Compositions and methods for producing cell culture supplements

Abstract

Various aspects of the present disclosure relate to a fermentation culture medium supplement that comprises a soluble fraction derived from a leguminous plant. Provided herein, in some aspects, are compositions comprising the soluble fraction derived from a leguminous plant described herein, and methods of making the soluble fraction derived from a leguminous plant.

Description

WSGR Docket No. 69505-701.601COMPOSITIONS AND METHODS FOR PRODUCING CELL CULTURE SUPPLEMENTSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 726,194, filed November 27, 2024, which application is incorporated herein by reference in its entirety.BACKGROUND

[0002] Cell culture media provide essential nutrients that support the growth and activity of microorganisms across various applications, from research to industry. These media are formulated to deliver various macro- and micro- nutrients like carbohydrates, proteins, vitamins, and minerals. Optimizing culture media is key to achieving high microbial yield and functionality for its applicability in areas like industrial fermentation, agriculture, and probiotic production.SUMMARY

[0003] Provided herein is a fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant, wherein the soluble fraction comprises carbohydrates that are free or substantially free of glucose, wherein the carbohydrates are present in the soluble fraction at a concentration of at least about 50% w / w, and wherein the fermentation culture medium supplement provides nutrient substrates for microbial metabolism. In some embodiments, the soluble fraction further comprises proteins. In some embodiments, the proteins are present in the soluble fraction at a concentration of at least about 10% w / w. In some embodiments, the proteins are present in the soluble fraction at a concentration of from about 10.0% to about 40% w / w. In some embodiments, the carbohydrates are present in the soluble fraction at a concentration of from about 50% to about 90% w / w. In some embodiments, the carbohydrates comprise galactose, sucrose, stachyose, or a combination thereof. In some embodiments, the galactose is present in the soluble fraction at a concentration of at least about 10% w / w. In some embodiments, the sucrose is present in the soluble fraction at a concentration of at least about 13% w / w. In some embodiments, the soluble fraction further comprises nitrogen, ashes, mineral(s), or a combination thereof. In some embodiments, the mineral comprises potassium, sodium, magnesium, calcium, phosphorus, sulfur, iron, zinc, copper, manganese, molybdenum, or a combination thereof. In some embodiments, the nitrogen is present in the soluble fraction at a concentration of at least about 1.5 % w / w. In some embodiments, the microbial metabolism supports growth or activity of microorganisms. In some embodiments, the microorganisms are selected from the group consisting of Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense, Bradyrhizobium diazoefficiens,WSGR Docket No. 69505-701.601Rhizobium leguminosarum, Rhizobium etli, Azospirillum brasilense, Azospirillum lipoferum, Pseudomonas fhiorescens. Pseudomonas putida, Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus megalerium. Bacillus pumilus,, Bacillus licheniformis Bacillus velezensis, Trichoderma harzianum. Trichoderma viride. Streptomyces griseus. Streptomyces lydicus. Actinomyces israelii, Micromonospora lupini, Metarhizium anisopliae, Metarhizium robertsii, Metarhizium brunneum, Metarhizium flavoviride, Metarhizium guizhouense. Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus reuteri, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Bacillus coagulans, Bacillus subtilis, Lactobacillus bulgaricus, Propionibacterium freudenreichii, E.coli, Saccharomyces pastorianus, Saccharomyces cerevisiae, Aspergillus niger, Fusarium venenatum, Bacillus licheniformis, Trichoderma reesei, Pichia pastoris, Corynebacterium glutamicum, Aspergillus oryzae, Candida antarctica, and Micrococcus luteus. In some embodiments, the growth or the activity of the microorganisms is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant. In some embodiments, the growth or the activity of the microorganisms is measured by an optical density. In some embodiments, the soluble fraction induces sporulation of the microorganisms. In some embodiments, the soluble fraction enhances sporulation of the microorganisms. In some embodiments, the sporulation is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant. In some embodiments, the leguminous plant is selected from the group consisting of yellow pea, chickpea, mung bean, white bean, pinto bean, red kidney bean, lentil, black bean, cowpea, caupi bean, broad bean, and aduki bean. In some embodiments, the carbohydrates are present in the soluble fraction at an amount of at most 5% glucose. In some embodiments, the carbohydrates are present in the soluble fraction at an amount of at most 1% glucose. In some embodiments, the soluble fractionWSGR Docket No. 69505-701.601 further comprises a carrier. In some embodiments, the carrier comprises protein concentrates obtained from the leguminous plant. In some embodiments, the carrier comprises silicon dioxides, salts, sugars, or a combination thereof. In some embodiments, the carrier comprises a biopolymer. In some embodiments, the biopolymer is selected from the group consisting of a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, and maltodextrin. In some embodiments, the carrier is added to the soluble fraction at a dose of from about 0.1% to about 50%. In some embodiments, the soluble fraction is not liquefied or treated with a proteolytic enzyme. In some embodiments, the soluble fraction is liquefied or treated with a proteolytic enzyme. In some embodiments, the proteolytic enzyme comprises an endoprotease, an exoprotease, or a combination thereof. In some embodiments, the proteolytic enzyme treatment or liquefaction results in a hydrolyzed soluble fraction. In some embodiments, the hydrolyzed soluble fraction further comprises the carrier. In some embodiments, the soluble fraction replaces a yeast extract. In some embodiments, the soluble fraction replaces meat peptones or meat extracts. In some embodiments, the soluble fraction is processed by spray drying to produce a dry powder. In some embodiments, the soluble fraction is characterized by a moisture content of less than 50%.

[0004] Further provided herein is a fermentation culture medium supplement comprising: a soluble fraction derived from a leguminous plant, wherein the soluble fraction comprises at least one carbon source and at least one nitrogen source for microbial metabolism, wherein the at least one carbon source is free or substantially free of glucose. In some embodiments, the at least one nitrogen source is derived from an unhydrolyzed protein of the soluble fraction derived from the leguminous plant. In some embodiments, the at least one nitrogen source is present in an amount of at least about 10% by weight of the soluble fraction. In some embodiments, the at least one carbon source is carbohydrate present at a concentration of at least about 50% by weight of the soluble fraction. In some embodiments, the carbohydrate comprises galactose, stachyose, sucrose, or a combination thereof. In some embodiments, the carbohydrate is free or substantially free of glucose. In some embodiments, the soluble fraction derived from the leguminous plant further comprises at least one mineral. In some embodiments, the at least one mineral comprises potassium, sodium, magnesium, calcium, phosphorus, sulfur, iron, zinc, copper, manganese, molybdenum, or a combination thereof. In some embodiments, the microbial metabolism supports growth or activity of microorganisms. In some embodiments, the microorganisms are selected from the group consisting of: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense, Bradyrhizobium diazoefficiens, Rhizobium leguminosarum, Rhizobium etli, Azospirillum brasilense, Azospirillum lipoferum, Pseudomonas fhiorescens. Pseudomonas putida, Bacillus subtilis, Bacillus ihuringiensis. Bacillus amyloliquefaciens, Bacillus megalerium. Bacillus pumilus,, Bacillus licheniformis BacillusWSGR Docket No. 69505-701.601 velezensis, Trichoderma harzianum, Trichoderma viride, Streptomyces griseus, Streptomyces lydicus. Actinomyces israelii, Micromonospora lupini, Metarhizium anisopliae, Metarhizium robertsii, Metarhizium brunneum, Metarhizium flavoviride, Metarhizium guizhouense, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus reuteri, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Bacillus coagulans, Bacillus subtilis, Lactobacillus bulgaricus, Propionibacterium freudenreichii, E.coli, Saccharomyces pastorianus, Saccharomyces cerevisiae, Aspergillus niger, Fusarium venenatum, Bacillus licheniformis, Trichoderma reesei, Pichia pastoris, Corynebacterium glutamicum, Aspergillus oryzae, Candida antarctica, and Micrococcus luteus. In some embodiments, the growth or the activity of the microorganisms is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant. In some embodiments, the growth or the activity of the microorganisms is measured by an optical density. In some embodiments, the soluble fraction induces sporulation of the microorganisms. In some embodiments, the soluble fraction enhances sporulation of the microorganisms. In some embodiments, the sporulation is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant. In some embodiments, the leguminous plant is selected from the group consisting of yellow pea, chickpea, mung bean, white bean, pinto bean, red kidney bean, lentil, black bean, cowpea, caupi bean, broad bean, and aduki bean. In some embodiments, the at least one carbon source comprises at most 5% glucose. In some embodiments, the at least one carbon source comprises at most 1% glucose. In some embodiments, the soluble fraction further comprises a carrier. In some embodiments, the carrier comprises a protein concentrate obtained from the leguminous plant. In some embodiments, the carrier comprises silicon dioxides, salts, sugars, or a combination thereof. In some embodiments, the carrier comprises a biopolymer. In some embodiments, the biopolymer isWSGR Docket No. 69505-701.601 selected from the group consisting of a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, and maltodextrin. In some embodiments, the carrier is added at a dose of from about 0.1% to about 50% of the soluble fraction. In some embodiments, the soluble fraction is not liquefied or treated with a proteolytic enzyme. In some embodiments, the soluble fraction is liquefied or treated with a proteolytic enzyme. In some embodiments, the proteolytic enzyme comprises an endoprotease, an exoprotease, or a combination thereof. In some embodiments, the proteolytic enzyme treatment or liquefaction results in a hydrolyzed soluble fraction.

[0005] Provided herein is a fermentation culture medium comprising a basal culture medium, a fermentation culture medium supplement described herein, wherein the fermentation culture medium supplement is added at a final concentration of from 0.05 g / L to about 30 g / L in the basal culture medium. In some embodiments, the fermentation culture medium supplement is added at a final concentration of about 14 g / L in the basal culture medium. In some embodiments, the fermentation culture medium lacks a yeast extract. In some embodiments, the fermentation culture medium is free or substantially free of glucose.

