MICROBIAL COMPOSITIONS AND METHODS FOR FERMENTED FOODS
20260096563 ยท 2026-04-09
Inventors
Cpc classification
A21D8/045
HUMAN NECESSITIES
C12R2001/01
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates to compositions comprising a purified population of one or more microorganisms for producing a dough or baked food product. Said compositions may improve fermentation of the dough or baked food product by enhancing rheological properties, improving nutritional quality, and/or increasing shelf-life of the dough or baked food product.
Claims
1. A composition comprising: (a) a purified population of microorganisms comprising: (i) a yeast with an ITS nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 207; (ii) a bacterium with a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 4; and/or (iii) a bacterium with a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 8; and (b) a food-grade carrier or excipient; and wherein the purified population of microorganisms is lyophilized.
2. The composition of claim 1, wherein the purified population of microorganisms comprises a yeast with an ITS nucleic acid sequence comprising SEQ ID NO: 207.
3. The composition of claim 1, wherein the purified population of microorganisms comprises a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 4.
4. The composition of claim 1, wherein the purified population of microorganisms comprises a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 8.
5. The composition of claim 1, wherein the purified population of microorganisms comprises a Saccharomyces cerevisiae as deposited as PTA-127804.
6. The composition of claim 1, wherein the purified population of microorganisms comprises a Fructilactobacillus sanfranciscensis as deposited as PTA-127806.
7. The composition of claim 1, wherein the purified population of microorganisms comprises a Levilactobacillus brevis as deposited as PTA-127808.
8. The composition of claim 1, wherein the purified population of microorganisms comprises: (a) a yeast with an ITS nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 207; (b) a bacterium with a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 4; and (c) a bacterium with a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 8.
9. The composition of claim 1, wherein the purified population of microorganisms comprises: (a) a yeast with an ITS nucleic acid sequence comprising SEQ ID NO: 207; (b) a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 4; and/or (c) a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 8.
10. The composition of claim 1, wherein the purified population of microorganisms comprises: (a) a yeast with an ITS nucleic acid sequence comprising SEQ ID NO: 207; (b) a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 4; and (c) a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 8.
11. The composition of claim 1, wherein the purified population of microorganisms comprises: (a) a Saccharomyces cerevisiae as deposited as PTA-127804; (b) a Fructilactobacillus sanfranciscensis as deposited as PTA-127806; and/or (c) a Levilactobacillus brevis as deposited as PTA-127808.
12. The composition of claim 1, wherein the purified population of microorganisms comprises: (a) a Saccharomyces cerevisiae as deposited as PTA-127804; (b) a Fructilactobacillus sanfranciscensis as deposited as PTA-127806; and (c) a Levilactobacillus brevis as deposited as PTA-127808.
13. The composition of claim 1, wherein each of the microorganisms is present in the composition at about 10.sup.2 CFU/g or more.
14. The composition of claim 1, wherein each of the microorganisms is present in the composition at about 10.sup.9 CFU/g, about 10.sup.10 CFU/g, about 10.sup.11 CFU/g, about 10.sup.12 CFU/g, about 10.sup.13 CFU/g, or about 10.sup.14 CFU/g.
15. The composition of claim 1, wherein the composition comprises one or more preservatives, flavoring agents, or leavening agents.
16. The composition of claim 1, wherein the composition comprises maltose, trehalose, sucrose, maltodextrin, ascorbic acid, or a combination thereof.
17. The composition of claim 1, wherein the composition is formulated as a dry powder, food ingredient, food preparation, food supplement, water additive, moisture-stabilized additive, consumable, solid, gel, liquid, or dough.
18. The composition of claim 1, wherein the composition is a starter culture for baked good production, a baked good, or a dough.
19. The composition of claim 1, wherein the composition is a further comprises a bacterium with a 16S nucleic acid sequence sharing at least 99% sequence identity to SEQ ID NO: 269.
20. The composition of claim 1, wherein the composition further comprises a Kazachstania humilis as deposited as PTA-127805.
21. A method for improving fermentation of a food product, the method comprising admixing an effective amount of the composition of claim 1 to the food product.
22. A method of producing a dough, the method comprising admixing an effective amount of the composition of claim 1 to the dough.
23. A method of increasing leavening ability of a dough, the method comprising admixing an effective amount of the composition of claim 1 to the dough.
24. A method of lowering pH of a dough, the method comprising admixing an effective amount of the composition of claim 1 to the dough.
25. A method of increasing shelf-life of a dough or baked good, the method comprising admixing an effective amount of the composition of claim 1 to the dough or baked good.
26. A method of enhancing rheological properties of a dough or baked good, the method comprising admixing an effective amount of the composition of claim 1 to the dough or baked good.
27. A method of increasing flavor or aroma of a dough or baked good, the method comprising admixing an effective amount of the composition of claim 1 to the dough or baked good.
28. A method of increasing nutritional quality of a dough or baked good, the method comprising admixing an effective amount of the composition of claim 1 to the dough or baked good.
29. A method of increasing degradation of: (a) complex and/or simple carbohydrates in a dough; (b) chemical compounds or chemical precursors in a dough; or (c) proteins in a dough; the method comprising admixing an effective amount of the composition of claim 1 to the dough.
30. A method of increasing production of organic acids, peptides, and/or proteins in a dough, the method comprising admixing an effective amount of the composition of claim 1 to the dough.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0043] The accompanying figures, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, example embodiments and/or features of the present invention. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.
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DETAILED DESCRIPTION
[0058] The present disclosure provides isolated microorganisms, including novel strains of the microorganisms, microbial consortia, and compositions comprising the same, which can replicate both the organoleptic and health benefits observed with sourdough microbial communities in a safe, affordable, and consistent way at scale.
[0059] These microorganisms can be used in methods that include, inter alia, the improved fermentation of dough resulting in improved sensory evaluation, health, and shelf-life of the fermented food product. In some embodiments, the disclosure provides methods of improving the digestion, glycemic index, immunogenic gluten content, texture, aroma, flavor, and shelf-life of fermented foods through improved dough fermentation.
Definitions
[0060] Unless otherwise defined herein, technical and scientific terms used in the present description have the meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa unless the content clearly dictates otherwise. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.
[0061] The terms a, an, and the, as used herein, include plural references unless the context clearly dictates otherwise. As such, the terms a, an, one or more, and at least one are used interchangeably herein. In addition, reference to an element by the indefinite article a or an does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.
[0062] Throughout this application, the term about is used to indicate that a value includes the inherent variation of error for the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term about means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term about applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms about and approximately are used as equivalents.
[0063] The term and/or, as used herein, refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
[0064] The term between, as used in a phrase as such between A and B or between A-B refers to a range including both A and B.
[0065] The terms comprise and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
[0066] The terms including, includes, included, and other forms, as used herein, are not limiting.
[0067] As used herein, the term sequence identity refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of residues, e.g., nucleotides or amino acids. An identity fraction for aligned segments of a test sequence and a reference sequence is the number of identical residues which are shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent identity is the identity fraction times 100. Comparison of sequences to determine percent identity can be accomplished by a number of well-known methods, including for example by using mathematical algorithms, such as, for example, those in the BLAST suite of sequence analysis programs. Unless noted otherwise, the term sequence identity in the claims refers to sequence identity as calculated by Clustal Omega using default parameters.
[0068] The term composition relates to a composition comprising one or more of the microorganisms described herein and optionally one or more acceptable carriers. In some embodiments, the composition is a non-naturally occurring composition. In some embodiments, the composition is a food-grade composition. In some embodiments, the composition comprises additional components, such as stabilizers, bread improvers, and preservatives. In some embodiments, the composition is a starter culture, a dough, or a food product (such as a baked good).
[0069] The term starter culture refers to a preparation of living microorganisms that are added to foods to help start fermentation. The starter culture can comprise a single type of microorganism or a mixture of two or more microorganisms. The microorganisms can be bacteria, yeasts, or molds. Starter cultures are used to produce fermented foods by accelerating the fermentation process and changing the chemical composition and sensory properties of the substrate to create a more homogeneous product.
[0070] The term food product refers to each and every product for human or animal food consumption. In some embodiments, the food product is food for human consumption. In some embodiments, the food product is a fermented food product, such as a dough or baked good. In some embodiments, the food product is a fermented dairy product.
[0071] The term baked good includes any and all goods made from a dough or batter and cooked by baking, frying, or deep frying, preferably by baking. In some embodiments, the baked product is produced with an acidic dough. In some embodiments, the baked good is a bread, including flatbreads, bagels, bread rolls, and the like; a cracker; a pastry product; a tart or pie; or a viennoiserie. In some embodiments, the baked product is made from a leavened dough, such as loaf bread such as toast, baguette, and the like; leavened flatbread; or viennoiserie. In some embodiments, the baked good is sourdough bread.
[0072] The term flour is generally known to the skilled person. In some embodiments, the flour is cereal flour, such as a wheat, rye, barley, oat, corn, rice, spelt, sorghum millet, emmer, einkorn, kamut, or buckwheat flour. In some embodiments, the flour is pastry flour, all-purpose flour, or bread flour. In some embodiments, the flour is a powder generated by grinding grains, roots, beans, nuts, or other fruits of edible plants or parts thereof.
[0073] The term dough is used herein in a broad sense relating to any and all mixtures comprising the components described herein. In some embodiments, the dough is a liquid dough such as a batter or a semisolid or solid dough. In some embodiments, the dough comprises at least one type of flour. In some embodiments, the dough comprises different types of flour such as a mixed wheat flour/rye flour dough. In some embodiments, the dough comprises at least 10% (w/w) flour, at least 20% (w/w) flour, at least 30% (w/w) flour, at least 40% (w/w) flour, or at least 50% (w/w) flour. In some embodiments, the dough comprises one or more of the microorganisms described herein. In some embodiments, the dough comprises glucose and/or sucrose. In some embodiments, the dough comprises one or more microorganisms described herein, flour, and water.
[0074] Th term admixing refers to mixing a component(s) with another component(s) to homogeneity. In some embodiments, admixing comprises kneading the components. In some embodiments, admixing comprises stirring the components.
[0075] The term shelf-life refers to the time that the fermented dough or baked good can be stored while maintaining properties including, but not limited to, organoleptic properties, nutritional value, and safety. The safety of the fermented dough or baked goods includes, but is not limited to, competitively excluding microbial pathogens, molds, or other organisms that lead to food spoilage.
[0076] The autolyse stage referred to herein is defined as the resting period between the initial mixing and addition of the salt and/or leaving agent.
[0077] The bulk stage referred to herein is defined as first primary fermentation after the salt and leaving agent have been added.
[0078] The portion stage referred to herein is the dividing of the dough into individual pieces and shaping them into the final form for secondary fermentation or baking.
[0079] The term food grade carrier or excipient means a non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation of any type.
Microorganisms of the Disclosure
[0080] In some embodiments, the present disclosure provides isolated microorganisms, including novel strains of microorganisms, presented in Table 1. In some embodiments, the present disclosure provides isolated whole microbial cultures of the microorganisms identified in Table 1.
[0081] In some embodiments, the present disclosure provides utilizing one or more microorganisms selected from Table 1 to produce a dough or food product (e.g. a baked good).
[0082] In some embodiments, the present disclosure provides microbial consortia comprising a combination of at least two microorganisms selected from the microorganisms identified in Table 1. In some embodiments, the consortia comprises two microorganisms, three microorganisms, four microorganisms, five microorganisms, six microorganisms, seven microorganisms, eight microorganisms, nine microorganisms, ten microorganisms, or more. In some embodiments, the microbial consortia are different microbial species or different strains of a microbial species.
[0083] The microorganisms of Table 1 were matched to their nearest taxonomic groups by utilizing classification tools such the Ribosomal Database Project (RDP) for 16S rRNA sequences and the User-friendly Nordic ITS Ectomycorrhiza (UNITE) database for ITS rRNA sequences. The 16S or 18S rRNA sequences or ITS sequences are often used for making distinctions between species and strains, in that if one of the aforementioned sequences share less than a specified percent sequence identity from a reference sequence, then the two organisms from which the sequences were obtained are said to be of different species or strains. It is known in the art that 16S rRNA contains hypervariable regions that can provide species/strain-specific signature sequences useful for bacterial identification, and that ITS sequences can also provide species/strain-specific signature sequences useful for fungal identification. In some embodiments, the microorganisms of the present disclosure are interpreted as belonging to the same species if one or more of the sequenced genes shares at least 97% sequence identity to the reference sequence.
