia coli K12 strain SMG 123 cells (PTA-7555, ATCC; Manassas, VA, United States) and incubated at 20 °C. Plates were examined under an inverted microscope every three days to probe for amoebae along the edges of filter paper. Amoebae were recovered by scraping and transferred to a Page’s Amoeba Saline Solution (PAS) (magnesium sulfate,4 mM; calcium chloride, 0.4 M; sodium citrate dihydrate, 0.1%; monosodium phosphate, 2.5 mM; monopotassium phosphate, 2.5 mM; and pH adjusted to 4) supplemented with5 x 108E. coli cells in Luria-Bertani broth for growth at 20 °C. Amoebae were then subcultured by dilution in PAS once they reached confluency.

[0038] Example 2; DNA Extraction and Identification of Amoeba Host

[0039] To determine the taxonomy of the isolated amoeba, we used the methods of Giddings et al. 2022 (Giddings Front Microbiol. 2022 May 23; 13:856908). Briefly, amoeba liquid culture (30 ml) was filtered through a 3 pm nitrocellulose filter paper and DNA was extracted using a High Pure PCR template preparation kit (Roche; Basel, Switzerland)followed by ethanol precipitation (Giddings Front Microbiol. 2022 May 23; 13 :856908). Using 566F (5'-CAGCAGCCGCGGTAATTCC-3') (SEQ ID NO: 1) and 1200R (5'- CCCGTGTTGAGTCAAATTAAGC-3') (SEQ ID NO: 2) primers, PCR was used to amplify an 18S rRNA gene from extracted DNA using the following thermocycling parameters: 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 50°C for 20 s, 72°C for 1 min, and a final extension at 72°C for 7 min. Sanger sequencing was performed and the resulting sequence information was compared to sequences in the NCBI nr database using BLAST. The taxonomy was detemiined to be Stemonitis sp. (Giddings Front Microbiol. 2022 May 23; 13 :856908) with 99.79% identity.

[0040] Example 3: Identification of Host-Associated Bacterium

[0041] Whole genome sequencing of isolated amoeba DNA was performed using a combination of Illumina and Oxford Nanopore sequencing methods described in Giddings et al. 2022 (Giddings Front Microbiol. 2022 May 23; 13 :856908). A complete bacterial genome was also identified in the resulting assembly of sequencing data. Furthermore, this bacterium was continuously isolated from amoebae that had undergone several passages in PAS medium. The 16S rDNA sequence in the bacterial genome was compared to sequences in the NCBI nt database for taxonomic identification and named Dyella terrae Ely Copper Mine ''Dyella"’). Its association with the amoeba was visualized using confocal microscopy to image fluorescent in situ hybridization (FISH) with fluorescently labeled 16S and 18S rDNA probes as outlined in Giddings et al. 2022 (Giddings Front Microbiol. 2022 May 23; 13 :856908).

[0042] Example 4; Isolation and Cultivation of Host- ssociated Bacterium

[0043] Dyella were isolated from subcultured amoebae on BD Difco R2A agar (East Rutherford, NJ) plates and incubated at 30 °C for at least 24—48 hours. These Gram-negative bacterium formed yellow colonies after 1-2 days of growth.

[0044] Example 5; Initial Culturing and Metabolomics of Host-Associated Bacterium

[0045] As isolation of the amoeba host from its associated Dyella proved impossible, we sought to identify secondary metabolites and chemical signaling agents involved in this association by conducting microbial cultivation and LC-MS metabolomics experiments.

[0046] Preliminary investigation focused on bacterial metabolites with longer wavelength absorbing chromophores. Secondary metabolites and bioactive molecules are often conjugated and display such absorbances (Sant’ Anna Dyes and Pigments. 2013 Sept; 98(3):601-608). Briefly, 100 mL of BD Difco R2A broth (East Rutherford, NJ) was inoculated with a colony of Dyella and incubated at 30 °C with shaking at 250 rpm until stationary phase was reached and cultures were yellow, indicating pigments were produced (Figure 1). Cultures were grown for 2-10 days and extracted with HPLC-grade methanol using a solid-phase resin. Diaion HP -20 solid resin (5 g; Millipore Sigma, St. Louis, MO) was pre -rinsed with HPLC-grade methanol (50 mL) twice, washed with HPLC-grade water and then 100 mL cultures were poured over the resin and sonicated for 20 minutes before being left to incubate overnight at room temperature with shaking at 100 rpm. HPLC-grade methanol and resin were centrifuged at 4000 rpm for 10 min. Then, the resulting supernatant was evaporated to dryness using a nitrogen evaporator.