[0006] Provided herein is a method of making a fermentation culture medium, comprising: (a) providing or obtaining a leguminous plant or a leguminous plant material, (b) extracting a soluble fraction of the leguminous plant or the leguminous plant material, wherein the soluble fraction comprises carbohydrates, wherein the carbohydrates are free or substantially free of glucose, and (c) drying the soluble fraction of the leguminous plant or the leguminous plant material. In some embodiments, the leguminous plant or the leguminous plant material comprises plant flours, dried plant powders, or fresh plant biomass. In some embodiments, the leguminous plant or the leguminous plant material is selected from the group consisting of yellow pea, chickpea, mung bean, white bean, pinto bean, red kidney bean, lentil, black bean, cowpea, caupi bean, broad bean, and aduki bean. In some embodiments, the extracting the soluble fraction comprises mixing the leguminous plant or the leguminous plant material into an aqueous solution. In some embodiments, the extracting the soluble fraction further comprises decanting, purifying, or filtering the aqueous solution, thereby obtaining the soluble fraction. In some embodiments, after the extracting, the method further comprises concentrating the soluble fraction. In some embodiments, the concentration of the soluble fraction produces a soluble fraction having a total solid content of at least about 20%, at least about 30%, or at least about 40% (w / w). In some embodiments, the method further comprises, before the drying the soluble fraction, adding one or more carriers into the soluble fraction. In some embodiments, the method further comprises, after drying the soluble fraction, adding one or more carriers into the soluble fraction. In some embodiments, the one or more carriers comprises a protein concentrate obtained from the leguminous plant. In some embodiments, the oneWSGR Docket No. 69505-701.601 or more carriers comprises silicon dioxides, salts, sugars, or a combination thereof. In some embodiments, the one or more carriers comprises a biopolymer. In some embodiments, the biopolymer is selected from the group consisting of a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, and maltodextrin. In some embodiments, the extracting further comprises treating the soluble fraction with a proteolytic enzyme, thereby producing a hydrolyzed soluble fraction. In some embodiments, the proteolytic enzyme comprises an endoprotease, an exoprotease, or a combination thereof.

[0007] Provided herein is a method for growing microorganisms, comprising: (a) providing a fermentation culture medium comprising: a basal culture medium and a soluble fraction derived from a leguminous plant, wherein the soluble fraction comprises carbohydrates that are free or substantially free of glucose, and wherein the carbohydrates are present in the soluble fraction at a concentration of at least about 50% w / w; and (b) inoculating the fermentation culture medium with the microorganisms to promote microbial growth or microbial activity. In some embodiments, the soluble fraction further comprises proteins. In some embodiments, the proteins are present in the soluble fraction at a concentration of at least about 10% w / w. In some embodiments, the proteins are present in the soluble fraction at a concentration of from about 10.0% to about 40% w / w. In some embodiments, the carbohydrates are present in the soluble fraction at a concentration of from about 50% to about 90% w / w. In some embodiments, the carbohydrates comprise galactose, sucrose, stachyose, or a combination thereof. In some embodiments, the galactose is present in the soluble fraction at a concentration of at least about 10% w / w. In some embodiments, the sucrose is present in the soluble fraction at a concentration of at least about 13% w / w. In some embodiments, the soluble fraction further comprises nitrogen, ashes, mineral, or a combination thereof. In some embodiments, the mineral comprises potassium, sodium, magnesium, calcium, phosphorus, sulfur, iron, zinc, copper, manganese, molybdenum, or a combination thereof. In some embodiments, the carbohydrates have at most 5% glucose. In some embodiments, the carbohydrates have at most 1% glucose. In some embodiments, the microbial metabolism supports growth or activity of the microorganisms. In some embodiments, the microorganisms are selected from the group consisting of: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense, Bradyrhizobium diazoefficiens, Rhizobium leguminosarum, Rhizobium etli, Azospirdlum brasilense, Azospirdlum lipoferum, Pseudomonas fhiorescens. Pseudomonas putida, Bacdlus subtilis, Bacdlus ihuringiensis. Bacdlus amyloliquefaciens, Bacdlus megalerium. Bacdlus pumilus,, Bacdlus licheniformis Bacdlus velezensis, Trichoderma harzianum. Trichoderma viride. Streptomyces griseus. Streptomyces lydicus. Actinomyces israelii, Micromonospora lupini, Metarhizium anisopliae, Metarhizium robertsii, Metarhizium brunneum, Metarhizium flavoviride, MetarhiziumWSGR Docket No. 69505-701.601 guizhouense, Lactobacillus acidophilus, Lactobacillus casei. Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus reuteri, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Bacillus coagulans, Bacillus subtilis, Lactobacillus bulgaricus, Propionibacterium freudenreichii, E.coli, Saccharomyces pastorianus, Saccharomyces cerevisiae, Aspergillus niger, Fusarium venenatum, Bacillus licheniformis, Trichoderma reesei, Pichia pastoris, Corynebacterium glutamicum, Aspergillus oryzae, Candida antarctica, and Micrococcus luteus. In some embodiments, the growth or the activity of the microorganisms is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant. In some embodiments, the growth or the activity of the microorganisms is measured by an optical density. In some embodiments, the soluble fraction induces sporulation of the microorganisms. In some embodiments, the soluble fraction enhances sporulation of the microorganisms. In some embodiments, the sporulation is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant. In some embodiments, the leguminous plant is selected from the group consisting of yellow pea, chickpea, mung bean, white bean, pinto bean, red kidney bean, lentil, black bean, cowpea, caupi bean, broad bean, and aduki bean. In some embodiments, the soluble fraction further comprises a carrier. In some embodiments, the carrier comprises a protein concentrate obtained from the leguminous plant. In some embodiments, the carrier comprises silicon dioxides, salts, sugars, or a combination thereof. In some embodiments, the carrier comprises a biopolymer. In some embodiments, the biopolymer is selected from the group consisting of a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, and maltodextrin.WSGR Docket No. 69505-701.601INCORPORATION BY REFERENCE

[0008] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0010] FIG. 1 shows a flow chart of manufacturing a fermentation culture medium supplement derived from a leguminous plant (e.g., pea soluble).

[0011] FIG. 2 shows optical density of microorganisms grown in Luria Broth (LB), 100% pea soluble (Supplement A), 50% pea soluble and 50% maltodextrin (Supplement B), 100% hydrolyzed pea soluble (Supplement C), and 50% hydrolyzed pea soluble and 50% maltodextrin (Supplement D).DETAILED DESCRIPTION

[0012] Disclosed herein is a fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant and methods of making the same. In some embodiments, the compositions and methods described herein can utilize upcycled byproducts from, for example, the food and agricultural industry as starting materials for producing cell culture medium supplements. For example, in some embodiments, a leguminous plant or an extract (e.g., a soluble fraction derived from a leguminous plant) can be used as a cell culture media supplement. In some embodiments, the fermentation culture medium supplement (e.g., comprising a soluble fraction derived from a leguminous plant) described herein can replace one or more supplements of the cell culture medium. In some embodiments, for example, the one or more supplements of the fermentation culture medium supplement can be yeast extracts, beef extracts, meat extracts, and / or meat peptone. In some embodiments, the fermentation culture medium supplement described herein can replace all or a portion of yeast extracts, beef extracts, meat peptones, and / or meat extracts. In some embodiments, the soluble fraction derived from a leguminous plant can replace at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% (w / w) ofWSGR Docket No. 69505-701.601 supplements added to the culture medium. In some embodiments, the soluble fraction derived from a leguminous plant can fully replace (e.g., 100%) yeast extracts, beef extracts, meat peptone, and / or meat extracts at equivalent concentrations. In some embodiments, the cells (e.g, microorganism) cultivated in the culture media disclosed herein (e.g., comprising a soluble fraction of a leguminous plant) may exhibit no reduction in growth rate relative to a medium lacking a soluble fraction of a leguminous plant or relative to a medium comprising yeast extracts, beef extracts, meat peptone, and / or meat extracts. In some cases, the cells (e.g, microorganism) cultivated in the culture media disclosed herein (e.g., comprising a soluble fraction of a leguminous plant) the growth rate can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more relative to the corresponding conventional culture medium (e.g., a medium lacking a soluble fraction of a leguminous plant or relative to a medium comprising yeast extracts, beef extracts, meat peptone, and / or meat extracts). In some cases, the cells (e.g., microorganism) cultivated in the culture media disclosed herein may exhibit comparable or improved performance relative to conventional media. In some embodiments, such performance can be reflected in biomass accumulation, maximum cell density, sporulation efficiency, spore viability, or metabolite yield. For example, in some embodiments, sporulation efficiency of the cultured cells (e.g., microorganism) in the culture media disclosed herein (e.g., comprising a soluble fraction of a leguminous plant) may be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more relative to cultured cells in the corresponding conventional culture medium (e.g., a medium lacking a soluble fraction of a leguminous plant or relative to a medium comprising yeast extracts, beef extracts, meat peptone, and / or meat extracts). In some embodiments, biomass accumulations of the cultured cells (e.g., microorganism) in the culture media disclosed herein (e.g., comprising a soluble fraction of a leguminous plant) may be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more relative to cultured cells in the corresponding conventional culture medium (e.g., a medium lacking a soluble fraction of a leguminous plant or relative to a medium comprising yeast extracts, beef extracts, meat peptone, and / or meat extracts). In some embodiments, spore viability of the cultured cells (e.g., microorganism) in the culture media disclosed herein (e.g., comprising a soluble fraction of a leguminous plant) may be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more relative to cultured cells in the corresponding conventional culture medium (e.g., a medium lacking a soluble fraction of aWSGR Docket No. 69505-701.601 leguminous plant or relative to a medium comprising yeast extracts, beef extracts, meat peptone, and / or meat extracts).

[0013] In some embodiments, the fermentation culture medium supplement can be used as sole components of the cell culture media (e.g., for growth of microorganisms). In some embodiments, the soluble fraction derived from a leguminous plant can provide nitrogen sources, vitamins and cofactors, minerals and trace elements, metabolic stimulants, growth factors, and / or metabolic boosters necessary to support microorganism growth and proliferation. In some embodiments, when culture media are formulated such that the total nitrogen concentration supplied by the soluble fraction derived from a leguminous plant is equal to the nitrogen concentration provided by yeast extract, beef extracts, meat peptone, and / or meat extract, the microorganism may exhibit a faster growth rate (e.g., shorter doubling time), greater biomass accumulation, and / or enhanced proliferation. For example, in some cases, the cells (e.g., microorganism) cultured in the fermentation culture medium disclosed herein (e.g., comprising a soluble fraction derived from a leguminous plant) can exhibit an increase in growth rate, biomass accumulation, and / or proliferation that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more relative to cultured cells in the corresponding conventional culture medium (e.g., a medium lacking a soluble fraction of a leguminous plant or relative to a medium comprising yeast extracts, beef extracts, meat peptone, and / or meat extracts). Without being bound by theory, the superior performance observed from the optimized and balanced of amino acids, peptides, vitamins, and micronutrients present in the soluble from a leguminous plant, which are more readily assimilated by microbial cells compared to conventional extracts.