[0084] In some embodiments, the microbial strains of the present disclosure include those that comprise a nucleic acid sequence that shares at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-272.
[0085] The isolation, identification, and culturing of microorganisms of the present disclosure can be performed using standard microbiological techniques known in the art.
[0086] In some embodiments, isolation of the microorganisms described herein is performed by streaking the specimen on a solid medium (e.g., nutrient agar plates) to obtain a single colony, which is then characterized by phenotypic traits (e.g., Gram positive/negative, cellular morphology, metabolic secretions, and the like). In some embodiments, pure isolates are obtained through repeated subculture of biological samples, each subculture followed by streaking onto solid media to obtain individual colonies or colony forming units.
[0087] In some embodiments, the microorganisms of the present disclosure are propagated through the use of a liquid growth medium under aerobic or anaerobic conditions. A medium for growing bacteria or fungi includes carbon, nitrogen, salts, minerals, vitamins, pH buffer, and other energy sources or selective agents such as those provided in (Ronald M. Atlas 2004). Suitable mediums for bacteria include, but are not limited to, MRS (De MAN, Rogosa, and Sharpe 1960) or TSB (Doyle, Mehrhof, and Ernst 1968) and suitable mediums for fungi include, but are not limited to, YPD (YPD Media 2010) or PDB (Eddleman n.d.).
[0088] In some embodiments, the microorganisms of the present disclosure are further preserved using common techniques in microbiology. These techniques include, but are not limited to, freeze-drying, desiccation, freeze-drying with cryoprotectants, fluid-bed drying, extrusion, spray drying, matrix encapsulation, vacuum drying, and protective packaging. In freeze-drying, also referred to as lyophilization, the microbial culture is frozen and then subjected to a vacuum to remove water by sublimation. In some embodiments, microorganisms of the present disclosure are protected by the addition of cryoprotectants including, but not limited to, glycerol, sucrose, or trehalose. In some embodiments, the microorganisms of the present disclosure are preserved through desiccation or by spray drying the microorganisms in a fine droplet followed by rapid drying by hot air or nitrogen gas. The dried microorganisms can then be stored in a protective matrix including, but not limited to, alginate or wax encapsulate beads, which can be further protected by packaging to prevent the reabsorption of moisture.
[0089] In some embodiments, the microorganisms described herein maintain viability across a broad range of environmental conditions. In some embodiments, the microorganisms described herein maintain viability in environmental conditions including, but not limited to, hypersaline, high sugar, low temperature, high temperature, and dry environments. In some embodiments, the microorganisms described herein maintain viability during a microbial preservation process such as cryopreservation, centrifugation, spray drying, or lyophilization.
[0090] In some embodiments, the microorganisms of the present disclosure, i.e., the microorganisms listed in Table 1, were obtained from sourdough starters, environments that contribute to the formation of a sourdough starter (e.g., human skin, human saliva, water, air or insects), grain-based fermentations, or grains before fermentation.
[0091] In some embodiments, a biological sample is collected from an environment or from an environment across time. In some embodiments, these environments include sourdough starters, environments that contribute to the formation of a sourdough starter (e.g., human skin, human saliva, water, air, insects), grain-based fermentations, or grains before fermentation. The biological sample is processed to detect the presence, abundance, and activity of one or more microorganisms. The sample is processed for the isolation, identification, and culturing of the microorganisms contained within. In some embodiments, isolation is performed by spreading the biological sample on solid medium to obtain single colonies, which are then propagated in a liquid medium under aerobic or anaerobic conditions. Isolated organisms are characterized by the phenotypic traits (e.g., acid tolerance, enzyme production, carbon source utilization). Each biological sample is processed to detect the presence and abundance of one or more metabolite in the sample. The environmental parameters or outcomes of the sample are recorded and included with microbial and metabolite detection information (e.g., pH, organoleptic properties, immunogenic gluten content, glycemic index). Dimensionality reduction analysis and multi-dimensional dimensionality reduction is performed individually and across all information collected, to determine the important organisms, metabolite, and environmental parameter relationships. Using the dimensionality reduction output, one or more microorganisms are selected for preparing products (e.g., ensembles, aggregates, and/or other synthetic groupings) containing the selected strains. The microorganism or group of microorganisms of interest are introduced to the environment (i.e., dough or food product). Samples are taken from the environment (i.e., dough or food product) in which the microorganisms were introduced, and the presence, abundance, and activity of the administered microorganisms are verified. From the additional samples, the presence and abundance of metabolites from the administered microorganisms are verified. From the additional samples, the desired outcomes are verified (e.g., pH, organoleptic properties, immunogenic gluten content, glycemic index).
[0092] In some embodiments, a sample or multiple samples are collected cross-sectionally or longitudinally from grain fermentations (e.g., dough) and processed to predict in silico the minimal and optimal microorganism or microbial consortia, needed to produce the desired food product, e.g., sourdough bread. In some embodiments, samples were obtained from publicly available databases and preprocessed datasets including, but not limited to, metabolomics, volatile organic compounds (VOCs), 16S rRNA, ITS rRNA, metagenomics, metatranscriptomics, ribosome profiling, and associated sample metadata. In some embodiments, the data is analyzed and integrated by tools including, but not limited to, alpha-diversity, beta-diversity, clustering analysis, network analysis, and differential abundance analysis.
[0093] In some embodiments, the sample is compared or correlated across metadata by the taxonomic or functional diversity within (alpha-diversity) each sample or across time using but not limited to the Shannon index (Tucker et al. 2017). In some embodiments, the sample is compared or correlated across metadata by the taxonomic or functional differences in composition (beta-diversity) across samples or across time by all samples using UniFrac (Lozupone and Knight 2005), Compositional Tensor Factorization (Martino et al. 2021), and/or Robust Aitchison PCA (Martino et al. 2019) performed across datatypes or within each datatype individually to form low-dimensional sample clusters (i.e. dimensionality reduction).
[0094] In some embodiments, network analysis is performed using the dimensionality reduction clusters and from network correlation analyses including all datatypes and metadata with Pearson correlations, Spearman correlations, canonical correspondence analysis (CCA), and/or partial least squares (PLS) (Meng et al. 2016; Rohart et al. 2017).
[0095] In some embodiments, the differential abundance of specific taxa or functional compositions across samples, within samples across time, or across samples and time was performed using ALDEx2 (Gloor et al. 2019), Linear Mixed Effects models (Rahman et al. 2023), and/or ANCOM-BC (Lin and Peddada 2020).
[0096] The microorganisms found below in Table 1 will be utilized in various embodiments of the present disclosure. The 16S or ITS sequences referred to in Table 1 were determined by ITS or 16S Illumina short-read sequencing. These microorganisms are predicted to produce a desired dough or food product, e.g., sourdough.
TABLE-US-00001 TABLE 1 Microorganisms of the Disclosure Predicted Genus Species 16S or ITS Latilactobacillus sakei.sup. SEQ ID NO: 1 Leuconostoc mesenteroides.sup. SEQ ID NO: 2 Latilactobacillus curvatus.sup. SEQ ID NO: 3 Lactobacillus sanfranciscensis SEQ ID NO: 4 Pediococcus inopinatus SEQ ID NO: 5 Lactobacillus paralimentarius SEQ ID NO: 6 Lactobacillus sakei SEQ ID NO: 7 Lactobacillus brevis SEQ ID NO: 8 Lactobacillus plantarum SEQ ID NO: 9 Pediococcus pentosaceus SEQ ID NO: 10 Lactobacillus brevis SEQ ID NO: 11 Pediococcus parvulus SEQ ID NO: 12 Acetobacter tropicalis SEQ ID NO: 13 Acetobacter orleanensis SEQ ID NO: 14 Lactobacillus hilgardii SEQ ID NO: 15 Lactobacillus parabuchneri SEQ ID NO: 16 Lactobacillus sanfranciscensis.sup. SEQ ID NO: 17 Lactobacillus coryniformis SEQ ID NO: 18 Leuconostoc mesenteroides SEQ ID NO: 19 Lactobacillus casei SEQ ID NO: 20 Lactobacillus sanfranciscensis.sup. SEQ ID NO: 21 Lactobacillus brevis SEQ ID NO: 22 Leuconostoc gelidum SEQ ID NO: 23 Lactococcus sp. SEQ ID NO: 24 Paracoccus laeviglucosivorans SEQ ID NO: 25 Acetobacter lovaniensis SEQ ID NO: 26 Lactobacillus sanfranciscensis.sup. SEQ ID NO: 27 Leuconostoc gelidum.sup. SEQ ID NO: 28 Lactobacillus sakei.sup. SEQ ID NO: 29 Leuconostoc gelidum.sup. SEQ ID NO: 30 Leuconostoc gelidum SEQ ID NO: 31 Lactobacillus sakei SEQ ID NO: 32 Lactobacillus sakei SEQ ID NO: 33 Lactobacillus perolens SEQ ID NO: 34 Lactobacillus sakei SEQ ID NO: 35 Secundilactobacillus sp. SEQ ID NO: 36 Lactobacillus sanfranciscensis.sup. SEQ ID NO: 37 Gluconobacter thailandicus SEQ ID NO: 38 Leuconostoc mesenteroides.sup. SEQ ID NO: 39 Lactobacillus rossiae SEQ ID NO: 40 Levilactobacillus brevis.sup. SEQ ID NO: 41 Gluconobacter roseus SEQ ID NO: 42 Leucobacter albus SEQ ID NO: 43 Acetobacter persici SEQ ID NO: 44 Lactobacillus sakei.sup. SEQ ID NO: 45 Lactobacillus plantarum.sup. SEQ ID NO: 46 Leuconostoc gelidum.sup. SEQ ID NO: 47 Paracoccus aminophilus SEQ ID NO: 48 Lactobacillus fermentum SEQ ID NO: 49 Leuconostoc gelidum.sup. SEQ ID NO: 50 Lactobacillus sanfranciscensis SEQ ID NO: 51 Lactobacillus sp. SEQ ID NO: 52 Psychrobacter fulvigenes SEQ ID NO: 53 Weissella confusa SEQ ID NO: 54 Lactobacillus sakei SEQ ID NO: 55 Lactobacillus sanfranciscensis SEQ ID NO: 56 Fenollaria massiliensis SEQ ID NO: 57 Lactobacillus brevis SEQ ID NO: 58 Peptoniphilus sp. SEQ ID NO: 59 Lactobacillus brevis.sup. SEQ ID NO: 60 Corynebacterium