[0047] Extracts were reconstituted in methanol (10 mg / ml) and analyzed on a Thermo Scientific (Waltham, MA) Q-Exactive HF-X Hybrid Quadrupole-Orbitrap mass spectrometer interfaced with a Vanquish Horizon UHPLC system and VH-D10-A UV detector using a Waters (Milford, MA) HSS T3 C18 column (1.8 pm, 2.1 x 100 mm) with a VanGuard FIT precolumn. Using a flow rate of 0.5 mL / min, samples (2 pL) were injected onto the column, which was warmed to 40 °C. UHPLC separation was achieved using the following parameters: 2% acetonitrile: 98% water with 0.1% formic acid for 1 min, 2-40% over 4 min, 40-98% over 3 min, 98-2% over 0.2 min, and 2% for 2 min. Based on the yellow pigmentation of the Dyella, UV-vis data were collected at 450 nm (Figure 2). The following ESI settings were used in probe position D: 40 sheath gas flow, 8 auxiliary gas flow, 1 sweep gas flow, 3.5 kV spray voltage, 380 °C capillary temperature, 50 radiofrequency (RF) funnel level, and 350 °C auxiliary gas temperature. The major peaks were serendipitously found to be flavin derivatives with m / z values corresponding to lumichrome (m / z 243.0882, [M+H]) and riboflavin (m / z, 377.1461, [M+H]) and compound identities were confirmed by comparison of MS / MS spectra with Pubchem reference spectra and injection with authentic standards.

[0048] A lumichrome standard was obtained according to the methods of Suzuki et al. using a riboflavin supplement capsule (Suzuki J Chrom A. 1979; 169:459-461). Thecapsule was opened and dissolved in 250 ml of MilliQ water and incubated in sunlight. After one week, lumichrome and derivatives were extracted with 5% w / v HP20 resin, eluted with chloroform, evaporated, and reconstituted in HPLC-grade methanol for LC / MS analysis. Additionally, a multivitamin standard containing, riboflavin, pantothenic acid, and pyridoxine, was crushed for LC / MS analysis. These standards (10 mg) were dissolved in 1 mL of HPLC-grade methanol, and the mixture was injected onto the column to be able to identify vitamin standards. The MS / MS spectrum for each compound was compared to those of known molecules in the literature (Figure 3). In a time-course experiment (Figure 4), we observed an accumulation of lumichrome, a degradation product of riboflavin. Therefore, we extracted cells at earlier growth stages to obtain more riboflavin, as it is more abundant in the log phase. These metabolites were also relevant since amoebae are known to exhibit chemotactic responses towards riboflavin and other vitamins (Maeda Zoolog Sei. 2009 Mar;26(3): 179-86; Vargas- Villareal Parasitology’. 2020 Apr; 147(4): 501-505). Amoebae have been shown to acquire riboflavin through symbionts (Kamm Sci Rep. 2019 Nov 26;9(1): 17561). Furthermore, Dyella have also been isolated from traditional fermented food preparations (Lackey J Microbiol Methods . 2017 Oct;141:73-81; Melkonian Front Microbiol. 2019 Jun 25 ; 10: 1347). Thus, these vitamin-producing symbionts can be exploited to make products enriched with essential nutrients.

[0049] Example 6: Production of Vitamin-Enriched Dairy and Nondairy Food Products

[0050] Given the color of the fermented product being yellow, metabolites with chromophores, including secondary metabolites and signaling agents, were considered. Sterilized whole, skim, oat, and almond milk (100 mL) were inoculated with a colony of Dyella terrae Ely Copper Mine from R2A agar and grown at 30 °C for five days with shaking at 250 rpm along with sterilized dairy controls. Cultured inoculated with Dyella terrae Ely Copper Mine began to display orange pigmentation on the third day of growth (Figure 5A-C). The orange pigment indicated the presence of flavins, which can vary in color due to solvation conditions (Kar Advanced Review. 2022 May 11 ; 12(2): el 541. Vitamins, including pantothenic acid and pyridoxine (vitamins B5 and B6) (Figures 6-7), were detected using the aforementioned organic extraction and LC / MS methods. Over a twofold increase in pantothenic acid and pyridoxine were observed in milk inoculated withDyella. Notably, this finding can be applied to other nondairy food products that lack the typical levels of vitamins found in dairy products. Ion suppression was observed likely due to the presence of coeluting peptides. Therefore, further studies quantifying riboflavin in dairy products were performed using a UV-visible spectrophotometer, as detailed below.