[0014] In some embodiments, the fermentation culture medium supplement can be combined with other supplements (e.g., yeast extracts, minerals, carbohydrates, proteins, amino acids etc.). Also disclosed herein are methods of culturing cells (e.g., microorganisms) in the disclosed fermentation culture medium supplement and utilizing the cultures to promote growth and activity of different microorganisms involved in agriculture application, industrial fermentation, and / or probiotics for human and animal consumption.

[0015] Described herein is a novel fermentation culture medium supplement comprising a soluble fraction derived from leguminous plants. The soluble fraction is obtained through the extraction of select leguminous plant materials using aqueous or compatible solvent-based processes under conditions optimized to yield a highly bioavailable and nutrient-rich supplement. This fraction is uniquely suited to support microbial growth and metabolic function in a variety of fermentation applications.WSGR Docket No. 69505-701.601Fermentation culture medium supplement

[0016] Described herein is a fermentation culture medium supplement comprising a soluble fraction derived from a plant. As used herein, a “soluble fraction” refers to a portion of a substance derived from a source material (e.g., a plant or a leguminous plant) that is capable of being dissolved in an aqueous solution or specific solvent (e.g., water) under defined condition, separated from an insoluble residue. In some embodiments, the soluble fraction (e.g., derived from a plant or a leguminous plant) can comprise various dissolved components, including, but not limited to, proteins, carbohydrates, amino acids, minerals, and other small molecules.

[0017] In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from a leguminous plant. As used herein a “leguminous plant” refers to any plant belonging to the Leguminosae (or Fabaceae) family, or the fruit or seeds of such plants. For example, in some embodiments, a leguminous plant can comprise yellow pea (Pisum sativum), chickpea Cicer arielinum), mung bean Vigna radiala), white bean, pinto bean, red kidney bean (Phaseolus vulgaris), lentil Vicia lens o Lens culinaris, black bean (Castanospermum australe), cowpea or caupi bean (Vigna unguiculata), broad bean Vicia faba , aduki bean (Vigna angularis), soybean (Glycine max , green pea Pisum sativum var. arvense), lupin (Lupinus albus, Lupinus luteus), pigeon pea (Cajanus cajan), fenugreek (Trigonella foenum-graecum), grass pea (Lathyrus sativus), bambara groundnut Vigna subterranea), velvet bean (Mucuna pruriens), winged bean (Psophocarpus tetragonolobus), or jack bean (Canavalia ensiformis). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from yellow pea Pisum sativum). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from chickpea Cicer arietinum). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from mung bean (Vigna radiata). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from white bean, pinto bean, or red kidney bean (Phaseolus vulgaris). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from lentils (Vicia lens ox Lens culinaris). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from black bean (Castanospermum australe). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from cowpea or caupi bean (Vigna unguiculata). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from broad bean (Vicia faba). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from aduki bean (Vigna angularis). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from soybean (Glycine max). InWSGR Docket No. 69505-701.601 some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from green pea (Pisum sativum var. arvense). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from lupin (Lupinus albus, Lupinus luteus). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from pigeon pea (Cajanus cajari). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from fenugreek (Trigonella foenum-graecum). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from grass pea (Lathyrus sativus). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from Bambara groundnut (Vigna sublerranea). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from velvet bean (Mucuna pruriens). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from winged bean (Psophocarpus tetragonolobus). In some embodiments, the fermentation culture medium supplement may comprise a soluble fraction derived from jack bean (Canavalia ensiformis).

[0018] In some embodiments, the fermentation culture medium supplement can comprise a soluble fraction derived from a leguminous plant, wherein the soluble fraction derived from the leguminous plant can comprise protein, nitrogen (e.g., amino acid), ashes, carbohydrates dietary fiber, lipids, or minerals. In some embodiments, the fermentation culture medium supplement (e.g., comprising a soluble fraction of a leguminous plant) can be provided as a hydrolyzed form. In some cases, the hydrolyzed form of the fermentation culture medium supplement disclosed herein can be prepared by enzymatic, acid, or alkaline hydrolysis. In some cases, the fermentation culture medium supplement (e.g., comprising a soluble fraction of a leguminous plant) can be formulated in combination with maltodextrin or similar carbohydrate carriers. For example, carbohydrate carriers may function as a stabilizer, a carrier (e.g., that facilitates spray-drying or freeze-drying into a free- flowing powder), and / or an auxiliary carbon source. In some embodiments, the carbohydrates (e.g., carbohydrates derived from a soluble fraction of a leguminous plant) of the fermentation culture medium supplement described herein can be free or substantially free of glucose. As used herein “free or substantially free” refers to the substance (e.g., glucose) that is either entirely absent ("free") or present only in trace amounts that do not materially affect the functionality, performance, or intended properties of the composition or process. For example, “substantially free” may refer to substance levels (e.g., glucose levels) that is undetectable or negligible as measured by, for example, High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), enzymatic assays (e.g., glucose oxidase, hexokinase, or fructose dehydrogenase assays), or capillary electrophoresis.WSGR Docket No. 69505-701.601

[0019] In some embodiments, the low glucose level (e.g., free or substantially free of glucose) can be advantageous in fermentation culture medium supplement by minimizing or avoiding Maillard reaction occurring during the sterilization process of the culture media (e.g., culture media comprising the fermentation culture medium supplement described herein). As used herein, the “Maillard reaction” is a chemical reaction between amino acids and reducing sugars (e.g., glucose) that typically occurs when heat is applied, producing complex molecules that can affect the composition, color, and nutritional value of the medium. In some embodiments, the fermentation culture medium supplement described herein can avoid the Maillard reaction, thereby minimizing nutrient degradation, production of toxic byproducts, or pH changes. In some embodiments, Maillard compounds (e.g., chemical compounds created during the Maillard reaction including but are not limited to melanoidins, acylpyridines, alkylpyridines, pyrroles, furans and furanoses) can reduce growth rate or inhibit growth of the microorganisms. In some embodiments, the fermentation culture medium supplement described herein can minimize the preparation steps by avoiding a need to sterilize the amino acids and glucose separately (e.g., two-steps of sterilization). In some embodiments, this change allows the “low glucose level” to be potentially applied to different fermentation culture media or supplements. For example, the fermentation culture medium or supplement can be modified, treated, or purified such that the fermentation culture medium or supplement can be free or substantially free of glucose (e.g., as compared to that of a starting culture medium or supplements prior to one or more modifications, treatments, or purifications as described herein). In some embodiments, the fermentation culture medium or supplement can be modified, treated, or purified to achieve low glucose level (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L, or lower). For example, the fermentation culture medium or supplement can undergo enzymatic treatments (e.g., glucose oxidase or catalase), filiations (e.g., membrane filtration or ultra-filtration), chemical treatments (e.g., chemical treatment that neutralize glucose), activated carbon adsorptions, chromatography (e.g., ion-exchange chromatography or size-exclusion chromatography), or others (e.g., fermentation process, antibody that binds to glucose, chemical precipitation, or adsorption) that selectively reduce glucose level of the fermentation culture medium or supplement. For example, in some cases, the fermentation culture medium or supplement disclosed herein can undergo a spontaneous fermentation process. In some embodiments, the fermentation culture medium or supplement disclosed herein can undergo a controlled fermentation process. In some embodiments, a controlled fermentation may include adjusting parameters such as pH, temperature, and / or oxygenWSGR Docket No. 69505-701.601 availability. In some embodiments, the glucose level of the fermentation culture medium or supplements, upon treatment with one or methods described herein (e.g., enzymatic treatments, chemical treatments, or a spontaneous or controlled fermentation process), can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 95%, or about 100% as compared to the glucose level of a starting fermentation culture medium or supplements (e.g., prior to the one or more treatments, purifications, or modifications described herein). In some embodiments, the glucose level of the fermentation culture medium or supplements, upon treatment with one or methods described herein (e.g., enzymatic treatments, chemical treatments, or a spontaneous or controlled fermentation process), can be reduced by at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 95%, or about 100% as compared to the glucose level of a starting fermentation culture medium or supplements (e.g., prior to the one or more treatments, purifications, or modifications described herein).

[0020] In some embodiments, the fermentation culture medium supplement described herein (e.g., a soluble fraction derived from a leguminous plant) can be free or substantially free of fructose. In some embodiments, the “low fructose level” can be potentially applied to different fermentation culture medium or supplement, for example, the fermentation culture medium or supplement can be modified, treated, or purified such that the fermentation culture medium or supplement can be free or substantially free of fructose (e.g., as compared to that of a starting culture medium or supplement prior to one or more modifications, treatments, or purifications as described herein). In some embodiments, the fermentation culture medium or supplement can be modified, treated, or purified to achieve low fructose level (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower). For example, the fermentation culture medium or supplement can undergo enzymatic treatments (e.g., fructokinase, hexokinase, or fructose isomerase), filtrations (e.g., membrane filtration, ultra-filtration), chemical treatments (e.g., chemical treatment that neutralize fructose), activated carbon adsorptions, chromatography (e.g., ion-exchange chromatography or size-exclusion chromatography), or others (e.g., antibody that binds to fructose, chemical precipitations, or adsorptions) that selectively reduceWSGR Docket No. 69505-701.601 fructose level of the fermentation culture medium or supplement. In some embodiments, the fructose level of the fermentation culture medium or supplement, upon treatment with one or methods described herein (e.g., enzymatic treatments, chemical treatments, or a spontaneous or controlled fermentation process), can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 95%, or about 100% as compared to the fructose level of a starting fermentation culture medium or supplements (e.g., prior to the one or more treatments described herein). In some embodiments, the fructose level of the fermentation culture medium or supplement, upon treatment with one or methods described herein (e.g., enzymatic treatments, chemical treatments, or a spontaneous or controlled fermentation process), can be reduced by at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about70%, at most about 75%, at most about 80%, at most about 95%, or about 100% as compared to the fructose level of a starting fermentation culture medium or supplements (e.g., prior to the one or more treatments described herein).