tuberculostearicum SEQ ID NO: 61 Lactobacillus sakei SEQ ID NO: 62 Lactobacillus acetotolerans SEQ ID NO: 63 Lactobacillus brevis SEQ ID NO: 64 Leuconostoc mesenteroides.sup. SEQ ID NO: 65 Ezakiella coagulans SEQ ID NO: 66 Leuconostoc mesenteroides.sup. SEQ ID NO: 67 Lactobacillus brevis.sup. SEQ ID NO: 68 Pediococcus parvulus SEQ ID NO: 69 Eubacterium sp. SEQ ID NO: 70 Pediococcus parvulus SEQ ID NO: 71 Corynebacterium sp. SEQ ID NO: 72 Lactobacillus plantarum SEQ ID NO: 73 Lactobacillus pontis SEQ ID NO: 74 Micrococcus antarcticus SEQ ID NO: 75 Paracoccus marinus SEQ ID NO: 76 Lactobacillus brevis SEQ ID NO: 77 Anaerococcus nagyae SEQ ID NO: 78 Lactobacillus manihotivorans SEQ ID NO: 79 Lactobacillus acidophilus SEQ ID NO: 80 Lactobacillus sakei SEQ ID NO: 81 Lactobacillus sanfranciscensis SEQ ID NO: 82 Lactobacillus sanfranciscensis SEQ ID NO: 83 Lactobacillus helveticus SEQ ID NO: 84 Gluconobacter albidus SEQ ID NO: 85 Ezakiella peruensis SEQ ID NO: 86 Lactococcus sp. SEQ ID NO: 87 Lactobacillus paralimentarius SEQ ID NO: 88 Ruminococcus bicirculans SEQ ID NO: 89 Corynebacterium macginleyi SEQ ID NO: 90 Faecalibacterium sp. SEQ ID NO: 91 Lagierella massiliensis SEQ ID NO: 92 Corynebacterium kroppenstedtii SEQ ID NO: 93 Rothia sp. SEQ ID NO: 94 Lactobacillus perolens SEQ ID NO: 95 Pediococcus parvulus SEQ ID NO: 96 Lactobacillus sakei SEQ ID NO: 97 Lactobacillus siliginis SEQ ID NO: 98 Lactobacillus sakei SEQ ID NO: 99 Corynebacterium pseudodiphtheriticum SEQ ID NO: 100 Peptoniphilus sp. SEQ ID NO: 101 Pediococcus parvulus SEQ ID NO: 102 Lactobacillus hilgardii SEQ ID NO: 103 Lactobacillus sakei SEQ ID NO: 104 Lactobacillus brevis SEQ ID NO: 105 Clostridium sp. SEQ ID NO: 106 Lactobacillus sp. SEQ ID NO: 107 Corynebacterium simulans SEQ ID NO: 108 Rothia sp. SEQ ID NO: 109 Brevibacterium luteolum SEQ ID NO: 110 Lactobacillus sakei SEQ ID NO: 111 Acidiphilium organovorum SEQ ID NO: 112 Lactobacillus plantarum SEQ ID NO: 113 Peptoniphilus senegalensis SEQ ID NO: 114 Lactobacillus paralimentarius SEQ ID NO: 115 Anaerococcus octavius SEQ ID NO: 116 Pediococcus parvulus SEQ ID NO: 117 Lactobacillus plantarum SEQ ID NO: 118 Rothia dentocariosa SEQ ID NO: 119 Lactobacillus coryniformis SEQ ID NO: 120 Lactobacillus casei SEQ ID NO: 121 Corynebacterium sp. SEQ ID NO: 122 Leuconostoc carnosum SEQ ID NO: 123 Lactobacillus selangorensis SEQ ID NO: 124 Lactobacillus sakei SEQ ID NO: 125 Lactobacillus sakei SEQ ID NO: 126 Lactobacillus sanfranciscensis SEQ ID NO: 127 Weissella cibaria SEQ ID NO: 128 Pediococcus pentosaceus.sup. SEQ ID NO: 129 Lactobacillus sakei SEQ ID NO: 130 Lactobacillus fuchuensis SEQ ID NO: 131 Corynebacterium casei SEQ ID NO: 132 Pediococcus argentinicus SEQ ID NO: 133 Lactobacillus rossiae SEQ ID NO: 134 Lactobacillus siliginis SEQ ID NO: 135 Pediococcus pentosaceus.sup. SEQ ID NO: 136 Corynebacterium efficiens SEQ ID NO: 137 Anaerococcus sp. SEQ ID NO: 138 Dermacoccus profundi SEQ ID NO: 139 Eubacterium yurii SEQ ID NO: 140 Cutibacterium acnes SEQ ID NO: 141 Corynebacterium glucuronolyticum SEQ ID NO: 142 Lactobacillus sanfranciscensis SEQ ID NO: 143 Collinsella sp. SEQ ID NO: 144 Stomatobaculum longum SEQ ID NO: 145 Bacteriovorax stolpii SEQ ID NO: 146 Rothia sp. SEQ ID NO: 147 Corynebacterium faecale SEQ ID NO: 148 Lactococcus sp. SEQ ID NO: 149 Armatimonas rosea SEQ ID NO: 150 Lactobacillus sakei SEQ ID NO: 151 Acidiphilium organovorum SEQ ID NO: 152 Lactobacillus sp. SEQ ID NO: 153 Lactobacillus sakei SEQ ID NO: 154 Anaerotignum lactatifermentans SEQ ID NO: 155 Brachybacterium conglomeratum SEQ ID NO: 156 Saccharomyces sp. SEQ ID NO: 157 Kazachstania exigua SEQ ID NO: 158 Kazachstania humilis SEQ ID NO: 159 Saccharomyces paradoxus SEQ ID NO: 160 Saccharomyces sp. SEQ ID NO: 161 Kazachstania servazzii SEQ ID NO: 162 Saccharomyces cerevisiae.sup. SEQ ID NO: 163 Kazachstania humilis SEQ ID NO: 164 Pichia fermentans SEQ ID NO: 165 Naumovozyma castellii SEQ ID NO: 166 Candida sake SEQ ID NO: 167 Kazachstania humilis SEQ ID NO: 168 Kazachstania unispora SEQ ID NO: 169 Saccharomyces sp. SEQ ID NO: 170 Kazachstania barnettii SEQ ID NO: 171 Kazachstania humilis SEQ ID NO: 172 Pichia fermentans SEQ ID NO: 173 Saccharomyces pastorianus SEQ ID NO: 174 Kazachstania bulderi SEQ ID NO: 175 Saccharomyces cerevisiae.sup. SEQ ID NO: 176 Wickerhamomyces anomalus SEQ ID NO: 177 Wickerhamomyces anomalus SEQ ID NO: 178 Pichia membranifaciens SEQ ID NO: 179 Wickerhamomyces anomalus SEQ ID NO: 180 Candida sake SEQ ID NO: 181 Wickerhamomyces anomalus SEQ ID NO: 182 Saccharomyces cerevisiae.sup. SEQ ID NO: 183 Pichia kudriavzevii SEQ ID NO: 184 Wickerhamomyces anomalus SEQ ID NO: 185 Saccharomyces paradoxus SEQ ID NO: 186 Hanseniaspora sp. SEQ ID NO: 187 Pichia membranifaciens SEQ ID NO: 188 Penicillium sp. SEQ ID NO: 189 Hanseniaspora sp. SEQ ID NO: 190 Meyerozyma guilliermondii SEQ ID NO: 191 Saccharomyces sp. SEQ ID NO: 192 Saccharomyces pastorianus SEQ ID NO: 193 Wickerhamomyces anomalus SEQ ID NO: 194 Candida glabrata SEQ ID NO: 195 Debaryomyces delbrueckii SEQ ID NO: 196 Kazachstania naganishii SEQ ID NO: 197 Pichia cephalocereana SEQ ID NO: 198 Saccharomyces paradoxus SEQ ID NO: 199 Saccharomyces sp. SEQ ID NO: 200 Pichia kudriavzevii SEQ ID NO: 201 Pichia kudriavzevii SEQ ID NO: 202 Kazachstania servazzii SEQ ID NO: 203 Xeromyces bisporus SEQ ID NO: 204 Aspergillus sp. SEQ ID NO: 205 Xeromyces bisporus SEQ ID NO: 206 Saccharomyces cerevisiae.sup. SEQ ID NO: 207 Pichia cephalocereana SEQ ID NO: 208 Kazachstania humilis SEQ ID NO: 209 Saccharomyces cerevisiae.sup. SEQ ID NO: 210 Saccharomyces paradoxus SEQ ID NO: 211 Pichia kudriavzevii SEQ ID NO: 212 Kazachstania servazzii SEQ ID NO: 213 Penicillium sp. SEQ ID NO: 214 Penicillium sp. SEQ ID NO: 215 Aspergillus intermedius SEQ ID NO: 216 Pichia kudriavzevii SEQ ID NO: 217 Penicillium sp. SEQ ID NO: 218 Candida glabrata SEQ ID NO: 219 Penicillium tarraconense SEQ ID NO: 220 Penicillium sp. SEQ ID NO: 221 Candida parapsilosis SEQ ID NO: 222 Candida glabrata SEQ ID NO: 223 Aspergillus sp. SEQ ID NO: 224 Nakazawaea holstii SEQ ID NO: 225 Aspergillus sp. SEQ ID NO: 226 Phaeococcomyces sp. SEQ ID NO: 227 Pichia kudriavzevii SEQ ID NO: 228 Debaryomyces sp. SEQ ID NO: 229 Cephaloascus albidus SEQ ID NO: 230 Pichia kudriavzevii SEQ ID NO: 231 Saccharomyces cerevisiae.sup. SEQ ID NO: 232 Saccharomyces cerevisiae.sup. SEQ ID NO: 233 Aspergillus sp. SEQ ID NO: 234 Saccharomyces cerevisiae.sup. SEQ ID NO: 235 Penicillium sp. SEQ ID NO: 236 Eremothecium coryli SEQ ID NO: 237 Hanseniaspora mollemarum SEQ ID NO: 238 Cyberlindnera fabianii SEQ ID NO: 239 Kazachstania humilis SEQ ID NO: 240 Saccharomyces paradoxus SEQ ID NO: 241 Saccharomyces cerevisiae.sup. SEQ ID NO: 242 Saccharomycetales sp. SEQ ID NO: 243 Saccharomyces sp. SEQ ID NO: 244 Penicillium sp. SEQ ID NO: 245 Saccharomyces pastorianus SEQ ID NO: 246 Saccharomyces paradoxus SEQ ID NO: 247 Candida sp. SEQ ID NO: 248 Wickerhamomyces anomalus SEQ ID NO: 249 Aspergillus penicillioides SEQ ID NO: 250 Candida sake SEQ ID NO: 251 Pichia norvegensis SEQ ID NO: 252 Saccharomyces paradoxus SEQ ID NO: 253 Aspergillus sp. SEQ ID NO: 254 Candida sake.sup. SEQ ID NO: 255 Candida sp..sup. SEQ ID NO: 256 Kazachstania sp. SEQ ID NO: 257 Pichia cephalocereana SEQ ID NO: 258 Saccharomyces paradoxus SEQ ID NO: 259 Candida sake.sup. SEQ ID NO: 260 Pichia kudriavzevii SEQ ID NO: 261 Kazachstania humilis SEQ ID NO: 262 Penicillium sp. SEQ ID NO: 263 Wickerhamomyces anomalus SEQ ID NO: 264 Penicillium sp. SEQ ID NO: 265 Penicillium sp. SEQ ID NO: 266 .sup.Isolate .sup.Enrichment
[0097] Some microorganisms described in the present disclosure were deposited with the ATCC Patent Depository located at 10801 University Boulevard, Manassas, Virginia 20110, USA. The deposits were made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The ATCC Patent Depository accession numbers and corresponding dates of deposit for the aforementioned Budapest Treaty deposits are provided in Table 2 below.
TABLE-US-00002 TABLE 2 Budapest Treaty Deposits of the Disclosure Short Long 16S/ITS 16S/ITS Patent Patent Taxonomic Strain SEQ SEQ Deposit Deposit Designation Designation ID NO.sup.1 ID NO.sup.2 No. Date Saccharomyces A5Y 207 267 PTA- Aug. 8, cerevisiae 127804 2024 Kazachstania S1-3 269* 268 PTA- Aug. 8, humilis 127805 2024 Fructilactobacillus H11B 4 270 PTA- Aug. 8, sanfranciscensis 127806 2024 Pediococcus S1-1 10 271 PTA- Aug. 8, pentosaceus 127807 2024 Levilactobacillus S1-2 8 272 PTA- Aug. 8, brevis 127808 2024 .sup.116S/ITS sequences were determined by ITS or 16S Illumina short-read sequencing .sup.216S/ITS sequences were determined amplicon 16S and ITS long Sanger sequencing *SEQ ID NO: 269 is the reverse complement of SEQ ID NO: 159
[0098] In some embodiments, the isolated microbial strain is selected from any one of the microbial strains listed in Table 1. In some embodiments, the isolated microbial strain is selected from the group consisting of: Saccharomyces cerevisiae deposited as PTA-127804, Kazachstania humilis deposited as PTA-127805, Fructilactobacillus sanfranciscensis deposited as PTA-127806, Pediococcus pentosaceus deposited as PTA-127807, and Levilactobacillus brevis deposited as PTA-127808. In some embodiments, the isolated microbial strain is used to produce a starter culture, dough, or food product (e.g., a baked good).
Compositions
[0099] In some embodiments, the present disclosure provides compositions comprising the microorganisms described herein. In some embodiments, the composition is a starter culture, a dough, or a food product (e.g., a baked good). In some embodiments, the composition is a sourdough.
[0100] In some embodiments, the composition is a starter culture for bakery good production comprising one or more of the microorganisms described in Table 1. In some embodiments, the composition is a starter culture for bakery good production comprising one or more microorganisms with a nucleic acid sequence sharing at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-272. In some embodiments, the composition is a starter culture for bakery good production comprising one or more microorganisms with a nucleic acid sequence selected from any one of SEQ ID NOs: 1-272.
[0101] In some embodiments, the composition is a dough product comprising one or more of the microorganisms described in Table 1. In some embodiments, the composition is a dough product comprising one or more microorganisms with a nucleic acid sequence sharing at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-272. In some embodiments, the composition is dough product comprising one or more microorganisms with a nucleic acid sequence selected from any one of SEQ ID NOs: 1-272.