[0051] Example 7: Production of Vitamin-Enriched Greek Yogurt

[0052] Sterilized skim milk (100 mL) was inoculated with a colony of Dyella terrae Ely Copper Mine for two days and then 25 mL of either this starter culture or sterilized skim milk was added to 125 mL of sterilized milk containing a tablespoon of plain Greek yogurt and left to incubate overnight at 40 °C. After 24 hours, a yellow whey was observed on top of the bacterium-inoculated Greek yogurt (Figure 5D).

[0053] Example 8; Detection and Quantification of Riboflavin using UV-visible Spectroscopy

[0054] The lipid content of dairy products can complicate LC / MS analyses, thereby warranting another method for riboflavin quantification. Thus, in order to quantify riboflavin, riboflavin standards (0-2.5 mg / ml) were prepared in water to prepare a standard curve in a 96-well microplate. Then, 100 pL of each standard were used to spike whey samples (100 pL) from Greek yogurt with or without D. terrae Ely Copper Mine (Figure 5D). The absorbance of samples were measured at 450 nm using an Epoch BioTek (Agilent; Santa Clara, CA) microplate spectrophotometer to quantify the levels of riboflavin in each sample. Spiked samples were used to quantify the riboflavin concentration to be 0.025 mg / ml based on extrapolating concentrations from non-spiked samples (Figure 8).

[0055] Example 9; Production of Vitamin-Enriched Hot Sauce

[0056] A 3% (w / w) saline solution with completely submerged green bell and jalapeno peppers (included in the total weight with water for % sodium chloride) was inoculated in a 1-L flask with and without 10 mL of Dyella and left to ferment for 1 month. The resulting solution can be extracted using the aforementioned method with other dairy and nondairy products.

[0057] The publications (including patent publications), web sites, company names, books, manuals, treatise, and scientific literature referred to herein establish the knowledge that is available to those with skill in the art and are hereby incorporated by reference in their entirety to the same extent as if each was specifically and individually indicated to beincorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter.

[0058] Various embodiments of the present invention may be characterized by the potential claims listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims form a part of the written description of this application. Accordingly, subject matter of the following potential claims may be presented as actual claims in later proceedings involving this application or any application claiming priority based on this application. Inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Thus, a decision to not present these potential claims in later proceedings should not be construed as a donation of the subject matter to the public.