[0021] In some embodiments, the fermentation culture medium supplement described herein (e.g., a soluble fraction derived from a leguminous plant) can be free or substantially free of raffinose. In some embodiments, the “low raffinose level” can be applied to different fermentation culture medium or supplement. For example, the fermentation culture medium or supplement can be modified, treated, or purified such that the fermentation culture medium or supplement can be free or substantially free of raffinose (e.g., as compared to that of a starting culture medium or supplement prior to one or more modifications, treatments, or purifications as described herein). In some embodiments, the fermentation culture medium or supplement can be modified, treated, or purified to achieve low raffinose level (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower). For example, the fermentation culture medium or supplement can undergo enzymatic treatments (e.g., alphagalactosidase, invertase in combination with alpha-galactosidase), filtration (e.g., membrane filtration, ultra-filtration), chemical treatments (e.g., chemical treatment that neutralize raffinose), activated carbon adsorptions, chromatography (e.g., ion-exchange chromatography or size-exclusion chromatography), or others (e.g., antibody that binds to raffinose, chemical precipitations, orWSGR Docket No. 69505-701.601 adsorptions) that selectively reduce raffinose level of the fermentation culture medium or supplement. In some embodiments, the raffinose level of the fermentation culture medium or supplements, upon treatment with one or methods described herein (e.g., en enzymatic treatments, chemical treatments, or a spontaneous or controlled fermentation process), can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 95%, or about 100% as compared to the raffinose level of a starting fermentation culture medium or supplement (e.g., prior to the one or more treatments described herein). In some embodiments, the raffinose level of the fermentation culture medium or supplements, upon treatment with one or methods described herein (e.g., enzymatic treatments, chemical treatments, or a spontaneous or controlled fermentation process), can be reduced by at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 95%, or about 100% as compared to the raffinose level of a starting fermentation culture medium or supplement (e.g., prior to the one or more treatments described herein).

[0022] In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise carbohydrates. In some embodiments, the carbohydrates can include monosaccharides, disaccharides, oligosaccharides, and polysaccharide. In some embodiments, carbohydrate profile of the soluble fraction can comprise, for example, fructose, sucrose, galactose, raffinose, stachyose, other naturally occurring sugars, or a combination thereof. In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise carbohydrates, wherein the carbohydrates are free or substantially free of glucose. In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise monosaccharides, wherein monosaccharides comprise fructose or galactose. In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise disaccharides, wherein disaccharides comprise sucrose, maltose, or lactose. In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise oligosaccharides, wherein oligosaccharides comprise raffinose family oligosaccharides (RFOs) such as raffinose, stachyose, or verbascose, fructo-oligosaccharides (FOS), or galactic-oligosaccharides. In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise polysaccharides such as pectins and inulin. In some embodiments, the soluble fraction derived from a leguminousWSGR Docket No. 69505-701.601 plant described herein can comprise sugar alcohol such as sorbitol, mannitol, or xylitol. In some embodiments, the carbohydrate content of the soluble fraction can be quantified using various analytical methods, including High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), or colorimetric assays such as the phenol-sulfuric acid method. Additionally, total carbohydrate content may be calculated by the difference method, where the sum of protein, fat, moisture, and ash is subtracted from the total sample weight. In some embodiments, the presence of these carbohydrates in the soluble fraction can provide energy, support digestion, or serve as prebiotic fiber, depending on the specific carbohydrate composition and its intended application.

[0023] In some embodiments, the carbohydrate content in the soluble fraction derived from a leguminous plant may contribute to the functional, nutritional (e.g., energy storage), or bioactive properties of the microorganisms grown in culture media comprising the fermentation culture medium supplement described herein. In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight to about 90 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight to about 50 % by weight, about 45 % by weight to about 55 % by weight, about 45 % by weight to about 60 % by weight, about 45 % by weight to about 65 % by weight, about 45 % by weight to about 70 % by weight, about 45 % by weight to about 75 % by weight, about 45 % by weight to about 80 % by weight, about 45 % by weight to about 85 % by weight, about 45 % by weight to about 90 % by weight, about 50 % by weight to about 55 % by weight, about 50 % by weight to about 60 % by weight, about 50 % by weight to about 65 % by weight, about 50 % by weight to about 70 % by weight, about 50 % by weight to about 75 % by weight, about 50 % by weight to about 80 % by weight, about 50 % by weight to about 85 % by weight, about 50 % by weight to about 90 % by weight, about 55 % by weight to about 60 % by weight, about 55 % by weight to about 65 % by weight, about 55 % by weight to about 70 % by weight, about 55 % by weight to about 75 % by weight, about 55 % by weight to about 80 % by weight, about 55 % by weight to about 85 % by weight, about 55 % by weight to about 90 % by weight, about 60 % by weight to about 65 % by weight, about 60 % by weight to about 70 % by weight, about 60 % by weight to about 75 % by weight, about 60 % by weight to about 80 % by weight, about 60 % by weight to about 85 % by weight, about 60 % by weight to about 90 % by weight, about 65 % by weight to about 70 % by weight, about 65 % by weight to about 75 % by weight, about 65 % by weight to about 80 % by weight, about 65 % by weight to about 85 % by weight, about 65 % by weight to about 90 % by weight, about 70 % by weight to about 75 % by weight, about 70 % by weight to about 80 % by weight, about 70 % by weight to about 85 % by weight, about 70 % byWSGR Docket No. 69505-701.601 weight to about 90 % by weight, about 75 % by weight to about 80 % by weight, about 75 % by weight to about 85 % by weight, about 75 % by weight to about 90 % by weight, about 80 % by weight to about 85 % by weight, about 80 % by weight to about 90 % by weight, or about 85 % by weight to about 90 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight, about 50 % by weight, about 55 % by weight, about 60 % by weight, about 65 % by weight, about 70 % by weight, about 75 % by weight, about 80 % by weight, about 85 % by weight, or about 90 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of at least about 45 % by weight, about 50 % by weight, about 55 % by weight, about 60 % by weight, about 65 % by weight, about 70 % by weight, about 75 % by weight, about 80 % by weight, or about 85 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of at most about 50 % by weight, about 55 % by weight, about 60 % by weight, about 65 % by weight, about 70 % by weight, about 75 % by weight, about 80 % by weight, about 85 % by weight, or about 90 % by weight (e.g., % w / w dry basis).

[0024] In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight to about 90 % by weight (e.g., % w / w dry basis), wherein the carbohydrate content is free or substantially free of glucose. For example, in some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight to about 90 % by weight (e.g., % w / w dry basis), wherein the glucose content of the soluble fraction can be at most about 0.01%, at most about 0.05%, at most about 0.1%, at most about 0.15%, at most about 0.2%, at most about 0.25%, at most about 0.3%, at most about 0.4%, at most about 0.5%, at most about 0.6%, at most about 0.7%, at most about 0.8%, at most about 0.9%, at most about 1.0%, at most about 1.2%, at most about 1.4%, at most about 1.6%, at most about 1.8%, at most about 2.0%, at most about 2.5%, at most about 3.0%, at most about 3.5%, at most about 4.0%, at most about 4.5%, or at most about 5.0% by weight (e.g., % w / w dry basis).

[0025] In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight to about 90 % by weight (e.g., % w / w dry basis), wherein the carbohydrate content is free or substantially free of fructose. For example, in some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight to about 90 % by weight (e.g., % w / w dry basis), wherein the fructose content of the soluble fraction can be at most aboutWSGR Docket No. 69505-701.6010.01%, at most about 0.05%, at most about 0.1%, at most about 0.2%, at most about 0.3%, at most about 0.4%, at most about 0.5%, at most about 0.6%, at most about 0.7%, at most about 0.8%, at most about 0.9%, at most about 1.0%, at most about 1.2%, at most about 1.4%, at most about 1.6%, at most about 1.8%, at most about 2.0%, at most about 2.5%, at most about 3.0%, at most about 3.5%, at most about 4.0%, at most about 4.5%, or at most about 5.0% by weight (e.g., % w / w dry basis).

[0026] In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight to about 90 % by weight (e.g., % w / w dry basis), wherein the carbohydrate content is free or substantially free of raffinose. For example, in some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a carbohydrate content of about 45 % by weight to about 90 % by weight (e.g., % w / w dry basis), wherein the raffinose content of the soluble fraction can be at most about 0.01%, at most about 0.05%, at most about 0.1%, at most about 0.2%, at most about 0.3%, at most about 0.4%, at most about 0.5%, at most about 0.6%, at most about 0.7%, at most about 0.8%, at most about 0.9%, at most about 1.0%, at most about 1.2%, at most about 1.4%, at most about 1.6%, at most about 1.8%, at most about 2.0%, at most about 2.5%, at most about 3.0%, at most about 3.5%, at most about 4.0%, at most about 4.5%, or at most about 5.0% by weight (e.g., % w / w dry basis).

[0027] In some embodiments, the carbohydrate content of the soluble fraction can comprise fructose, wherein fructose is present at an amount of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, or at least about 15% by weight (e.g., % w / w dry basis). In some embodiments, the carbohydrate content of the soluble fraction can comprise fructose, wherein fructose is present at an amount of at most about 1%, at most about 2%, at most about 3%, at most about 4%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, or at most about 15% by weight (e.g., % w / w dry basis).

[0028] In some embodiments, the carbohydrate content of the soluble fraction can comprise galactose, wherein galactose is present at an amount of at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19% by weight, or at least about 20% by weight (e.g., % w / w dry basis). In some embodiments, the carbohydrate content of the soluble fraction can compriseWSGR Docket No. 69505-701.601 galactose, wherein galactose is present at an amount of at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, at most about 15%, at most about 16%, at most about 17%, at most about 18%, at most about 19% by weight, or at most about 20% by weight (e.g., % w / w dry basis).

[0029] In some embodiments, the carbohydrate content of the soluble fraction can comprise sucrose, wherein sucrose is present at an amount of at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19% by weight, or at least about 20% by weight (e.g., % w / w dry basis). In some embodiments, the carbohydrate content of the soluble fraction can comprise sucrose, wherein sucrose is present at an amount of at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, at most about 15%, at most about 16%, at most about 17%, at most about 18%, at most about 19% by weight, or at most about 20% by weight (e.g., % w / w dry basis).

[0030] In some embodiments, the carbohydrate content of the soluble fraction can comprise raffinose, wherein raffinose is present at an amount of at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, or at least about 15% by weight (e.g., % w / w dry basis). In some embodiments, the carbohydrate content of the soluble fraction can comprise raffinose, wherein raffinose is present at an amount of at most about 1%, at most about 2%, at most about 3%, at most about 4%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 11%, at most about 12%, at most about 13%, at most about 14%, or at most about 15% by weight (e.g., % w / w dry basis).