[0102] In some embodiments, the composition is a food product comprising one or more of the microorganisms described in Table 1. In some embodiments, the composition is a food product comprising one or more microorganisms with a nucleic acid sequence sharing at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-272. In some embodiments, the composition is food product comprising one or more microorganisms with a nucleic acid sequence selected from any one of SEQ ID NOs: 1-272.
[0103] In some embodiments, the composition is a sourdough comprising one or more of the microorganisms described in Table 1. In some embodiments, the composition is a sourdough comprising one or more microorganisms with a nucleic acid sequence sharing at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1-272. In some embodiments, the composition is sourdough comprising one or more microorganisms with a nucleic acid sequence selected from any one of SEQ ID NOs: 1-272.
[0104] In some embodiments, the composition comprises one or more isolated microbial strains selected from the group consisting of: Saccharomyces cerevisiae deposited as PTA-127804, Kazachstania humilis deposited as PTA-127805, Fructilactobacillus sanfranciscensis deposited as PTA-127806, Pediococcus pentosaceus deposited as PTA-127807, and Levilactobacillus brevis deposited as PTA-127808. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0105] In some embodiments, the composition comprises Saccharomyces cerevisiae. In some embodiments, the composition comprises Saccharomyces cerevisiae, wherein the Saccharomyces cerevisiae comprises an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 207. In some embodiments, the composition comprises Saccharomyces cerevisiae as deposited as PTA-127804. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0106] In some embodiments, the composition comprises Saccharomyces cerevisiae. In some embodiments, the composition comprises Saccharomyces cerevisiae, wherein the Saccharomyces cerevisiae comprises an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 267. In some embodiments, the composition comprises Saccharomyces cerevisiae as deposited as PTA-127804. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0107] In some embodiments, the composition comprises Kazachstania humilis. In some embodiments, the composition comprises Kazachstania humilis, wherein the Kazachstania humilis comprises an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 159. In some embodiments, the composition comprises Kazachstania humilis as deposited as PTA-127805. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0108] In some embodiments, the composition comprises Kazachstania humilis. In some embodiments, the composition comprises Kazachstania humilis, wherein the Kazachstania humilis comprises an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 268. In some embodiments, the composition comprises Kazachstania humilis as deposited as PTA-127805. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0109] In some embodiments, the composition comprises Kazachstania humilis. In some embodiments, the composition comprises Kazachstania humilis, wherein the Kazachstania humilis comprises an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 269. In some embodiments, the composition comprises Kazachstania humilis as deposited as PTA-127805. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0110] In some embodiments, the composition comprises Fructilactobacillus sanfranciscensis. In some embodiments, the composition comprises Fructilactobacillus sanfranciscensis, wherein the Fructilactobacillus sanfranciscensis comprises a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the composition comprises Fructilactobacillus sanfranciscensis as deposited as PTA-127806. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0111] In some embodiments, the composition comprises Fructilactobacillus sanfranciscensis. In some embodiments, the composition comprises Fructilactobacillus sanfranciscensis, wherein the Fructilactobacillus sanfranciscensis comprises a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 270. In some embodiments, the composition comprises Fructilactobacillus sanfranciscensis as deposited as PTA-127806. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0112] In some embodiments, the composition comprises Pediococcus pentosaceus. In some embodiments, the composition comprises Pediococcus pentosaceus, wherein the Pediococcus pentosaceus comprises a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the composition comprises Pediococcus pentosaceus as deposited as PTA-127807. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0113] In some embodiments, the composition comprises Pediococcus pentosaceus. In some embodiments, the composition comprises Pediococcus pentosaceus, wherein the Pediococcus pentosaceus comprises a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 271. In some embodiments, the composition comprises Pediococcus pentosaceus as deposited as PTA-127807. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0114] In some embodiments, the composition comprises Levilactobacillus brevis. In some embodiments, the composition comprises Levilactobacillus brevis, wherein the Levilactobacillus brevis comprises a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. In some embodiments, the composition comprises Levilactobacillus brevis as deposited as PTA-127808. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0115] In some embodiments, the composition comprises Levilactobacillus brevis. In some embodiments, the composition comprises Levilactobacillus brevis, wherein the Levilactobacillus brevis comprises a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 272. In some embodiments, the composition comprises Levilactobacillus brevis as deposited as PTA-127808. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0116] In some embodiments, the composition comprises: (a) Saccharomyces cerevisiae with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 207 or SEQ ID NO: 267; (b) Kazachstania humilis with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 159, SEQ ID NO: 268, or SEQ ID NO: 269; (c) Fructilactobacillus sanfranciscensis with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 270; (d) Pediococcus pentosaceus with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 271; and/or (e) Levilactobacillus brevis with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 272. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0117] In some embodiments, the composition comprises: (a) a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 207 or SEQ ID NO: 267; (b) a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 159, SEQ ID NO: 268, or SEQ ID NO: 269; (c) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 270; (d) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 271; and/or (e) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 272. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0118] In some embodiments, the composition comprises: (a) a Saccharomyces cerevisiae as deposited as PTA-127804; (b) a Kazachstania humilis as deposited as PTA-127805; (c) a Fructilactobacillus sanfranciscensis as deposited as PTA-127806; (d) a Pediococcus pentosaceus as deposited as PTA-127807; and/or (e) a Levilactobacillus brevis as deposited as PTA-127808. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0119] In some embodiments, the composition comprises: (a) a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 207 or SEQ ID NO: 267; and/or (b) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 270. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0120] In some embodiments, the composition comprises: (a) a Saccharomyces cerevisiae as deposited as PTA-127804; and/or (b) a Fructilactobacillus sanfranciscensis as deposited as PTA-127806. In some embodiments, the composition is a starter culture, dough, or food product (e.g., a baked good).
[0121] In some embodiments, the composition comprising the microorganisms described herein is formulated as a liquid, a powder, a pill, a tablet, or a pellet. In some embodiments, the powder is a temperature-stable powder or a cold-requiring powder. In some embodiments, the composition is a time-controlled released product or a pH-controlled release product.
[0122] In some embodiments, the compositions comprising microorganisms of the present disclosure include combinations of microorganisms as spores and/or vegetative cells from bacteria and/or fungi. In some embodiments, the compositions comprise bacteria and/or fungi in the form of spores. In some embodiments, the compositions comprise bacteria and/or fungi in the form of vegetative cells.
[0123] In some embodiments, the composition comprises viable cells of the microorganisms described herein. In some embodiments, the composition comprises at least 25%, at least 50%, at least 75%, at least 85%, or at least 95% viable cells from one or more microorganisms described herein.
[0124] In some embodiments, the composition comprises a microbe with a viable cell count of at least 10.sup.2 cells/g, at least 10.sup.3 cells/g, at least 10.sup.4 cells/g, at least 10.sup.5 cells/g, at least 10.sup.6 cells/g, at least 10.sup.7 cells/g, at least 10.sup.8 cells/g, at least 10.sup.9 cells/g, at least 10.sup.10 cells/g, at least 10.sup.11 cells/g, at least 10.sup.12 cells/g, at least 10.sup.13 cells/g, at least 10.sup.14 cells/g, at least 10.sup.15 cells/g, at least 10.sup.16 cells/g, at least 10.sup.17 cells/g, or at least 10.sup.18 cells/g.
[0125] In some embodiments, the composition comprises microorganisms with a total viable cell count of at least 10.sup.2 cells/g, at least 10.sup.3 cells/g, at least 10.sup.4 cells/g, at least 10.sup.5 cells/g, at least 10.sup.6 cells/g, at least 10.sup.7 cells/g, at least 10.sup.8 cells/g, at least 10.sup.9 cells/g, or at least 10.sup.10 cells/g, at least 10.sup.11 cells/g, at least 10.sup.12 cells/g, at least 10.sup.13 cells/g, at least 10.sup.14 cells/g, at least 10.sup.15 cells/g, at least 10.sup.16 cells/g, at least 10.sup.17 cells/g, or at least 10.sup.18 cells/g.
[0126] In some embodiments, the composition comprises a microbe with a viable cell count of at least 10.sup.2 CFU/g, at least 10.sup.3 CFU/g, at least 10.sup.4 CFU/g, at least 10.sup.5 CFU/g, at least 10.sup.6 CFU/g, at least 10.sup.7 CFU/g, at least 10.sup.8 CFU/g, at least 10.sup.9 CFU/g, at least 10.sup.10 CFU/g, at least 10.sup.11 CFU/g, at least 10.sup.12 CFU/g, at least 10.sup.13 CFU/g, at least 10.sup.14 CFU/g, at least 10.sup.15 CFU/g, at least 10.sup.16 CFU/g, at least 10.sup.17 CFU/g, or at least 10.sup.18 CFU/g.
[0127] In some embodiments, the composition comprises microorganisms with a total viable cell count of at least 10.sup.2 CFU/g, at least 10.sup.3 CFU/g, at least 10.sup.4 CFU/g, at least 10.sup.5 CFU/g, at least 10.sup.6 CFU/g, at least 10.sup.7 CFU/g, at least 10.sup.8 CFU/g, at least 10.sup.9 CFU/g, or at least 10.sup.10 CFU/g, at least 10.sup.11 CFU/g, at least 10.sup.12 CFU/g, at least 10.sup.13 CFU/g, at least 10.sup.14 CFU/g, at least 10.sup.15 CFU/g, at least 10.sup.16 CFU/g, at least 10.sup.17 CFU/g, or at least 10.sup.18 CFU/g.
[0128] In some embodiments, the composition comprising one or more microorganisms described herein further comprises one or more additional compounds. In some embodiments, the compound is an enzyme (e.g., amylase or a gluten-degrading enzyme), salt (e.g., sodium chloride, an acetate salt, a fumarate salt, a citrate salt, and/or a carbonate salt), an antioxidant (e.g., ascorbic acid or ascorbate salts), or a growth-promoting ingredient for select microorganisms.
[0129] In some embodiments, the composition comprising one or more microorganisms described herein comprises one or more preservatives such as calcium propionate, sorbic acid, sodium benzoate, potassium sorbate, ascorbic acid, azodicarbonamide, or sugar.
[0130] In some embodiments, the composition comprising one or more microorganisms described herein comprises one or more leavening agents such as baking powder, baking soda, cream of tartar, air, steam, or baker's yeast.
[0131] In some embodiments, the composition comprising one or more microorganisms described herein comprises one or more flavoring agents. Flavoring agents include, but are not limited to, organic acids such as lactic, acetic, or ascorbic acid. Dehydrated grain or dairy products such as malt powder, milk powder, or malted milk powder. Liquid dairy products such as milk or cream.
[0132] Sugars including glucose, maltose, sucrose. Fats such as butter, olive oil, canola oil, and peanut oil. Herbs spices, and their extracts such as vanilla, rosemary, thyme, pepper, and garlic. Fermented foods including yogurt, chocolate, fermented grains or fermented vegetables. Whole Fruit, nuts, dehydrated fruits, and/or their extracts such as cherry, raisin, almond, pistachio, or apple.
[0133] In some embodiments, the composition comprising one or more microorganisms described herein is encapsulated. In some embodiments, the encapsulating composition is a matrix selected from a sugar matrix, gelatin matrix, polymer matrix, silica matrix, or foam matrix. In some embodiments, the encapsulating composition is a gelatin, chitosan, pectin, carrageenan, maltodextrin, starch, cyclodextrins, polyvinyl alcohol, liposomes, polyhydroxyalkanoates, silica, polyethylene glycol, whey protein, soy protein, casein, alginate, sugars, fats, or wax.
[0134] In some embodiments, the composition comprising one or more microorganisms described herein is incorporated into dough as a single dose. In some embodiments, the composition comprising one or more microorganisms described herein is incorporated into dough in multiple doses.
[0135] In some embodiments, the composition comprising one or more microorganisms described herein is shelf-stable in a refrigerator (2-4 C.) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the composition comprising one or more microorganisms described herein is shelf-stable in a refrigerator (2-4 C.) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks.
[0136] In some embodiments, the composition comprising one or more microorganisms described herein is shelf-stable at room temperature (20-22 C.) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days. In some embodiments, the composition comprising one or more microorganisms described herein is shelf-stable at room temperature (20-22 C.) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks.
[0137] In some embodiments, the composition comprising one or more microorganisms described herein is shelf-stable in a freezer (18 to 20 C.) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days.
[0138] In some embodiments, the composition comprising one or more microorganisms described herein is shelf-stable in a freezer (18 to 20 C.) for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or 52 weeks.