[0059] Without limitation, potential subject matter that may be claimed (prefaced with the letter “P” so as to avoid confusion with the actual claims presented below) includes:Pl. A cultured food product enriched in a nutrient, the cultured food product comprising a host-associated bacterium and the nutrient, the nutrient being produced by the host- associated bacterium when cultured with a food, thereby forming the cultured food product enriched in the nutrient.P2. The cultured food product of potential claim Pl, wherein the host-associated bacterium is Dyella terrae.P3. The cultured food product according to any one of potential claims P1-P2, wherein the host-associated bacterium is Dyella terrae Ely Copper Mine.P4. The cultured food product according to any one of potential claims P1-P3, wherein the nutrient is riboflavin, (vitamin B2), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), and combinations thereof.P5. The cultured food product according to any one of potential claims P1-P4, wherein the food is selected from the group consisting of a dairy product, a milk alternative, a fruit, a meat, a fish, and a vegetable.P6. The cultured food product according to any one of potential claims P1-P5, wherein the cultured food product is selected from the group consisting of kombucha, a cultured dairy product, a cultured milk alternative, a fermented fruit product, a fermented meat, a fermented fish, and a fermented vegetable product.P7. The cultured food product of potential claim P6, wherein the cultured dairy product is selected from the group consisting of cultured milk and a yogurt.P8. The cultured food product of potential claim P6, wherein the cultured milk substitute is selected from the group consisting of cultured almond milk, cultured oat milk, and cultured soy milk.P9. The cultured food product of potential claim P6, wherein the fermented vegetable product is selected from the group consisting of kimchi and sauerkraut.PIO. The cultured food product of potential claim P6, wherein the fermented fruit product is selected from the group consisting of a fermented apple product and a pepper sauce.Pl 1. The cultured food product of potential claim P5, wherein the dairy product is milk.P12. The cultured food product of potential claim P5, wherein the milk substitute is selected from the group consisting of almond milk, oat milk, and soy milk.P13. The cultured food product of potential claim P5, wherein the vegetable is a cabbage, a carrot, an onion, a radish, and a garlic.P14. The cultured food product of potential claim P5, wherein the fruit is an apple and a pepper.Pl 5. A method of producing a cultured food product enriched in a nutrient, the method comprising culturing a food with a host-associated bacterium, wherein the host-associated bacterium produces the nutrient during the culturing, thereby forming the cultured food product enriched in the nutrient.Pl 6. The method of potential claim Pl 5, wherein the host-associated bacterium is Dyella terrae.Pl 7. The method according to any one of potential claims P15-P16, wherein the host- associated bacterium is Dyella terrae Ely Copper Mine.P18. The method according to any one of potential claims P15-P17, wherein the nutrient is riboflavin, (vitamin B2), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), and combinations thereof.P19. The method according to any one of potential claims P15-P18, wherein the food is selected from the group consisting of a dairy product, a milk alternative, a fruit, a meat, a fish, and a vegetable.P20. The method according to any one of potential claims P15-P19, wherein the cultured food product is selected from the group consisting of kombucha, a cultured dairy product, a cultured milk alternative, a fermented fruit product, a fermented meat, a fermented fish, and a fermented vegetable product.P21. The method of potential claim P20, wherein the cultured dairy product is selected from the group consisting of cultured milk and a yogurt.P22. The method of potential claim P20, wherein the cultured milk substitute is selected from the group consisting of cultured almond milk, cultured oat milk, and cultured soy milk.P23. The method of potential claim P20, wherein the fermented vegetable product is selected from the group consisting of kimchi and sauerkraut.P24. The method of potential claim P20, wherein the fermented fruit product is a fermented apple product or a pepper sauce.P25. The cultured food product of potential claim Pl 9, wherein the dairy product is milk.P26. The cultured food product of potential claim Pl 9, wherein the milk substitute is selected from the group consisting of almond milk, oat milk, and soy milk.P27. The cultured food product of potential claim Pl 9, wherein the vegetable is a cabbage, a carrot, an onion, a radish, and a garlic.P28. The cultured food product of potential claim P5, wherein the fruit is an apple or a pepper.P29. The cultured food product according to any one of potential claims P1-P5, wherein the cultured food product is a cultured dairy product selected from the group consisting of cultured milk and a yogurt.P30. The cultured food product according to any one of potential claims P1-P5, wherein the cultured food product is a cultured milk substitute selected from the group consisting of cultured almond milk, cultured oat milk, and cultured soy milk.P31. The cultured food product according to any one of potential claims P1-P5, wherein the cultured food product is a fermented vegetable product selected from the group consisting of kimchi and sauerkraut.P32. The cultured food product according to any one of potential claims P1-P5, wherein the cultured food product is a fermented fruit product selected from the group consisting of a fermented apple product and a pepper sauce.P33. The cultured food product according to any one of potential claims P1-P4 and P29, wherein the food is milk.P34. The cultured food product according to any one of potential claims P1-P4 and P30, wherein the food is a milk substitute selected from the group consisting of almond milk, oat milk, and soy milk.P35. The cultured food product according to any one of potential claims P1-P4 and P31, wherein the food is a vegetable selected from the group consisting of a cabbage, a carrot, an onion, a radish, a garlic, and combinations thereof.P36. The cultured food product according to any one of potential claims P1-P4 and P32, wherein the food is a fruit selected from the group consisting of an apple and a pepper.P37. The method according to any one of potential claims P15-P19, wherein the cultured food product is a cultured dairy product selected from the group consisting of cultured milk and a yogurt.P38. The method according to any one of potential claims P15-P19, wherein the cultured food product is a cultured milk substitute selected from the group consisting of cultured almond milk, cultured oat milk, and cultured soy milk.P39. The method according to any one of potential claims P15-P19, wherein the cultured food product is a fermented vegetable product selected from the group consisting of kimchi and sauerkraut.P40. The method according to any one of potential claims P15-P19, wherein the cultured food product is a fermented fruit product selected from the group consisting of a fermented apple product and a pepper sauce.P41. The cultured food product according to any one of potential claims P15-P18 and P37, wherein the food is milk.P42. The cultured food product according to any one of potential claims P15-P18 and P38, wherein the food is a milk substitute selected from the group consisting of almond milk, oat milk, and soy milk.P43. The cultured food product according to any one of potential claims P15-P18 and P39, wherein the food is a vegetable selected from the group consisting of a cabbage, a carrot, an onion, a radish, a garlic, and combinations thereof.P44. The cultured food product according to any one of potential claims P15-P18 and P40, wherein the food is a fruit selected from the group consisting of an apple and a pepper.