[0031] In some embodiments, the carbohydrate content of the soluble fraction can comprise stachyose, wherein stachyose is present at an amount of at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34% by weight, at least about 35% by weight, at least about 36% by weight, at least about 37% by weight, at least about 38% by weight, at least about 39% by weight, or at least about 40% by weight (e.g., % w / w dry basis). In some embodiments, the carbohydrate content of the soluble fraction can comprise stachyose, wherein stachyose is present atWSGR Docket No. 69505-701.601 an amount of at most about 20%, at most about 21%, at most about 22%, at most about 23%, at most about 24%, at most about 25%, at most about 26%, at most about 27%, at most about 28%, at most about 29%, at most about 30%, at most about 31%, at most about 32%, at most about 33%, at most about 34% by weight, at most about 35% by weight, at most about 36% by weight, at most about 37% by weight, at most about 38% by weight, at most about 39% by weight, or at most about 40% by weight (e.g., % w / w dry basis).

[0032] In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise proteins. In some embodiments, the protein content in the soluble fraction derived from a leguminous plant may contribute to the functional, nutritional, or bioactive properties of the microorganisms grown in culture media comprising the fermentation culture medium supplement described herein. In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a protein content of about 8 % by weight to about 40 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a protein content of about 8 % by weight to about 10 % by weight, about 8 % by weight to about 12 % by weight, about 8 % by weight to about 14 % by weight, about 8 % by weight to about 16 % by weight, about 8 % by weight to about 18 % by weight, about 8 % by weight to about 20 % by weight, about 8 % by weight to about 24 % by weight, about 8 % by weight to about 28 % by weight, about 8 % by weight to about 32 % by weight, about 8 % by weight to about 36 % by weight, about 8 % by weight to about 40 % by weight, about 10 % by weight to about 12 % by weight, about 10 % by weight to about 14 % by weight, about 10 % by weight to about 16 % by weight, about 10 % by weight to about 18 % by weight, about 10 % by weight to about 20 % by weight, about 10 % by weight to about 24 % by weight, about 10 % by weight to about 28 % by weight, about 10 % by weight to about 32 % by weight, about 10 % by weight to about 36 % by weight, about 10 % by weight to about 40 % by weight, about 12 % by weight to about 14 % by weight, about 12 % by weight to about 16 % by weight, about 12 % by weight to about 18 % by weight, about 12 % by weight to about 20 % by weight, about 12 % by weight to about 24 % by weight, about 12 % by weight to about 28 % by weight, about 12 % by weight to about 32 % by weight, about 12 % by weight to about 36 % by weight, about 12 % by weight to about 40 % by weight, about 14 % by weight to about 16 % by weight, about 14 % by weight to about 18 % by weight, about 14 % by weight to about 20 % by weight, about 14 % by weight to about 24 % by weight, about 14 % by weight to about 28 % by weight, about 14 % by weight to about 32 % by weight, about 14 % by weight to about 36 % by weight, about 14 % by weight to about 40 % by weight, about 16 % by weight to about 18 % by weight, about 16 % by weight to about 20 % by weight, about 16 % by weight to about 24 % byWSGR Docket No. 69505-701.601 weight, about 16 % by weight to about 28 % by weight, about 16 % by weight to about 32 % by weight, about 16 % by weight to about 36 % by weight, about 16 % by weight to about 40 % by weight, about 18 % by weight to about 20 % by weight, about 18 % by weight to about 24 % by weight, about 18 % by weight to about 28 % by weight, about 18 % by weight to about 32 % by weight, about 18 % by weight to about 36 % by weight, about 18 % by weight to about 40 % by weight, about 20 % by weight to about 24 % by weight, about 20 % by weight to about 28 % by weight, about 20 % by weight to about 32 % by weight, about 20 % by weight to about 36 % by weight, about 20 % by weight to about 40 % by weight, about 24 % by weight to about 28 % by weight, about 24 % by weight to about 32 % by weight, about 24 % by weight to about 36 % by weight, about 24 % by weight to about 40 % by weight, about 28 % by weight to about 32 % by weight, about 28 % by weight to about 36 % by weight, about 28 % by weight to about 40 % by weight, about 32 % by weight to about 36 % by weight, about 32 % by weight to about 40 % by weight, or about 36 % by weight to about 40 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a protein content of about 8 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 24 % by weight, about 28 % by weight, about 32 % by weight, about 36 % by weight, or about 40 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a protein content of at least about 8 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 24 % by weight, about 28 % by weight, about 32 % by weight, or about 36 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a protein content of at most about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 24 % by weight, about 28 % by weight, about 32 % by weight, about 36 % by weight, or about 40 % by weight (e.g., % w / w dry basis). In some embodiments, the protein content or level is based on the total weight of the composition and may be measured using any suitable protein quantification method, including, but not limited to, the Kjeldahl method, Dumas combustion, or colorimetric assays such as the Bradford assay.

[0033] In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise nitrogen. In some embodiments, the nitrogen content in the soluble fraction derived from a leguminous plant may contribute to the functional, nutritional, or bioactive properties of the fermentation culture medium supplement described herein. In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a nitrogen content of aboutWSGR Docket No. 69505-701.6011 % by weight to about 7 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a nitrogen content of about1 % by weight to about 1.5 % by weight, about 1 % by weight to about 2 % by weight, about 1 % by weight to about 2.5 % by weight, about 1 % by weight to about 3 % by weight, about 1 % by weight to about 3.5 % by weight, about 1 % by weight to about 4 % by weight, about 1 % by weight to about 4.5 % by weight, about 1 % by weight to about 5 % by weight, about 1 % by weight to about5.5 % by weight, about 1 % by weight to about 6 % by weight, about 1 % by weight to about 7 % by weight, about 1.5 % by weight to about 2 % by weight, about 1.5 % by weight to about 2.5 % by weight, about 1.5 % by weight to about 3 % by weight, about 1.5 % by weight to about 3.5 % by weight, about 1.5 % by weight to about 4 % by weight, about 1.5 % by weight to about 4.5 % by weight, about 1.5 % by weight to about 5 % by weight, about 1.5 % by weight to about 5.5 % by weight, about 1.5 % by weight to about 6 % by weight, about 1.5 % by weight to about 7 % by weight, about 2 % by weight to about 2.5 % by weight, about 2 % by weight to about 3 % by weight, about 2 % by weight to about 3.5 % by weight, about 2 % by weight to about 4 % by weight, about2 % by weight to about 4.5 % by weight, about 2 % by weight to about 5 % by weight, about 2 % by weight to about 5.5 % by weight, about 2 % by weight to about 6 % by weight, about 2 % by weight to about 7 % by weight, about 2.5 % by weight to about 3 % by weight, about 2.5 % by weight to about 3.5 % by weight, about 2.5 % by weight to about 4 % by weight, about 2.5 % by weight to about 4.5 % by weight, about 2.5 % by weight to about 5 % by weight, about 2.5 % by weight to about 5.5 % by weight, about 2.5 % by weight to about 6 % by weight, about 2.5 % by weight to about 7 % by weight, about 3 % by weight to about 3.5 % by weight, about 3 % by weight to about 4 % by weight, about 3 % by weight to about 4.5 % by weight, about 3 % by weight to about 5 % by weight, about 3 % by weight to about 5.5 % by weight, about 3 % by weight to about 6 % by weight, about 3 % by weight to about 7 % by weight, about 3.5 % by weight to about 4 % by weight, about3.5 % by weight to about 4.5 % by weight, about 3.5 % by weight to about 5 % by weight, about3.5 % by weight to about 5.5 % by weight, about 3.5 % by weight to about 6 % by weight, about3.5 % by weight to about 7 % by weight, about 4 % by weight to about 4.5 % by weight, about 4 % by weight to about 5 % by weight, about 4 % by weight to about 5.5 % by weight, about 4 % by weight to about 6 % by weight, about 4 % by weight to about 7 % by weight, about 4.5 % by weight to about 5 % by weight, about 4.5 % by weight to about 5.5 % by weight, about 4.5 % by weight to about 6 % by weight, about 4.5 % by weight to about 7 % by weight, about 5 % by weight to about5.5 % by weight, about 5 % by weight to about 6 % by weight, about 5 % by weight to about 7 % by weight, about 5.5 % by weight to about 6 % by weight, about 5.5 % by weight to about 7 % by weight, or about 6 % by weight to about 7 % by weight e.g., % w / w dry basis). In someWSGR Docket No. 69505-701.601 embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a nitrogen content of about 1 % by weight, about 1.5 % by weight, about 2 % by weight, about 2.5 % by weight, about 3 % by weight, about 3.5 % by weight, about 4 % by weight, about 4.5 % by weight, about 5 % by weight, about 5.5 % by weight, about 6 % by weight, or about 7 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a nitrogen content of at least about 1 % by weight, about 1.5 % by weight, about 2 % by weight, about 2.5 % by weight, about 3 % by weight, about 3.5 % by weight, about 4 % by weight, about 4.5 % by weight, about 5 % by weight, about 5.5 % by weight, or about 6 % by weight (e.g., % w / w dry basis). In some embodiments, the soluble fraction derived from a leguminous plant described herein can comprise a nitrogen content of at most about 1.5 % by weight, about 2 % by weight, about 2.5 % by weight, about 3 % by weight, about 3.5 % by weight, about 4 % by weight, about 4.5 % by weight, about 5 % by weight, about 5.5 % by weight, about 6 % by weight, or about 7 % by weight (e.g., % w / w dry basis). In some embodiments, the nitrogen content or level is based on the total weight of the composition and may be measured using any suitable quantification method, including, but not limited to, the Kjeldahl method, Dumas combustion, or an elemental analyzer (CHN Analyzer).

[0034] In some embodiments, the nitrogen content of the soluble fraction derived from a leguminous plant may be present in the form of one or more amino acids. These amino acids can contribute to the overall nitrogen profile, providing essential and / or non-essential amino acids that may enhance the functional or nutritional properties of the microorganism grown in culture media comprising the fermentation culture medium supplement described herein. In some embodiments, the nitrogen content can be present in the form of histidine, isoleucine, leucine, lysine, threonine, valine, methionine, cysteine, phenylalanine, tyrosine, arginine, alanine, aspartic acids, glutamic acids, or glycine.

[0035] In some embodiments, the nitrogen content of the soluble fraction can comprise histidine, wherein histidine can be present in an amount of at least about 100 mg (per g Nitrogen). In some embodiments, histidine can be present in an amount of at least about 100 mg, at least about 110 mg, at least about 120 mg, at least about 130 mg, at least about 140 mg, at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, or at least about 200 mg. In some embodiments, histidine may be present in an amount of at most about 110 mg, at most about 120 mg, at most about 130 mg, at most about 140 mg, at most about 150 mg, at most about 160 mg, at most about 170 mg, at most about 180 mg, at most about 190 mg, or at most about 200 mg.WSGR Docket No. 69505-701.601

[0036] In some embodiments, the nitrogen content of the soluble fraction can comprise isoleucine, wherein isoleucine can be present in an amount of at least about 150 mg (per g Nitrogen). In some embodiments, isoleucine can be present in an amount of at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, or at least about 250 mg. In some embodiments, isoleucine can be present in an amount of at most about 150 mg, at most about 160 mg, at most about 170 mg, at most about 180 mg, at most about 190 mg, at most about 200 mg, at most about 210 mg, at most about 220 mg, at most about 230 mg, at most about 240 mg, or at most about 250 mg.