[0139] In some embodiments, the composition comprising one or more microorganisms described herein is shelf-stable at a relative humidity of at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%.
Methods and Uses
[0140] In some embodiments, the present disclosure relates to a method of producing a dough, the method comprising admixing one or more of the microorganisms described herein to the dough and incubating the admixture for a period of time. In some embodiments, the method comprises a further step of baking the dough. The present disclosure also relates to a food product (e.g., a baked good) produced by the methods described herein.
[0141] In some embodiments, the present disclosure provides a method of producing fermented dough or baked goods with improved organoleptic or human health impacts comprising administering a composition comprising one or more microorganisms described herein to a dough in an amount effective to produce the desired effects in the dough administered compared to a dough that was not administered the composition or administered a non-defined microbial community or administered baker's yeast alone.
[0142] In some embodiments, the disclosure provides methods of improving the rheological properties of dough and/or the baked good by using one or more of the microorganisms described herein. Rheological properties include, but are not limited to, reduced time of fermentation, bread texture, bread shape, bread or dough volume, sweet taste, sour taste, aroma, bread crust and bread crumb color, bread moisture retention, and bread crumb structure.
[0143] In some embodiments, the method comprises incubating the admixture comprising the one or more microorganisms for a period of time. In some embodiments, the admixture is incubated for at least at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, or at least 120 hours.
[0144] In some embodiments, the method comprises incubating the admixture comprising the one or more microorganisms at different temperatures. In some embodiments, the admixture is incubated at least 5 degrees Celsius, at least 0 degrees Celsius, at least 5 degrees Celsius, at least 10 degrees Celsius, at least 15 degrees Celsius, at least 20 degrees Celsius, at least 25 degrees Celsius, at least 30 degrees Celsius, at least 40 degrees Celsius, at least 50 degrees Celsius, at least 60 degrees Celsius, at least 70 degrees Celsius, at least 80 degrees Celsius, at least 90 degrees Celsius, or at least 100 degrees Celsius.
[0145] In some embodiments, the composition comprising one or more microorganisms described herein reduces time of fermentation. In some embodiments, the one or more microorganisms reduce time of fermentation by at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, or at least 120 hours.
[0146] In some embodiments, the composition comprising one or more microorganisms described herein improves the texture of the dough and/or baked good. The term texture includes, but is not limited to, hardness, adhesiveness, resilience, cohesiveness, chewiness, springiness, and gumminess of the dough and/or baked good.
[0147] In some embodiments, the composition comprising one or more microorganisms described herein increases leavening of dough. In some embodiments, the increased leavening of dough during fermentation results in a baked good that has the desired volume, texture, and/or crumb.
[0148] In some embodiments, the composition comprising one or more microorganisms described herein increases rise rate of dough. In some embodiments, the increased rise rate of dough during fermentation results in a baked good that has the desired volume, texture, and/or crumb.
[0149] In some embodiments, the composition comprising one or more microorganisms described herein increases elasticity of the dough and/or baked good. In some embodiments, the composition comprising one or more microorganisms described herein decreases elasticity of the dough and/or baked good.
[0150] In some embodiments, the composition comprising one or more microorganisms described herein increases firmness of the dough and/or baked good. In some embodiments, the composition comprising one or more microorganisms described herein decreases firmness of the dough and/or baked good.
[0151] In some embodiments, the composition comprising one or more microorganisms described herein affects proteins in the dough and/or baked good. Proteins in the dough or baked good include, but are not limited to, gliadins, glutenin, secalins, albumin, globulins, and prolamins. In some embodiments, the composition comprising one or more microorganisms described herein increases protein degradation in the dough and/or baked good. In some embodiments, the composition comprising one or more microorganisms described herein decreases protein degradation in the dough and/or baked good.
[0152] In some embodiments, the composition comprising one or more microorganisms described herein produces peptides in the dough and/or baked good. Some peptide classes produced include, but are not limited to, antioxidant peptides, antihypertensive peptides, and anti-tumoral peptides.
[0153] In some embodiments, the composition comprising one or more microorganisms described herein degrades chemical compounds and/or chemical precursors in the dough and/or baked good. Chemical compounds include, but are not limited to, phytic acid, tannins, biogenic amines, acrylamide, furosine and 5-hydroxymethylfurfural.
[0154] In some embodiments, the composition comprising one or more microorganisms described herein generates compounds that enhance flavor and/or aroma of the dough and/or baked good. Flavor and aroma compounds include, but are not limited to, phenolics, esters, aldehydes, ketones, alcohols, and lipids.
[0155] In some embodiments, the composition comprising one or more microorganisms described herein generates compounds that enhance nutritional quality of the dough and/or baked good. Compounds that enhance the nutritional value of bread and dough include, but are not limited to, polyphenols, vitamins, and minerals.
[0156] In some embodiments, the composition comprising one or more microorganisms described herein degrades simple and/or complex carbohydrates in the dough and/or baked good. In some embodiments, the composition comprising one or more microorganisms described herein utilizes simple and/or complex carbohydrates in the dough and/or baked good. Carbohydrates include, but are not limited to, fructans, dextrins, maltose, glucose, fructose, melibiose, starch, sorbitol, mannitol, and raffinose.
[0157] In some embodiments, the composition comprising one or more microorganisms described herein modulates the pH of dough over the length of fermentation (i.e., from addition of the microbial composition to the baked product). In some embodiments, the composition comprising one or more microorganisms described herein maintains the pH of dough over the length of fermentation. In some embodiments, the composition comprising one or more microorganisms described herein maintains the pH of dough at the mixing stage, autolyse stage, bulk stage, portion stage, and/or baked/final stage of fermentation.
[0158] In some embodiments, the composition comprising one or more microorganisms described herein lowers the pH of dough over the length of fermentation (i.e., from addition of the microbial composition to the baked product). In some embodiments, the composition comprising one or more microorganisms described herein lowers the pH of dough at the mixing stage, autolyse stage, bulk stage, portion stage, and/or baked/final stage of fermentation. In some embodiments, the composition comprising one or more microorganisms described herein lowers the pH of the baked good. In some embodiments, the composition comprising one or more microorganisms described herein lowers the pH of dough by about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0.
[0159] In some embodiments, the composition comprising one or more microorganisms described herein increases the pH of dough over the length of fermentation (i.e., from addition of the microbial composition to the baked product). In some embodiments, the composition comprising one or more microorganisms described herein increases the pH of dough at the mixing stage, autolyse stage, bulk stage, portion stage, and/or baked/final stage of fermentation. In some embodiments, the composition comprising one or more microorganisms described herein increases the pH of the baked good. In some embodiments, the composition comprising one or more microorganisms described herein increases the pH of dough by about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0.
[0160] In some embodiments, use of the composition comprising one or more microorganisms described herein results in a dough and/or food product with a pH of about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0 of the dough or the baked good. In some embodiments, use of the composition comprising one or more microorganisms described herein results in a dough and/or food product with a pH between about 4 and about 5. In some embodiments, use of the composition comprising one or more microorganisms described herein results in a dough and/or food product with a pH between about 4.0 and about 4.5. In some embodiments, use of the composition comprising one or more microorganisms described herein results in a dough and/or food product with a pH between about 4.5 and about 5.0. In some embodiments, use of the composition comprising one or more microorganisms described herein results in a dough and/or food product with a pH between about 4.25 and about 4.5. In some embodiments, use of the composition comprising one or more microorganisms described herein results in a dough and/or food product with a pH between about 4.5 and about 4.75. In some embodiments, use of the composition comprising one or more microorganisms described herein results in a dough and/or food product with a pH between about 4.75 and about 5.0.
[0161] In some embodiments, the composition comprising one or more microorganisms described herein generates organic acids during fermentation. These acids include but are not limited to lactic, acetic, propionic, butyric, isobutyric, valeric, and isovaleric acids. The single organism, set, or ensemble of organisms' ability to produce organic acids both in dough and other mediums can be assessed using measurements of total titratable acidity (TTA) or more specifically using analytical chemistry techniques such as high-performance liquid chromatography (HPLC).
[0162] In some embodiments, the composition comprising one or more microorganisms described herein degrades dough substrates during the fermentation process that impact human health. Substrates degraded by microorganisms include allergens such as gluten, high glycemic carbohydrates, and those which impact the digestibility of breads. Gluten is a widely dispersed protein found in grains such as wheat, barley, and rye. While gluten is essential to maintain the structure of bread, it is also an allergen for a portion of the population. The microorganisms in the present disclosure act to degrade dough proteins including gluten through enzymatic digestion, specifically those glutens that cause allergic reactions (i.e., the immunogenic gluten epitopes). The glycemic index attempts to quantitatively measure how quickly and significantly the carbohydrates in food consumed will affect blood sugar. Catabolism of carbohydrates, such as glucose, by the microorganisms described herein act to lower the glycemic index of breads through the fermentation of blood glucose elevating sugars before they are ingested. Digestibility enhancement is a process in which foods are transformed to make them easier to be broken down and for the nutrients contained within to be absorbed. In some embodiments, the compositions comprising one or more microorganisms described herein increase digestibility of breads. In some embodiments, digestibility of breads is enhanced through degradation of proteins, carbohydrates, organic acids, and the liberation of nutrients essential for effective digestion.
[0163] In some embodiments, the composition comprising one or more microorganisms described herein has a lower glycemic index compared to the same composition produced without the microorganisms of the present disclosure.
[0164] In some embodiments, consumption of a composition comprising one or more microorganisms described herein results in a lower blood glucose level in a subject compared to the same composition produced without the microorganisms of the present disclosure.
[0165] In some embodiments, the composition comprising one or more microorganisms described herein increase the shelf-life of the dough or baked good. In some embodiments, the composition comprising one or more microorganisms described herein increases shelf-life by at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, at least ten days, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, or at least 8 weeks compared to the same dough or baked good produced without the microorganisms of the present disclosure.
[0166] In some embodiments, the composition comprising one or more microorganisms described herein competitively excludes microbial pathogens, such as molds or other microorganisms that lead to food spoilage.
[0167] In some embodiments, the composition comprising one or more microorganisms described herein modulates the metabolism or activity of other microorganisms in the dough, baked good, or other medium.
Examples
[0168] The following is a description of various methods and materials used in the studies. They are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.
Example 1: In Silico Isolation of Microorganisms
Sample Collection
[0169] Samples of sourdough starters were obtained through collection from outside sources or produced in-house through spontaneous fermentation. Sourdough starters were produced by backslopping, which involves mixing whole wheat flour, rye, all-purpose flour, or some mixture of the flours with water at room temperature (e.g., 70-75 F. or 21-24 C.) for 24 hours or at colder temperatures (e.g., 40-69 F. or 4.5-20.6 C.). Then, on the second day, and proceeding days, the mixture was refreshed with part of the previous day's fermented mixture with additional flour and water. Starters obtained from an outside source were similarly refreshed daily, weekly, monthly, or bi-monthly. For each starter, one or more samples were obtained across passages from an epoch or across a fermentation cycle (e.g., across 24 hours or up to two weeks).
[0170] The environmental conditions and metadata were collected for each sample. Sample metadata included sample history, pH, temperature, flour type, leavening time, age, time of collection, the total number of bacterial or fungal cells (e.g., CFUs), organoleptic properties, immunogenic gluten content, and glycemic index.
[0171]
[0172]
[0173]
Sample Processing and Data Production
[0174] DNA was extracted from a sample, a set of two samples, or more than two samples containing multiple organisms. Extracted DNA was subjected to PCR to enrich the copy number of a selected conserved target gene (i.e., 16S rRNA or internal transcribed spacer (ITS) sequencing). Amplified DNA of the enriched genes was sequenced using selected sequencing technology. Resulting sequences were trimmed, quality filtered, and clustered into operational taxonomic units (OTUs). Taxonomy was assigned to OTUs by sequence alignment to a reference database for the target gene of interest. Extracted DNA was subjected to fragmentation with subsequent short-read sequencing (e.g., 20-300 base pairs per sequence) and direct sequencing of long-reads (e.g., 300 to greater than 1 million base pairs per sequence) using selected sequencing technology (i.e. shotgun metagenomics). The resulting sequences were trimmed, quality filtered, and directly aligned to known sequence databases or reconstructed into genomes (e.g., metagenome-assembled genomes (MAG)). The taxonomic abundance and functional capacity abundance of each MAG and sample were quantified through a reference database. RNA was extracted from a sample, a set of two samples, or more than two samples containing multiple organisms of the same or similar samples to those from which DNA was extracted (i.e., metatranscriptomics). The RNA was enriched for mRNA and sequenced using selected sequencing technology. The resulting RNA sequences were then trimmed, quality filtered, in silico enriched for mRNA, and then directly aligned to functional gene databases or aligned to MAGs constructed from the DNA extracted samples. The transcriptional regulation and abundance were then quantified from the alignments for each sample and MAG. Only the RNA contained within the ribosome at the time of sampling was extracted from a sample, a set of two samples, or more than two samples containing multiple organisms of the same or similar samples to those from which DNA was extracted (i.e., ribosome profiling). The RNA was enriched for mRNA and sequenced using selected sequencing technology. The resulting RNA sequences were then trimmed, quality filtered, in silico enriched for mRNA, and then directly aligned to functional gene databases or aligned to MAGs constructed from the DNA extracted samples. The translational regulation and abundance were then quantified from the alignments for each sample and MAG.