[0060] The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

What is claimed is:

1. A cultured food product enriched in a nutrient, the cultured food product comprising a host-associated bacterium and the nutrient, the nutrient being produced by the host- associated bacterium when cultured with a food, thereby forming the cultured food product enriched in the nutrient.

2. The cultured food product of claim 1, wherein the host-associated bacterium is Dyella terrae.

3. The cultured food product according to any one of the preceding claims, wherein the host- associated bacterium is Dyella terrae Ely Copper Mine.

4. The cultured food product according to any one of the preceding claims, wherein the nutrient is riboflavin, (vitamin B2), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), and combinations thereof.

5. The cultured food product according to any one of the preceding claims, wherein the food is selected from the group consisting of a dairy product, a milk alternative, a fruit, a meat, a fish, and a vegetable.

6. The cultured food product according to any one of the preceding claims, wherein the cultured food product is selected from the group consisting of kombucha, a cultured dairy product, a cultured milk alternative, a fermented fruit product, a fermented meat, a fermented fish, and a fermented vegetable product.

7. The cultured food product according to any one of claims 1-5, wherein the cultured food product is a cultured dairy product selected from the group consisting of cultured milk and a yogurt.

8. The cultured food product according to any one of claims 1—5, wherein the cultured food product is a cultured milk substitute selected from the group consisting of cultured almond milk, cultured oat milk, and cultured soy milk.

9. The cultured food product according to any one of claims 1-5, wherein the cultured food product is a fermented vegetable product selected from the group consisting of kimchi and sauerkraut.

10. The cultured food product according to any one of claims 1-5, wherein the cultured food product is a fermented fruit product selected from the group consisting of a fermented apple product and a pepper sauce.

11. The cultured food product according to any one of claims 1-4 and 7, wherein the food is milk.

12. The cultured food product according to any one of claims 1-4 and 8, wherein the food is a milk substitute selected from the group consisting of almond milk, oat milk, and soy milk.

13. The cultured food product according to any one of claims 1-4 and 9, wherein the food is a vegetable selected from the group consisting of a cabbage, a carrot, an onion, a radish, a garlic, and combinations thereof.

14. The cultured food product according to any one of claims 1-4 and 10, wherein the food is a fruit selected from the group consisting of an apple and a pepper.

15. A method of producing a cultured food product enriched in a nutrient, the method comprising culturing a food with a host-associated bacterium, wherein the host-associated bacterium produces the nutrient during the culturing, thereby forming the cultured food product enriched in the nutrient.

16. The method of claim 15, wherein the host-associated bacterium is Dyella terrae.

17. The method according to any one of claims 15-16, wherein the host-associated bacterium is Dyella terrae Ely Copper Mine.

18. The method according to any one of claims 15-17, wherein the nutrient is riboflavin, (vitamin B2), pantothenic acid (vitamin B5), pyridoxine (vitamin B6), and combinations thereof.

19. The method according to any one of claims 15-18, wherein the food is selected from the group consisting of a dairy product, a milk alternative, a fruit, a meat, a fish, and a vegetable.

20. The method according to any one of claims 15-19, wherein the cultured food product is selected from the group consisting of kombucha, a cultured dairy product, a cultured milk alternative, a fermented fruit product, a fermented meat, a fermented fish, and a fermented vegetable product.

21. The method according to any one of claims 15-19, wherein the cultured food product is a cultured dairy product selected from the group consisting of cultured milk and a yogurt.

22. The method according to any one of claims 15-19, wherein the cultured food product is a cultured milk substitute selected from the group consisting of cultured almond milk, cultured oat milk, and cultured soy milk.

23. The method according to any one of claims 15-19, wherein the cultured food product is a fermented vegetable product selected from the group consisting of kimchi and sauerkraut.

24. The method according to any one of claims 15-19, wherein the cultured food product is a fermented fruit product selected from the group consisting of a fermented apple product and a pepper sauce.

25. The cultured food product according to any one of claims 15—18 and 21, wherein the food is milk.

26. The cultured food product according to any one of claims 15-18 and 22, wherein the food is a milk substitute selected from the group consisting of almond milk, oat milk, and soy milk.

27. The cultured food product according to any one of claims 15-18 and 23, wherein the food is a vegetable selected from the group consisting of a cabbage, a carrot, an onion, a radish, a garlic, and combinations thereof.

28. The cultured food product according to any one of claims 15-18 and 24, wherein the food is a fruit selected from the group consisting of an apple and a pepper.

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