[0037] In some embodiments, the nitrogen content of the soluble fraction can comprise leucine, wherein leucine can be present in an amount of at least about 350 mg (per g Nitrogen). In some embodiments, leucine can be present in an amount of at least about 350 mg, at least about 360 mg, at least about 370 mg, at least about 380 mg, at least about 390 mg, at least about 400 mg, at least about 410 mg, at least about 420 mg, at least about 430 mg, at least about 440 mg, or at least about 450 mg. In some embodiments, leucine can be present in an amount of at most about 350 mg, at most about 360 mg, at most about 370 mg, at most about 380 mg, at most about 390 mg, at most about 400 mg, at most about 410 mg, at most about 420 mg, at most about 430 mg, at most about 440 mg, or at most about 450 mg.

[0038] In some embodiments, the nitrogen content of the soluble fraction can comprise lysine, wherein lysine can be present in an amount of at least about 450 mg (per g Nitrogen). In some embodiments, lysine is present in an amount of at least about 450 mg, at least about 460 mg, at least about 470 mg, at least about 480 mg, at least about 490 mg, at least about 500 mg, at least about 510 mg, at least about 520 mg, at least about 530 mg, at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 570 mg, or at least about 580 mg. In some embodiments, lysine can be present in an amount of at most about 450 mg, at most about 460 mg, at most about 470 mg, at most about 480 mg, at most about 490 mg, at most about 500 mg, at most about 510 mg, at most about 520 mg, at most about 530 mg, at most about 540 mg, at most about 550 mg, at most about 560 mg, at most about 570 mg, or at most about 580 mg.

[0039] In some embodiments, the nitrogen content of the soluble fraction can comprise threonine, wherein threonine can be present in an amount of at least about 200 mg (per g Nitrogen). In some embodiments, threonine can be present in an amount of at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, or at least about 330 mg. In someWSGR Docket No. 69505-701.601 embodiments, threonine can be present in an amount of at most about 200 mg, at most about 210 mg, at most about 220 mg, at most about 230 mg, at most about 240 mg, at most about 250 mg, at most about 260 mg, at most about 270 mg, at most about 280 mg, at most about 290 mg, at most about 300 mg, at most about 310 mg, at most about 320 mg, or at most about 330 mg.

[0040] In some embodiments, the nitrogen content of the soluble fraction can comprise valine, wherein valine can be present in an amount of at least about 200 mg (per g Nitrogen). In some embodiments, valine can be present in an amount of at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, or at least about 330 mg. In some embodiments, valine can be present in an amount of at most about 200 mg, at most about 210 mg, at most about 220 mg, at most about 230 mg, at most about 240 mg, at most about 250 mg, at most about 260 mg, at most about 270 mg, at most about 280 mg, at most about 290 mg, at most about 300 mg, at most about 310 mg, at most about 320 mg, or at most about 330 mg.

[0041] In some embodiments, the nitrogen content of the soluble fraction can comprise methionine, wherein methionine can be present in an amount of at least about 30 mg (per g Nitrogen). In some embodiments, methionine can be present in an amount of at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 55 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 85 mg, at least about 90 mg, or at least about 95 mg. In some embodiments, methionine can be present in an amount of at most about 30 mg, at most about 35 mg, at most about 40 mg, at most about 45 mg, at most about 50 mg, at most about 55 mg, at most about 60 mg, at most about 65 mg, at most about 70 mg, at most about 75 mg, at most about 80 mg, at most about 85 mg, at most about 90 mg, or at most about 95 mg.

[0042] In some embodiments, the nitrogen content of the soluble fraction can comprise phenylalanine, wherein phenylalanine can be present in an amount of at least about 200 mg (per g Nitrogen). In some embodiments, phenylalanine can be present in an amount of at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, or at least about 330 mg. In some embodiments, phenylalanine can be present in an amount of at most about 200 mg, at most about 210 mg, at most about 220 mg, at most about 230 mg, at most about 240 mg, at most about 250 mg, at most about 260 mg, at most about 270 mg, at most about 280 mg, at most about 290 mg, at most about 300 mg, at most about 310 mg, at most about 320 mg, or at most about 330 mg.WSGR Docket No. 69505-701.601

[0043] In some embodiments, the nitrogen content of the soluble fraction can comprise tyrosine, wherein tyrosine can be present in an amount of at least about 150 mg (per g Nitrogen). In some embodiments, tyrosine can be present in an amount of at least about 150 mg, at least about 160 mg, at least about 170 mg, at least about 180 mg, at least about 190 mg, at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, or at least about 250 mg. In some embodiments, tyrosine can be present in an amount of at most about 150 mg (per g Nitrogen). In some embodiments, isoleucine is present in an amount of at most about 150 mg, at most about 160 mg, at most about 170 mg, at most about 180 mg, at most about 190 mg, at most about 200 mg, at most about 210 mg, at most about 220 mg, at most about 230 mg, at most about 240 mg, or at most about 250 mg.

[0044] In some embodiments, the nitrogen content of the soluble fraction can comprise arginine, wherein arginine can be present in an amount of at least about 480 mg (per g Nitrogen). In some embodiments, arginine can be present in an amount of at least about 480 mg, at least about 490 mg, at least about 500 mg, at least about 510 mg, at least about 520 mg, at least about 530 mg, at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 570 mg, at least about 580 mg, at least about 590 mg, at least about 600 mg, at least about 610 mg, at least about 620 mg, at least about 630 mg, at least about 640 mg, or at least about 650 mg. In some embodiments, arginine can be present in an amount of at most about 480 mg, at most about 490 mg, at most about 500 mg, at most about 510 mg, at most about 520 mg, at most about 530 mg, at most about 540 mg, at most about 550 mg, at most about 560 mg, at most about 570 mg, at most about 580 mg, at most about 590 mg, at most about 600 mg, at most about 610 mg, at most about 620 mg, at most about 630 mg, at most about 640 mg, or at most about 650 mg.

[0045] In some embodiments, the nitrogen content of the soluble fraction can comprise alanine, wherein alanine can be present in an amount of at least about 250 mg (per g Nitrogen). In some embodiments, alanine can be present in an amount of at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, at least about 330 mg, at least about 340 mg, at least about 350 mg, at least about 360 mg, at least about 370 mg, or at least about 380 mg. In some embodiments, alanine can be present in an amount of at most about 250 mg, at most about 260 mg, at most about 270 mg, at most about 280 mg, at most about 290 mg, at most about 300 mg, at most about 310 mg, at most about 320 mg, at most about 330 mg, at most about 340 mg, at most about 350 mg, at most about 360 mg, at most about 370 mg, or at most about 380 mg.

[0046] In some embodiments, the nitrogen content of the soluble fraction can comprise aspartic acid, wherein aspartic acid can be present in an amount of at least about 700 mg (per g Nitrogen). InWSGR Docket No. 69505-701.601 some embodiments, aspartic acid can be present in an amount of at least about 700 mg, at least about 710 mg, at least about 720 mg, at least about 730 mg, at least about 740 mg, at least about 750 mg, at least about 760 mg, at least about 770 mg, at least about 780 mg, at least about 790 mg, at least about 800 mg, at least about 810 mg, at least about 820 mg, at least about 830 mg, at least about 840 mg, at least about 850 mg, at least about 860 mg, at least about 870 mg, at least about 880 mg, at least about 890 mg, at least about 900 mg, at least about 910 mg, at least about 920 mg, at least about 930 mg, at least about 940 mg, or at least about 950 mg. In some embodiments, aspartic acid can be present in an amount of at most about 700 mg, at most about 710 mg, at most about 720 mg, at most about 730 mg, at most about 740 mg, at most about 750 mg, at most about 760 mg, at most about 770 mg, at most about 780 mg, at most about 790 mg, at most about 800 mg, at most about 810 mg, at most about 820 mg, at most about 830 mg, at most about 840 mg, at most about 850 mg, at most about 860 mg, at most about 870 mg, at most about 880 mg, at most about 890 mg, at most about 900 mg, at most about 910 mg, at most about 920 mg, at most about 930 mg, at most about 940 mg, or at most about 950 mg.

[0047] In some embodiments, the nitrogen content of the soluble fraction can comprise glutamic acid, wherein glutamic acid can be present in an amount of at least about 1000 mg (per g Nitrogen). In some embodiments, glutamic acid can be present in an amount of at least about 1000 mg, at least about 1100 mg, at least about 1120 mg, at least about 1140 mg, at least about 1160 mg, at least about 1180 mg, at least about 1200 mg, at least about 1220 mg, at least about 1240 mg, at least about 1260 mg, at least about 1280 mg, at least about 1300 mg, at least about 1320 mg, at least about 1340 mg, at least about 1360 mg, at least about 1380 mg, or at least about 1400 mg. In some embodiments, glutamic acid can be present in an amount of at most about 1000 mg, at most about 1100 mg, at most about 1120 mg, at most about 1140 mg, at most about 1160 mg, at most about 1180 mg, at most about 1200 mg, at most about 1220 mg, at most about 1240 mg, at most about 1260 mg, at most about 1280 mg, at most about 1300 mg, at most about 1320 mg, at most about 1340 mg, at most about 1360 mg, at most about 1380 mg, or at most about 1400 mg.

[0048] In some embodiments, the nitrogen content of the soluble fraction can comprise glycine, wherein glycine can be present in an amount of at least about 200 mg (per g Nitrogen). In some embodiments, glycine can be present in an amount of at least about 200 mg, at least about 210 mg, at least about 220 mg, at least about 230 mg, at least about 240 mg, at least about 250 mg, at least about 260 mg, at least about 270 mg, at least about 280 mg, at least about 290 mg, at least about 300 mg, at least about 310 mg, at least about 320 mg, at least about 330 mg, at least about 340 mg, at least about 350 mg, at least about 360 mg, at least about 370 mg, at least about 380 mg, at least about 390 mg, or at least about 400 mg. In some embodiments, glycine can be present in an amount of at most aboutWSGR Docket No. 69505-701.601200 mg, at most about 210 mg, at most about 220 mg, at most about 230 mg, at most about 240 mg, at most about 250 mg, at most about 260 mg, at most about 270 mg, at most about 280 mg, at most about 290 mg, at most about 300 mg, at most about 310 mg, at most about 320 mg, at most about 330, at most about 340, at most about 350, at most about 360, at most about 370, at most about 380, at most about 390, or at most about 400mg.