[0175] Metabolites were quantified in a sample, a set of two samples, or more than two samples that are the same or similar samples to those from which DNA was extracted. Metabolites were measured using either Liquid Chromatography-Mass Spectrometry (LC-MS/MS), Nuclear Magnetic Resonance (NMR) Spectroscopy, chromatograph/mass spectrometer (GC/MS), Mass Spectrometry (MS), or High-Performance Liquid Chromatography (HPLC). For LC-MS/MS, metabolites were first extracted using liquid-liquid extraction or solid-phase extraction, then Liquid Chromatography (LC) was used to separate the metabolites within the sample based on their chemical properties followed by MS or Triple Quadrupole MS to detect and quantify metabolites based on their mass-to-charge ratio (m/z). Next, the raw data was processed for peak picking, deconvolution, retention time alignment, spectral matching or molecular networking to known metabolites, and quantification by peak areas or intensities. For GC/MS, volatile organic compounds (VOCs) were captured from each sample and then introduced into the GC/MS system through direct injection, headspace sampling, or thermal desorption. The carrier in the GC was either helium or nitrogen, which leads to the separation of compounds based on volatility. After separation, the MS detected and quantified metabolites based on their mass-to-charge ratio (m/z). Data was processed in a similar way to LC-MS/MS.
In Silico Identification of Microbe Combinations
[0176] Across samples and for each sample across time, each data type (e.g., metabolite, VOCs, 16S, ITS, metagenomics, metatranscriptomics, metadata, and ribosome profiling). Each sample was compared or correlated across metadata by the taxonomic or functional diversity within (alpha-diversity) each sample or across time using the Shannon index (Tucker et al. 2017). Each sample was compared or correlated across metadata by the taxonomic or functional differences in composition (beta-diversity) across samples or across time by all samples using UniFrac (Lozupone and Knight 2005), Compositional Tensor Factorization (Martino et al. 2021), and/or Robust Aitchison PCA (Martino et al. 2019) performed across datatypes or within each datatype individually to form low-dimensional sample clusters (i.e. dimensionality reduction). Network analysis was performed using the dimensionality reduction clusters and from network correlation analyses including all datatypes and metadata with Pearson correlations, Spearman correlations, canonical correspondence analysis (CCA), and partial least squares (PLS) (Meng et al. 2016; Rohart et al. 2017). The differential abundance of specific taxa or functional compositions across samples, within samples across time, or across samples and time was performed through ALDEx2 (Gloor et al. 2019), Linear Mixed Effects models (Rahman et al. 2023), and ANCOM-BC (Lin and Peddada 2020).
[0177]
[0178]
Example 2: Microbial Identification for Exclusion of Undesirable Organisms
[0179] This example aims to identify the impact of select microbial compositions to competitively exclude microbial pathogens, molds, or other organisms that lead to food spoilage.
Method for Identifying Antimycotic Capability of Supernatant Using Disk Diffusion
[0180] Susceptibility of mold to experimental organisms was conducted using the disk diffusion method (aka Kirby-Bauer method). Experimental organisms were grown to maximum optical density. Experimental cultures were centrifuged at 4,000 g for 10 minutes and the supernatant was harvested and applied to 6 mm sterile filter paper disks. Select molds were densely plated to form a lawn on solid media. The supernatant-saturated disks were applied to the plates with the selected molds and allowed to incubate for 24 hours at 32 C. Plates were removed from the incubator and clearing of the mold around each disk was observed and measured to determine the antimycotic capability of each experimental sample.
[0181]
Example 3: Microbial Identification for Modulation of Organisms in a Consortia
[0182] This example aims to identify the impact of select microbial compositions to modulate the metabolism or activity of other organisms in a dough or other medium.
Method to Assess Microbial Growth as Part of a Consortia in Dough
[0183] Each organism to be tested was cultured in isolation in growth media and incubated at 32 C. for 24 hours. Cells were harvested by centrifugation and 0.1 g of centrifuged cell concentrate was retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate was diluted in 1 mL of sterile water and then was serially diluted. Each serial dilution was spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis was incubated for 24 hours at 32 C. and colonies were counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture were calculated using colony count and back calculated using the dilution factor to get the total CFU/g for each organism tested. Wheat flour dough was prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each set or ensemble of organisms to be tested. Co-culture experiments were conducted using methods outlined by Carbonetto et al. (Carbonetto et al. 2020). Each set or ensemble of organisms was mixed into their respective dough at 10.sup.7 cells per organism per gram of dough. Control doughs were prepared by adding 10.sup.7 total cells per dough using each organism to be tested in isolation. Doughs were incubated at a selected temperature of interest for 24 hours. At the conclusion of 24 hours, organisms in the dough were CFU counted by resuspending 2 g of dough in 40 mL of tryptone/NaCl solution (1 g of tryptone and 8.5 g of NaCl in 1000 mL of distilled water). The dough/tryptone/NaCl mix was serial diluted and each dilution was spread on solid media optimal for the growth of each organism in the set or consortia. Cell counts were conducted on the dilution, which resulted in 30-100 total colonies on the respective plate. Cell counts for each culture were calculated using colony count and back calculated using the dilution factor to get the total CFU/g for each organism tested. The CFU counts were compared between co-culture and controls to make inferences about the interaction between the organisms.
[0184]
Example 4: Isolation, Propagation and Formulation of Microorganisms
[0185] This example aims to isolate microorganisms from a consortium and propagate and formulate the microorganisms into a stable product using standard microbiology techniques.
Method for Isolation and Storage of Microorganisms
[0186] Microorganisms were isolated from a sample containing a collection of different microorganisms (i.e., a microbial consortium). First, when the sample contained a high level of biomass, a dilution was made by pipetting 100 L of sample into 900 L of sterile several times serially. Next, the diluted sample was streaked onto a solid medium to promote the growth of the microbe of interest, including MRS (De MAN, Rogosa, and Sharpe 1960), YPD, or TSB (Doyle, Mehrhof, and Ernst 1968) then incubated in both aerobic and anaerobic conditions between 20-50 C. After incubation, single colonies, which represent a single strain of microorganism, were chosen by color, shape, and size and transferred to a liquid medium of the same type using aseptic techniques. To ensure the purity of the strain, this process was repeated on the colony grown in the liquid medium. Once purified, the single strain is then transferred to a medium suited for long-term storage, such as a solution containing glycerol, and frozen between 40 C. to 80 C. (Gherna and Reddy 2014).
[0187] The microorganisms were propagated through use of growth medium. Medium for growing bacteria or fungi includes carbon, nitrogen, salts, minerals, vitamins, pH buffer, and other energy sources or selective agents such as those provided in (Ronald M. Atlas 2004). Suitable mediums for bacteria include but are not limited to MRS (De MAN, Rogosa, and Sharpe 1960) or TSB (Doyle, Mehrhof, and Ernst 1968) and suitable mediums for fungi include but are not limited to YPD (YPD Media 2010) or PDB (Eddleman n.d.).
[0188] The microorganisms were preserved using common techniques of microbiology. These techniques include but are not limited to freeze-drying, desiccation, freeze-drying with cryoprotectants, spray drying, matrix encapsulation, vacuum drying, and protective packaging.
[0189]
Example 5: Identification of Microbes
[0190] This example aims to identify microorganisms from a consortium using molecular or microbiological techniques including but not limited to DNA sequencing, qPCR, carbohydrate metabolism, or microscopy.
Method for Identification of Microorganisms by Conserved Target Gene DNA Sequencing
[0191] DNA was extracted from a single organism, a set of two organisms, or an ensemble comprised of more than two organisms. Extracted DNA was subjected to PCR to enrich the copy number of a selected conserved target gene (i.e., 16S rRNA or internal transcribed spacer). Amplified DNA of the enriched genes was sequenced using selected sequencing technology. Resulting sequences were trimmed, quality filtered, and clustered into operational taxonomic units (OTUs). Taxonomy was assigned to OTUs by sequence alignment to a reference database for the target gene of interest. The identity of microorganisms in each sample was inferred using the OTUs with assigned taxonomy.
Example 6: Microbial Identification for pH Modification
[0192] This example aims to identify the impact of select microbial compositions on their ability to lower, raise, or maintain the pH of the dough or baked good. pH can be assessed by a pH meter by measuring a single time point or over the length of the fermentation.
Method for Assaying pH Using a pH Meter or Probe
[0193] Each organism to be tested was cultured in isolation or as a part of a consortium in growth media and incubated at 32 C. for 24 hours. Cells were harvested by centrifugation and 0.1 g of centrifuged cell concentrate was retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate was diluted in 1 mL of sterile water and then was serially diluted. Each serial dilution was spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis was incubated for 24 hours at 32 C. and colonies were counted for the dilution factor that resulted in 30-100 total colonies on the respective plate. Cell counts for each culture were calculated using the colony count and back calculated using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough was prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms tested. Each organism, set, or ensemble of organisms was mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Samples were taken for each dough at the conclusion of mixing to act as a baseline measurement. The dough was then left to ferment in an incubated or unincubated environment for 24-100 hours. The pH was continuously or periodically measured using a calibrated Pro2Go Portable pH meter (Mettler Toledo, USA) by interesting the probe into the dough. In other cases, pH was assessed using colorimetric or fluorescence-based assays in which dough or other medium was inoculated with the organism(s) and samples were evaluated at a single time point or over the course of fermentation.
Method of Assaying pH Using a Colorimetric Assay
[0194] Each organism to be tested was cultured in isolation or as a part of a consortium in liquid growth media and incubated at 32 C. for 24 hours. The indicator dye that fits the pH range to be tested was selected. For sourdough organisms, two of the most relevant are bromocresol green with a pH detection range between 3.1-6.5, and bromocresol purple with a pH range from 4.9-7.6. The indicator dye was diluted by adding 0.5 g of the dye to 500 mL of deionized water. Standards were made to generate a standard curve. For each standard required, 10 mL of deionized water was mixed with the appropriate levels of acid or base to generate standards that span the breadth of the range to be tested in regular intervals. For instance, if an eight-point standard curve was desired for testing with bromocresol green dye, the eight-point standard covered pH 3.1-6.5, using pH 0.5 as the interval between standards. 10 L of diluted dye was added to the 10 mL standards just prior to conducting the experiment. In a clear, flat bottom, 96-well plate, 100 L of each standard with dye was added to the appropriate number of wells. 1 L of each culture to be tested was inoculated into 99 L of fresh liquid growth media (pH 7) with 0.1% diluted dye to the remaining empty wells in the plate. The plate was incubated at 32 C. for 24-100 hours in a BioTek Synergy plate reader (Agilent Technologies, USA) with shaking. The plate reader was set to read the samples every 15 minutes. After the experiment was concluded, the standard curve was used to determine the absolute pH change and rate of pH change for each experimental well.
[0195]
Example 7: Microbial Identification for Rheological Benefit
[0196] This example aims to identify the impact of the microbial compositions described herein on rheological properties, such as extensibility or firmness, of dough and bread.
Method for Measuring Dough Extensibility by Rheometer
[0197] Each microorganism to be tested was cultured in isolation or as a part of a consortium in growth media and incubated at 32 C. for 24 hours. Cells were harvested by centrifugation and 0.1 g of centrifuged cell concentrate was retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate was diluted in 1 mL of sterile water and then was serially diluted. Each serial dilution was spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis was incubated for 24 hours at 32 C. and colonies are counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture were calculated using the colony count and back calculating using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough was prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested. Each organism, set, or ensemble of organisms was mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Samples were taken for each dough at the conclusion of mixing to act as a baseline measurement. The dough was incubated to allow for fermentation with samples taken periodically or at the fermentation endpoint.
[0198] Experimental dough samples were tested for small amplitude oscillatory shear using an MCR502 rheometer equipped with a 40 mm sand-blasted parallel plate geometry (Anton Paar, AUT). Dough loaded into the rheometer was allowed to sit for 5 minutes and measurements evaluating elasticity of the dough were collected in triplicate at 25 C.
[0199]
Example 8: Microbial Identification for Leavening Ability
[0200] This example aims to identify the impact of select microbial compositions on their ability to leaven dough or bread.
Method for Determining Dough Leavening
[0201] Each organism to be tested was cultured in isolation or as a part of consortia in growth media and incubated at 32 C. for 24 hours. Cells were harvested by centrifugation and 0.1 g of centrifuged cell concentrate was retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate was diluted in 1 mL of sterile water and then was serially diluted. Each serial dilution was spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis was incubated for 24 hours at 32 C. and colonies were counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture were calculated using the colony count and back calculated using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough was prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested. Each organism, set, or ensemble of organisms was mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough.
[0202] A 5 g portion of dough from each experimental group was put into a 15 mL conical tube and centrifuged at 4,000 g for 2 minutes. Experimental doughs were left to ferment in an incubated or unincubated environment for 24-48 hours. A camera was preset to take photographs of the dough in the tubes every 10 minutes to generate a timelapse. The method for evaluation of total rise and rise rate was calculated as outlined by Landis et al. using the R package GrowthCurver (Landis et al. 2021).
[0203]
Example 9: Microbial Identification for Organic Acid Generation
[0204] This example aims to identify the ability of select microbial compositions to generate organic acids in dough during fermentation.
Method for Measuring Organic Acids in Dough by Assessing Titratable Acidity (TTA)
[0205] Each organism to be tested is cultured in isolation or as a part of a consortium in growth media and incubated at 32 C. for 24 hours. Cells are harvested by centrifugation and 0.1 g of centrifuged cell concentrate is retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate is diluted in 1 mL of sterile water and then is serially diluted. Each serial dilution is spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis is incubated for 24 hours at 32 C. and colonies are counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture are calculated using the colony count and back calculating using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough is prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested.
[0206] Each organism, set, or ensemble of organisms is mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Samples are taken for each dough at the conclusion of mixing to act as a baseline measurement. The dough is incubated to allow for fermentation with samples taken periodically or at the fermentation endpoint. For each experimental dough, 10 g of dough is added to 90 mL of deionized water and homogenized by vortexing for 3 minutes. While stirring the diluted dough mixture, 0.1 M NaOH is slowly added, and the pH of the mixture is recorded. Once the pH of the mixture equals 8.3, the experiment is concluded and TTA is reported as the total volume of NaOH added.
Method for Measuring Organic Acids in Dough by HPLC
[0207] Each organism to be tested is cultured in isolation or as a part of a consortium in growth media and incubated at 32 C. for 24 hours. Cells are harvested by centrifugation and 0.1 g of centrifuged cell concentrate is retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate is diluted in 1 mL of sterile water and then is serially diluted. Each serial dilution is spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis is incubated for 24 hours at 32 C. and colonies are counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture are calculated using the colony count and back calculating using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough is prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested.
[0208] Each organism, set, or ensemble of organisms is mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Samples are taken for each dough at the conclusion of mixing to act as a baseline measurement. The dough is incubated to allow for fermentation with samples taken periodically or at the fermentation endpoint.
[0209] For each experimental dough, lg of dough is homogenized in 10 mL of deionized water by vortex for 10 minutes and then centrifuged at 12,000 g for 10 minutes. The supernatant is harvested, and 7 mL of supernatant is added to 0.2 mL potassium II hexaferrocyanate, (0.085 mol/L) and 0.2 mL zinc sulfate (0.25 mol/L). Samples are vortexed, centrifuged at 12,000 g for 10 minutes, and filtered through a 0.22 m filter. The filtrate is then run on an HPLC/RI/UV system at 60 C. using 0.005 mol/L H2SO4 at a flow rate of 0.6 ml/min and analyzed for the presence of organic acids.
Example 10: Microbial Identification for Protein Degradation
[0210] This example aims to identify the ability of select microbial compositions to degrade proteins in the dough and/or baked good.
Method for Assaying Protein Hydrolysis in Dough
[0211] Each organism to be tested is cultured in isolation or as a part of a consortium in growth media and incubated at 32 C. for 24 hours. Cells are harvested by centrifugation and 0.1 g of centrifuged cell concentrate is retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate is diluted in 1 mL of sterile water and then is serially diluted. Each serial dilution is spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis is incubated for 24 hours at 32 C. and colonies are counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture are calculated using the colony count and back calculating using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough is prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested. Each organism, set, or ensemble of organisms is mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Samples are taken for each dough at the conclusion of mixing to act as a baseline measurement. Experimental doughs are left to ferment in an incubated or unincubated environment for 24-100 hours and samples are taken periodically across the fermentation. In addition, 8 samples for the generation of a standard curve are generated using leucine. The concentration of leucine for generating the standard curve at 0.125, 0.25, 0.375, 0.5, 0.625, 0.75, 0.875, 1.0 mmol/L. Protein hydrolysis is measured for each experimental and standard curve sample using the Trinitrobenzenesulfonic acid method to measure free amino groups (NH.sub.2) (Adler-Nissen 1979; Zotta et al. 2006). To do so, 0.25 g of each dough or leucine stand control sample to be tested is mixed with 2.0 mL of phosphate buffer (pH 8.5) in a tube to which 2.0 mL of 0.1% Trinitrobenzenesulfonic acid is added. The tube is vortexed, wrapped in aluminum foil to prevent light exposure, and incubated at 50 C. for 60 minutes. At the conclusion of the incubation, 4.0 mL of 0.1N HCL is added to stop the reaction, and the tube is incubated at room temperature for 30 minutes. 100 L from the experimental and standard curve samples are added to a clear, flat bottom 96 well plate and are read by a BioTek Synergy plate reader at 340 nM (Agilent Technologies, USA). After the experiment is concluded, the standard curve can be used to determine the protein degradation in each experimental well.
Example 11: Microbial Identification for Production of Peptides
[0212] This example aims to identify the ability of select microbial compositions to produce peptides in the dough and/or baked good.
Method for Identifying Antioxidant Peptides in Dough
[0213] Each organism to be tested is cultured in isolation or as a part of a consortium in growth media and incubated at 32 C. for 24 hours. Cells are harvested by centrifugation and 0.1 g of centrifuged cell concentrate is retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate is diluted in 1 mL of sterile water and then is serially diluted. Each serial dilution is spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis is incubated for 24 hours at 32 C. and colonies are counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture are calculated using the colony count and back calculating using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough is prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested. Each organism, set, or ensemble of organisms is mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Samples are taken for each dough at the conclusion of mixing to act as a baseline measurement. Experimental doughs are left to ferment in an incubated or unincubated environment for 24-100 hours and samples are taken periodically across the fermentation. Water/salt-soluble extracts are prepared from each dough using a method developed by Weiss et al. (Weiss, Vogelmeier, and Gorg 1993). Briefly, 7.5 g of each dough is diluted in 30 mL of 50 mM Tris-HCL (pH 8.8) and incubated at 4 C. for 1 hour. The incubated sample is centrifuged at 20,000 g for 20 minutes. The supernatant is retained for analysis of antioxidant peptide content. Inhibition of linoleic acid autoxidation is measured as described by Coda et al. (Coda et al. 2012) with the exception that the samples are not freeze-dried. For each sample 1 mL of supernatant is added to 1 mL of 50 mM linoleic acid which was diluted in 100% ethanol. The sample is incubated in the dark at 60 C. for 8 days. Positive antioxidant controls, butylated hydroxytoluene (1 mg/ml), and -tocopherol (1 mg/ml) were treated in the same way as the extracted supernatants. After incubation, 100 L of sample is mixed with 4.7 mL of 75% (vol/vol) ethanol, 100 L of 30% (wt/vol) ammonium thiocyanate, 100 L of 0.02 M ferrous chloride diluted in 1M HCL. The samples are incubated for 3 minutes, added to a clear, flat bottom 96 well plate, and read by a BioTek Synergy plate reader at 500 nM (Agilent Technologies, USA). The results for experimental samples are compared to the positive control references and relative antioxidant capacity is assessed.
Example 12: Microbial Identification for Degradation of Chemical Compounds
[0214] This example aims to identify the ability of select microbial compositions to degrade chemical compounds and chemical precursors in the dough and/or baked good.
Method for Identifying Acrylamide in Dough
[0215] Each organism to be tested is cultured in isolation or as a part of a consortium in growth media and incubated at 32 C. for 24 hours. Cells are harvested by centrifugation and 0.1 g of centrifuged cell concentrate is retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate is diluted in 1 mL of sterile water and then is serially diluted. Each serial dilution is spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis is incubated for 24 hours at 32 C. and colonies are counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture are calculated using the colony count and back calculating using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough is prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested. Each organism, set, or ensemble of organisms is mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Samples are taken for each dough at the conclusion of mixing to act as a baseline measurement. Experimental doughs are left to ferment in an incubated or unincubated environment for 24-100 hours and samples are taken periodically across the fermentation. Assessment of acrylamide in dough is done using the Acrylamide Starter Kit for UPLC MS/MS (Waters Corporation, USA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the methods outlined by the kit manual and Diana et al. (Free Amino Acids, Acrylamide and Biogenic Amines in Gamma-Aminobutyric Acid Enriched Sourdough and Commercial Breads 2014). Briefly, lg of dough is taken from each sample and an isotopically labeled internal standard (Acrylamide d3) is added to the samples prior to extraction in order to correct for any variability during extraction. Samples are extracted using the Acrylamide Starter Kit for UPLC MS/MS by the manufacturer-recommended protocol. Extracts are cleaned up using Dispersive solid phase extraction tubes containing 300 mg of secondary amine sorbent and 900 mg of MgSO4. The extract is then evaporated and reconstituted in 0.1% formic acid. The reconstituted extract is then evaluated by LC-MS/MS using parameters outlined in the Acrylamide Starter Kit for UPLC MS/MS manual.
Example 13: Microbial Identification for Degradation and/or Utilization of Carbohydrates
[0216] This example aims to identify the ability of select microbial compositions to degrade and/or utilize simple and/or complex carbohydrates in the dough and/or baked good.
Method for Evaluating Carbohydrate Utilization
[0217] Each organism to be tested is cultured in isolation or as a part of a consortium in growth media and incubated at 32 C. for 24 hours. Each culture is centrifuged at 3,000 g for 10 minutes, cells are harvested and resuspended in a carbon source and electron donor free medium with 0.017% (w/v) bromocresol purple as an indicator of acidification of carbohydrates. Add the resuspended culture dropwise to each well of the 50 wells on an API50 CH test strip (bioMerieux SA, FRA) and incubate the strip at 32 C. for 24 to 48 hours. Carbohydrates tested by the assay include d-galactose, d-glucose, d-fructose, maltose, glycogen, aesculin/ferric citrate, starch, glycerol, erythritol, d-arabinose, 1-arabinose, d-ribose, d-xylose, 1-xylose, methyl 3-d-xylopyranoside, d-cellobiose, d-adonitol, d-lactose, d-saccharose, d-trehalose, d-melibiose, d-mannose, 1-arabitol, 1-sorbose, 1-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl d-mannopyranoside, methyl d-glucopyranoside, N-acetyl glucosamine, amygdalin, arbutin, melezitose, raffinose, xylitol, inulin, salicin, gentiobiose, turanose, d-lyxose, d-tagatose, d-fucose, 1-fucose, d-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. At the conclusion of the incubation check the strip for qualitative acidification of the growth media by color change, if the carbohydrate has been utilized the media in the well will be yellow, if it has not been utilized the media will remain purple.
Example 14: Microbial Identification for Generation of Aroma and Flavor Compounds
[0218] This example aims to identify the ability of select microbial compositions that generate compounds that enhance the flavor or aroma in the dough and/or baked good.