[0049] In some embodiments, the soluble fraction derived from a leguminous plant may comprise one or more minerals. In some embodiments, an amount of one or more minerals present in the soluble fraction derived from a leguminous plant may be measured using any suitable quantification method, including, but not limited to, inductively coupled plasma optical emission spectroscopy (ICP-OES), inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectroscopy (AAS), or X-ray fluorescence (XRF), colorimetric methods (e.g., molybdenum blue methods or calcium and magnesium colorimetric assays), or ion chromatography (IC).

[0050] In some embodiments, the one or more mineral present in the soluble fraction can comprise potassium, sodium, magnesium, calcium, phosphorus, sulfur, iron, zinc, copper, manganese or molybdenum. In some embodiments, potassium can be present in an amount from about 5000 mg to about 7000 mg (mg per 100 g product). For example, in some embodiments, potassium can be present in an amount of at least about 5000 mg, at least about 5500 mg, at least about 6000 mg, at least about 6500 mg. In some embodiments, sodium can be present in an amount from about 1000 mg to about 3000 mg (mg per 100 g product). For example, in some embodiments, sodium can be present in an amount of at least about 1000 mg, at least about 1500 mg, at least about 2000 mg, or at least about 2500 mg. In some embodiments, magnesium can be present in an amount from about 300 mg to about 600 mg (mg per 100 g product). For example, in some embodiments, magnesium can be present in an amount of at least about 300 mg, at least about 400 mg, or at least about 500 mg. In some embodiments, calcium can be present in an amount from about 100 mg to about 400 mg (mg per 100 g product). For example, in some embodiments, calcium can be present in an amount of at least about 100 mg, at least about 200 mg, or at least about 300 mg. In some embodiments, phosphorus can be present in an amount from about 500 mg to about 1000 mg (mg per 100 g product). For example, in some embodiments, phosphorus can be present in an amount of at least about 500 mg, at least about 600 mg, at least about 700 mg, at least about 800 mg, or at least about 900 mg. In some embodiments, sulfur can be present in an amount from about 300 mg to about 800 mg (mg per 100 g product). For example, in some embodiments, sulfur can be present in an amount of at least about 300 mg, at least about 400 mg, at least about 500 mg, at least about 600 mg, or at least about 700 mg. In some embodiments, iron can be present in an amount from about 2 mg toWSGR Docket No. 69505-701.601 about 7 mg (mg per 100 g product). For example, in some embodiments, iron can be present in an amount of at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, or at least about 6 mg. In some embodiments, zinc can be present in an amount from about 2 mg to about 7 mg (mg per 100 g product). For example, in some embodiments, zinc can be present in an amount of at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, or at least about 6 mg. In some embodiments, copper can be present in an amount from about 2 mg to about 7 mg (mg per 100 g product). For example, in some embodiments, copper can be present in an amount of at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, or at least about 6 mg. In some embodiments, manganese can be present in an amount from about 2 mg to about 7 mg (mg per 100 g product). For example, in some embodiments, manganese can be present in an amount of at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, or at least about 6 mg. In some embodiments, molybdenum can be present in an amount from about 2 mg to about 7 mg (mg per 100 g product). For example, in some embodiments, molybdenum can be present in an amount of at least about 2 mg, at least about 3 mg, at least about 4 mg, at least about 5 mg, or at least about 6 mg.

[0051] In some embodiments, the soluble fraction derived from a leguminous plant may comprise ashes. In some embodiments, an amount of ashes present in the soluble fraction derived from a leguminous plant may be measured using any suitable quantification method, including, but not limited to, gravimetric methods (e.g., dry ashing), acid digestion (e.g., wet ashing), sulfated ash methods (e.g., by treating with sulfuric acid), spectroscopic methods, or thermogravimetric analysis (TGA). In some embodiments, the amount of ash present in the soluble fraction derived from a leguminous plant may be about 5 % by weight to about 26 % by weight. In some embodiments, the soluble fraction derived from a leguminous plant may have about 5 % by weight to about 7 % by weight, about 5 % by weight to about 9 % by weight, about 5 % by weight to about 10 % by weight, about 5 % by weight to about 12 % by weight, about 5 % by weight to about 14 % by weight, about 5 % by weight to about 16 % by weight, about 5 % by weight to about 18 % by weight, about 5 % by weight to about 20 % by weight, about 5 % by weight to about 22 % by weight, about 5 % by weight to about 24 % by weight, about 5 % by weight to about 26 % by weight, about 7 % by weight to about 9 % by weight, about 7 % by weight to about 10 % by weight, about 7 % by weight to about 12 % by weight, about 7 % by weight to about 14 % by weight, about 7 % by weight to about 16 % by weight, about 7 % by weight to about 18 % by weight, about 7 % by weight to about 20 % by weight, about 7 % by weight to about 22 % by weight, about 7 % by weight to about 24 % by weight, about 7 % by weight to about 26 % by weight, about 9 % by weight to about 10 % by weight, about 9 % by weight to about 12 % by weight, about 9 % by weight to about 14 % byWSGR Docket No. 69505-701.601 weight, about 9 % by weight to about 16 % by weight, about 9 % by weight to about 18 % by weight, about 9 % by weight to about 20 % by weight, about 9 % by weight to about 22 % by weight, about 9 % by weight to about 24 % by weight, about 9 % by weight to about 26 % by weight, about 10 % by weight to about 12 % by weight, about 10 % by weight to about 14 % by weight, about 10 % by weight to about 16 % by weight, about 10 % by weight to about 18 % by weight, about 10 % by weight to about 20 % by weight, about 10 % by weight to about 22 % by weight, about 10 % by weight to about 24 % by weight, about 10 % by weight to about 26 % by weight, about 12 % by weight to about 14 % by weight, about 12 % by weight to about 16 % by weight, about 12 % by weight to about 18 % by weight, about 12 % by weight to about 20 % by weight, about 12 % by weight to about 22 % by weight, about 12 % by weight to about 24 % by weight, about 12 % by weight to about 26 % by weight, about 14 % by weight to about 16 % by weight, about 14 % by weight to about 18 % by weight, about 14 % by weight to about 20 % by weight, about 14 % by weight to about 22 % by weight, about 14 % by weight to about 24 % by weight, about 14 % by weight to about 26 % by weight, about 16 % by weight to about 18 % by weight, about 16 % by weight to about 20 % by weight, about 16 % by weight to about 22 % by weight, about 16 % by weight to about 24 % by weight, about 16 % by weight to about 26 % by weight, about 18 % by weight to about 20 % by weight, about 18 % by weight to about 22 % by weight, about 18 % by weight to about 24 % by weight, about 18 % by weight to about 26 % by weight, about 20 % by weight to about 22 % by weight, about 20 % by weight to about 24 % by weight, about 20 % by weight to about 26 % by weight, about 22 % by weight to about 24 % by weight, about 22 % by weight to about 26 % by weight, or about 24 % by weight to about 26 % by weight. In some embodiments, the amount of ash present in the soluble fraction derived from a leguminous plant may be about 5 % by weight, about 7 % by weight, about 9 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 22 % by weight, about 24 % by weight, or about 26 % by weight. In some embodiments, the amount of ash present in the soluble fraction derived from a leguminous plant may be at least about 5 % by weight, about 7 % by weight, about 9 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 22 % by weight, or about 24 % by weight. In some embodiments, the amount of ash present in the soluble fraction derived from a leguminous plant may be at most about 7 % by weight, about 9 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 22 % by weight, about 24 % by weight, or about 26 % by weight.WSGR Docket No. 69505-701.601

[0052] In some embodiments, the soluble fraction derived from a leguminous plant may comprise dietary fibers. In some embodiments, an amount of dietary fibers present in the solution fraction derived from a leguminous plant may be measured using any suitable quantification method, including, but not limited to, enzymatic-gravimetric methods, enzyme-chemical methods (e.g., englyst methods), or chemical methods. In some embodiments, the amount of dietary fibers present in the soluble fraction derived from a leguminous plant may be about 0.5 % by weight to about 30 % by weight. In some embodiments, the amount of dietary fiber present in the soluble fraction derived from a leguminous plant may be at least about 0.5 % by weight, about 1 % by weight, about 5 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 22 % by weight, about 24 % by weight, about 26 % by weight, about 28 % by weight, or about 30 % by weight.. In some embodiments, the amount of dietary fiber present in the soluble fraction derived from a leguminous plant may be at most about 0.5 % by weight, about 1 % by weight, about 5 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 22 % by weight, about 24 % by weight, about 26 % by weight, about 28 % by weight, or about 30 % by weight.

[0053] In some embodiments, the soluble fraction derived from a leguminous plant may comprise lipids. In some embodiments, an amount of lipid present in the soluble fraction derived from a leguminous plant may be measured using any suitable quantification method, including, but not limited to, gravimetric methods (e.g., solvent extraction methods or soxhlet extraction methods), spectrophotometric methods (e.g., colorimetric assay, turbidimetric methods), chromatographic methods (e.g., gas chromatography, high-performance liquid chromatography (HPLC)), or enzymatic methods (e.g., lipase-based assays). In some embodiments, the amount of lipid present in the soluble fraction derived from a leguminous plant may be about 0.5 % by weight to about 30 % by weight. In some embodiments, the amount of dietary fiber present in the soluble fraction derived from a leguminous plant may be at least about 0.5 % by weight, about 1 % by weight, about 5 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 22 % by weight, about 24 % by weight, about 26 % by weight, about 28 % by weight, or about 30 % by weight. In some embodiments, the amount of lipid present in the soluble fraction derived from a leguminous plant may be at most about 1 %, about 5 % by weight, about 10 % by weight, about 12 % by weight, about 14 % by weight, about 16 % by weight, about 18 % by weight, about 20 % by weight, about 22 % by weight, about 24 % by weight, about 26 % by weight, about 28 % by weight, or about 30 % by weight.WSGR Docket No. 69505-701.601

[0054] In some embodiments, a fermentation culture medium supplement described herein can comprise a mixture of unhydrolyzed soluble fraction derived from a leguminous plant and a hydrolyzed form of the soluble fraction derived from e.g., a leguminous plant. In some embodiments, the soluble fraction of the leguminous plant described herein may undergo hydrolysis to break down complex carbohydrates, proteins, or other macromolecules into simpler, more bioavailable forms. In some embodiments, hydrolysis can be performed enzymatically, chemically, or through a combination of both methods to achieve targeted breakdown of specific components. In some embodiments, the hydrolysis can be performed using specific proteolytic enzymes, such as exoproteases, endoproteases, or a combination thereof. In some embodiments, exoproteases may include, but are not limited to, aminopeptidases and carboxypeptidases. In some embodiments, endopeptidases may include, but are not limited to, pepsin, trypsin, chymotrypsin, and elastase. In some embodiments, proteolytic enzymes may include, but are not limited to, papain, bromelain, and collagenase. In some embodiments, these enzymes can target proteins within the soluble fraction, breaking them down into smaller peptides or free amino acids, thereby enhancing the fraction’s bioavailability and functional properties. In some embodiments, specific enzymes such as amylases, cellulases, or proteases can be used to selectively break down polysaccharides into monosaccharides or di saccharides, and proteins into peptides or amino acids. In some embodiments, the ratio between the unhydrolyzed soluble fraction and the hydrolyzed form of the soluble fraction of the fermentation culture medium supplement can be 1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:2, respectively.