Method for Identification and Quantitation of Volatile Flavor and Aroma Compounds in Bread
[0219] Each organism to be tested is cultured in isolation or as a part of consortia in growth media and incubated at 32 C. for 24 hours. Cells are harvested by centrifugation and 0.1 g of centrifuged cell concentrate is retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate is diluted in 1 mL of sterile water and then is serially diluted. Each serial dilution is spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis is incubated for 24 hours at 32 C. and colonies are counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture are calculated using the colony count and back calculating using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough is prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested. Each organism, set, or ensemble of organisms is mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Experimental doughs are left to ferment in an incubated or unincubated environment for 24-100 hours and then baked. Baked bread is then evaluated for volatile aroma and flavor compounds by headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS) as detailed by Aslankoohi et al. (Aslankoohi et al. 2016). For the solid-phase extraction, 5.0 g of baked bread is put in a sealed glass vial and the volatiles are sampled using a triphase DVB/Carboxen/PDMS 50/30 m SPME fiber. Volatiles from the fiber are injected into a gas chromatograph-mass spectrometer by heating the fiber at 250 C. for 5 minutes. The GC-MS is equipped with a non-polar column and 1.4 ml/min helium is used for the carrier. The temperature of the during measurement is held at 36 C. for 10 minutes and raised by 6 C. a minute until 220 C. is reached. The spectra of volatile compounds are then deconvoluted and matched to a reference library for identification.
Example 15: Microbial Identification for Enhancement of Nutritional Qualities in Bread and Dough
[0220] This example aims to identify the ability of select microbial compositions that enhance the nutritional quality in the dough and/or baked good.
Method to Assay Free Mineral Content in Bread
[0221] Each organism to be tested is cultured in isolation or as a part of consortia in growth media and incubated at 32 C. for 24 hours. Cells are harvested by centrifugation and 0.1 g of centrifuged cell concentrate is retained for colony-forming unit (CFU) plating. To conduct CFU plating, 0.1 g of the retained cell concentrate is diluted in 1 mL of sterile water and then is serially diluted. Each serial dilution is spread onto solid media optimal for the growth of the specific organism. Each plate for CFU analysis is incubated for 24 hours at 32 C. and colonies are counted for the dilution factor that results in 30-100 total colonies on the respective plate. Cell counts for each culture are calculated using the colony count and back calculating using the dilution factor to get the total CFU/g for each organism to be tested. Wheat flour dough is prepared by mixing 1000 g all-purpose flour (King Arthur Baking Company, USA) 700 g distilled water, and 30 g salt, using a FAMAG IM-8 mixer (Fabbrica Macchine Alimentari Grillo, Italy) for each organism, set, or ensemble of organisms to be tested. Each organism, set, or ensemble of organisms is mixed into their respective dough at a normalized amount across all experimental doughs at 10.sup.7 to 10.sup.13 total cells per dough. Experimental doughs are left to ferment in an incubated or unincubated environment for 24-100 hours and then baked. Baked bread is evaluated for free mineral content by methods outlined by Cizeikiene et al. (Cizeikiene et al. 2015). Soluble minerals are extracted from each experimental bread by combining 10 g baked bread with 50 mL deionized water and vortexed for 30 minutes to homogenize the sample. The samples are then centrifuged at 3,000 g for 5 minutes. The resulting supernatant is retained, and the pellet is washed with 50 mL of 0.9% NaCl (pH 2.0) and incubated at room temperature for 30 minutes. The samples are centrifuged, and the supernatant is retained. This process of washing is repeated twice more with 0.9% NaCl (pH 7.0) and the supernatant from each step is pooled together at completion. The pooled supernatant is then dried in a 105 C.-oven followed by washing in a muffle oven at 500 C. Calcium, zinc, iron, and manganese content is determined using a Spectr AA-Plus atomic absorption spectrophotometer by the Cereal and Grain Association AACC method 40-70.
Example 16: Microbial Identification for Environmental Condition Tolerance
[0222] This example aims to identify the ability of select microbial compositions that maintain viability across a broad range of environmental conditions.
Method to Assay Viability of Organisms Grown in Hypersaline Growth Media
[0223] Optimal growth media is determined for each organism to be tested and the organism is grown to max optical density in the optimal media. Solid media is prepared with a range of gradually increasing NaCl concentrations and 200 L of liquid culture is added to each plate. Plates are incubated for 24 hours at 32 C. and colonies are counted for each dilution to determine the salt tolerance of each organism.
Numbered Embodiments
[0224] Embodiment 1. A composition comprising a purified population of one or more microorganisms comprising a nucleic acid sequence that shares at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1-272; and a food-grade carrier or excipient.
[0225] Embodiment 2. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a nucleic acid sequence that shares at least 98% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1-272.
[0226] Embodiment 3. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a nucleic acid sequence that shares at least 99% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1-272.
[0227] Embodiment 4. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a nucleic acid sequence selected from SEQ ID NOs: 1-272.
[0228] Embodiment 5. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 207 or SEQ ID NO: 267.
[0229] Embodiment 6. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a yeast with an ITS nucleic acid sequence comprising SEQ ID NO: 207 or SEQ ID NO: 267.
[0230] Embodiment 7. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a Saccharomyces cerevisiae as deposited as PTA-127804.
[0231] Embodiment 8. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 159, SEQ ID NO: 268, or SEQ ID NO: 269.
[0232] Embodiment 9. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a yeast with an ITS nucleic acid sequence comprising SEQ ID NO: 159, SEQ ID NO: 268, or SEQ ID NO: 269.
[0233] Embodiment 10. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a Kazachstania humilis as deposited as PTA-127805.
[0234] Embodiment 11. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 4 or SEQ ID NO: 270.
[0235] Embodiment 12. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 4 or SEQ ID NO: 270.
[0236] Embodiment 13. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a Fructilactobacillus sanfranciscensis as deposited as PTA-127806.
[0237] Embodiment 14. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10 or SEQ ID NO: 271.
[0238] Embodiment 15. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 10 or SEQ ID NO: 271.
[0239] Embodiment 16. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a Pediococcus pentosaceus as deposited as PTA-127807.
[0240] Embodiment 17. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 8 or SEQ ID NO: 272.
[0241] Embodiment 18. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a bacterium with a 16S nucleic acid sequence comprising SEQ ID NO: 8 or SEQ ID NO: 272.
[0242] Embodiment 19. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises a Levilactobacillus brevis as deposited as PTA-127808.
[0243] Embodiment 20. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises: [0244] (a) a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 207 or SEQ ID NO: 267; [0245] (b) a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 159, SEQ ID NO: 268, or SEQ ID NO: 269; [0246] (c) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 270; [0247] (d) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 271; and/or [0248] (e) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 272.
[0249] Embodiment 21. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises: [0250] (a) a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 207 or SEQ ID NO: 267; and/or [0251] (b) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 270.
[0252] Embodiment 22. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises: [0253] (a) a yeast with an ITS nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 207 or SEQ ID NO: 267; and [0254] (b) a bacterium with a 16S nucleic acid sequence sharing at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 270.
[0255] Embodiment 23. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises: [0256] (a) a Saccharomyces cerevisiae as deposited as PTA-127804; [0257] (b) a Kazachstania humilis as deposited as PTA-127805; [0258] (c) a Fructilactobacillus sanfranciscensis as deposited as PTA-127806; [0259] (d) a Pediococcus pentosaceus as deposited as PTA-127807; and/or [0260] (e) a Levilactobacillus brevis as deposited as PTA-127808.
[0261] Embodiment 24. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises: [0262] (a) a Saccharomyces cerevisiae as deposited as PTA-127804; and/or [0263] (b) a Fructilactobacillus sanfranciscensis as deposited as PTA-127806.
[0264] Embodiment 25. The composition of embodiment 1, wherein the purified population of one or more microorganisms comprises: [0265] (a) a Saccharomyces cerevisiae as deposited as PTA-127804; and [0266] (b) a Fructilactobacillus sanfranciscensis as deposited as PTA-127806.
[0267] Embodiment 26. The composition of any one of embodiments 1-25, wherein the one or more microorganisms is lyophilized.
[0268] Embodiment 27. The composition of any one of embodiments 1-26, wherein the one or more microorganisms is encapsulated.
[0269] Embodiment 28. The composition of any one of embodiments 1-27, wherein the one or more microorganisms is present in the composition at about 10.sup.2 CFU/g or more.
[0270] Embodiment 29. The composition of embodiment 28, wherein the one or more microorganisms is present in the composition at about 10.sup.9 CFU/g, about 10.sup.10 CFU/g, about 10.sup.11 CFU/g, about 10.sup.12 CFU/g, about 10.sup.13 CFU/g, or about 10.sup.14 CFU/g.
[0271] Embodiment 30. The composition of any one of embodiments 1-29, wherein the composition comprises one or more additional preservatives.
[0272] Embodiment 31. The composition of any one of embodiments 1-30, wherein the composition comprises one or more additional flavoring agents.
[0273] Embodiment 32. The composition of any one of embodiments 1-31, wherein the composition comprises one or more additional leavening agents.
[0274] Embodiment 33. The composition of any one of embodiments 1-32, wherein the composition is formulated as a dry powder, food ingredient, food preparation, food supplement, water additive, moisture-stabilized additive, consumable, solid, gel, liquid, or dough.
[0275] Embodiment 34. The composition of any one of embodiments 1-32, wherein the composition is a food.
[0276] Embodiment 35. The composition of any one of embodiments 1-32, wherein the composition is a dough.
[0277] Embodiment 36. The composition of any one of embodiments 1-32, wherein the composition is a starter culture for bakery good production.
[0278] Embodiment 37. The composition of any one of embodiments 1-32, wherein the composition is a baked good.
[0279] Embodiment 38. The composition of embodiment 37, wherein the baked good is a bread.
[0280] Embodiment 39. The composition of embodiment 37, wherein the baked good is a sourdough bread.
[0281] Embodiment 40. A method for improving the fermentation of a food product, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the food product.
[0282] Embodiment 41. A method of producing a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0283] Embodiment 42. A method of increasing the shelf-life of a dough or baked good, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough or baked good.
[0284] Embodiment 43. A method of lowering the pH of a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0285] Embodiment 44. A method of increasing organic acids in a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0286] Embodiment 45. A method of enhancing rheological properties of a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0287] Embodiment 46. A method of increasing protein degradation in a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0288] Embodiment 47. A method of increasing peptide or protein production in a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0289] Embodiment 48. A method of increasing degradation of complex and/or simple carbohydrates in a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0290] Embodiment 49. A method of increasing degradation of chemical compounds or chemical precursors in a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0291] Embodiment 50. A method of increasing the flavor or aroma of a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0292] Embodiment 51. A method of increasing leavening ability of a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0293] Embodiment 52. A method of increasing nutritional quality of a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0294] Embodiment 53. The method of embodiment 52, wherein the method reduces the glycemic index of the dough or a baked food product of the dough.
[0295] Embodiment 54. The method of embodiment 52, wherein the method reduces glutens in the dough or a baked food product of the dough.
[0296] Embodiment 55. A method of modulating activity of one or more microorganisms in a dough, the method comprising admixing an effective amount of the composition of any one of embodiments 1-32 to the dough.
[0297] Embodiment 56. A method for improving fermentation of a dough, comprising: admixing an effective amount of a composition comprising a purified population of one or more microorganisms comprising a nucleic acid sequence that shares at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1-272 to the dough.
[0298] Embodiment 57. A method for increasing the shelf-life of a dough, comprising: admixing an effective amount of a composition comprising a purified population of one or more microorganisms comprising a nucleic acid sequence that shares at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1-272 to the dough.
[0299] Embodiment 58. A method of enhancing rheological properties of a dough, the method comprising: admixing an effective amount of a composition comprising a purified population of one or more microorganisms comprising a nucleic acid sequence that shares at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1-272 to the dough.
[0300] Embodiment 59. The method of any one of embodiments 55-58, wherein the admixture results in a baked food product.
[0301] Embodiment 60. The method of embodiment 59, wherein the baked food product is a sourdough bread.
[0302] Embodiment 61. A method for increasing the shelf-life of a baked food product, comprising: admixing an effective amount of a composition comprising a purified population of one or more microorganisms comprising a nucleic acid sequence that shares at least 97% sequence identity with a nucleic acid sequence selected from SEQ ID NOs: 1-272 to a dough, wherein the admixture results in the baked food product with an increased shelf-life.
[0303] Embodiment 62. The method of any one of embodiments 40 to 61, wherein the method comprises incubating the admixture for a period of time.
[0304] Embodiment 63. The method of embodiment 62, wherein the admixture is incubated for at least 15 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, or at least 120 hours.
INCORPORATION BY REFERENCE
[0305] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.
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