[0055] In some embodiments, a soluble fraction derived from a leguminous plant (e.g., unhydrolyzed or hydrolyzed soluble fraction), or a mixture of unhydrolyzed soluble and hydrolyzed form of the soluble fraction can further comprise a carrier. In some embodiments, the carrier can be added to assist in the drying process of the plant-derived soluble fraction (e.g., a soluble fraction of the leguminous plant). During drying process (e.g., spray-drying or freeze-drying), the carrier described herein can help facilitate moisture removal by reducing stickiness and enhancing the formation of a free-flowing powder. In some embodiments, the carrier can form a matrix around the plant soluble, reducing tackiness and preventing clumping. By improving the handling and flow characteristics of the dried fraction, the carrier can enhance powder yield and contributes to the stability and shelf life of the final product. For example, in some embodiments, the carrier can enhance powder yield by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.2-fold, at least about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold, at least about 2.0-fold, at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 6.0-fold, at least aboutWSGR Docket No. 69505-701.6017.0-fold, at least about 8.0-fold, at least about 9.0-fold, or at least about 10.0-fold higher than powder yield of a plant soluble undergoing the drying process (e.g., spray-drying or freeze-drying) without a carrier. In some embodiments, the carriers described herein can aid in stabilizing the active components, improving their solubility or dispersibility, and protecting them from degradation, thereby extending product shelf life. In some embodiments, the shelf life of the soluble fraction derived from a leguminous plant comprising carriers (e.g., in a solution or a dried-powder from) can be extended by at least about 1 day, at least about 7 days, at least about 2 weeks, at least about 4 weeks, at least about 2 months, at least about 4 months, at least about 6 months, at least about 8 months, at least about 10 months, at least about 12 months, at least about 1.5 years, at least about 2 years, at least about 4 years or at least about 5 years more than that of the soluble fraction derived from a leguminous plant without carriers. In some embodiments, the amount of carrier can be at least about 0.5 %, at least about 2 %, at least about 4 %, at least about 6 %, at least about 8 %, at least about 10 %, at least about 12 %, at least about 14 %, at least about 16 %, at least about 18 %, at least about 20 %, at least about 22 %, at least about 24 %, at least about 26 %, at least about 28 %, at least about 30 %, at least about 32 %, at least about 34 %, at least about 36 %, at least about 38 %, at least about 40 %, at least about 42 %, at least about 44 %, at least about 46 %, at least about 48 %, at least about 50 %, at least about 52 %, or at least about 54 % of the final product (e.g., a soluble fraction derived from a leguminous plant, a fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant, or a culture medium comprising a fermentation culture medium supplement). Additionally, the carriers described herein can contribute to consistent dosage and handling by enhancing the flow and recoverability of the powder during production. In some embodiments, the choice and amount of carrier can be optimized based on the desired characteristics of the final product, such as stability, texture, release profile, and compatibility with other ingredients, ensuring effective delivery of the active components in the soluble plant fraction. In some embodiments, the fermentation culture medium supplement can further comprise a carrier, which can facilitate the delivery, stability, and processing of active ingredients derived from the soluble fraction of a leguminous plant. Suitable carriers may include silicon dioxides, salts, sugars, or a combination thereof. In some embodiments, the carrier can be protein concentrates obtained from the leguminous plant. In some embodiments, the carrier can be a biopolymer. In some embodiments, the biopolymer can be a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, or maltodextrin.

[0056] In some embodiments, the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can provide nutrient substrates for microbial metabolism. In some embodiments, the fermentation culture medium supplement comprising a soluble fractionWSGR Docket No. 69505-701.601 derived from a leguminous plant can support growth or activity of microorganisms. The term "microorganism" as used herein refers to both individual microorganism cells and pluralities of such cells (e.g., a population of microorganisms). In some embodiments, the microorganism is a single taxonomic species. In some embodiments, the microorganism is a single strain of a species (e.g., a pure culture). In some embodiments, microorganism populations can also be consortia of microorganism cells of different strains of a common species and / or can be consortia comprising different taxonomic species or genera. In some embodiments, the microorganism may be a wild-type organism (e.g., a microorganism that is essentially unaltered from the organism that originally was isolated from nature). In some embodiments, the microorganism may be a modified microorganism (e.g., a modified microorganism having one or more mutagenesis (e.g., insertion, deletion, substitution of one or more nucleotides, one or more amino acids, or one or more functional / non- functional domains).

[0057] In some embodiments, the fermentation culture medium supplement can be added to a basal medium to produce a fermentation culture medium. As used herein, a "basal medium" or “basal culture medium” refers to a primary nutrient medium that independently provides essential nutrients required for basic microbial growth without additional supplements (e.g., yeast extracts, beef extracts, meat peptone, and / or meat extracts). In some embodiments, a basal medium can be peptone water. In some embodiments, a basal medium can be free or substantially free of glucose. In some embodiments, distilled water or water can be used as the primary medium (e.g., a basal medium) for dissolving and facilitating the availability of one or more supplements (e.g., the fermentation culture medium supplement described herein). In some embodiments, the fermentation culture medium described herein can comprise a soluble fraction derived from a leguminous plant described herein and an additional supplement (e.g., yeast extracts, beef extracts, meat peptone, and / or meat extracts). In some embodiments, the fermentation culture medium described herein can comprise a soluble fraction derived from a leguminous plant described herein without any additional supplements.

[0058] In some embodiments, the fermentation culture medium supplement (e.g., the fermentation culture medium supplement in dry powder form) can be added to a basal medium at a final concentration of about 0.05 g / L to about 60 g / L to produce the fermentation culture medium. In some embodiments, the fermentation culture medium supplement can be added to a basal medium at a final concentration of about 0.05 g / L to about 1 g / L, about 0.05 g / L to about 5 g / L, about 0.05 g / L to about 10 g / L, about 0.05 g / L to about 12.5 g / L, about 0.05 g / L to about 15 g / L, about 0.05 g / L to about 17.5 g / L, about 0.05 g / L to about 20 g / L, about 0.05 g / L to about 30 g / L, about 0.05 g / L to about 40 g / L, about 0.05 g / L to about 50 g / L, about 0.05 g / L to about 60 g / L, about 1 g / L to about 5 g / L, about 1 g / L to about 10 g / L, about 1 g / L to about 12.5 g / L, about 1 g / L to about 15 g / L, about 1 g / L to about 17.5WSGR Docket No. 69505-701.601 g / L, about 1 g / L to about 20 g / L, about 1 g / L to about 30 g / L, about 1 g / L to about 40 g / L, about 1 g / L to about 50 g / L, about 1 g / L to about 60 g / L, about 5 g / L to about 10 g / L, about 5 g / L to about 12.5 g / L, about 5 g / L to about 15 g / L, about 5 g / L to about 17.5 g / L, about 5 g / L to about 20 g / L, about 5 g / L to about 30 g / L, about 5 g / L to about 40 g / L, about 5 g / L to about 50 g / L, about 5 g / L to about 60 g / L, about 10 g / L to about 12.5 g / L, about 10 g / L to about 15 g / L, about 10 g / L to about 17.5 g / L, about 10 g / L to about 20 g / L, about 10 g / L to about 30 g / L, about 10 g / L to about 40 g / L, about 10 g / L to about 50 g / L, about 10 g / L to about 60 g / L, about 12.5 g / L to about 15 g / L, about 12.5 g / L to about 17.5 g / L, about 12.5 g / L to about 20 g / L, about 12.5 g / L to about 30 g / L, about 12.5 g / L to about 40 g / L, about 12.5 g / L to about 50 g / L, about 12.5 g / L to about 60 g / L, about 15 g / L to about 17.5 g / L, about 15 g / L to about 20 g / L, about 15 g / L to about 30 g / L, about 15 g / L to about 40 g / L, about 15 g / L to about 50 g / L, about 15 g / L to about 60 g / L, about 17.5 g / L to about 20 g / L, about 17.5 g / L to about 30 g / L, about 17.5 g / L to about 40 g / L, about 17.5 g / L to about 50 g / L, about 17.5 g / L to about 60 g / L, about 20 g / L to about 30 g / L, about 20 g / L to about 40 g / L, about 20 g / L to about 50 g / L, about 20 g / L to about 60 g / L, about 30 g / L to about 40 g / L, about 30 g / L to about 50 g / L, about 30 g / L to about 60 g / L, about 40 g / L to about 50 g / L, about 40 g / L to about 60 g / L, or about 50 g / L to about 60 g / L. In some embodiments, the fermentation culture medium supplement can be added to a basal medium at a final concentration of about 0.05 g / L, about 1 g / L, about 5 g / L, about 10 g / L, about 12.5 g / L, about 15 g / L, about 17.5 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, or about 60 g / L. In some embodiments, the fermentation culture medium supplement can be added to a basal medium at a final concentration of at least about 0.05 g / L, about 1 g / L, about 5 g / L, about 10 g / L, about 12.5 g / L, about 15 g / L, about 17.5 g / L, about 20 g / L, about 30 g / L, about 40 g / L, or about 50 g / L. In some embodiments, the fermentation culture medium supplement can be added to a basal medium at a final concentration of at most about 1 g / L, about 5 g / L, about 10 g / L, about 12.5 g / L, about 15 g / L, about 17.5 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, or about 60 g / L.

[0059] In some embodiments, the fermentation culture medium comprising the fermentation culture medium supplement described herein (e.g., a soluble fraction of leguminous plant) can be free or substantially free of glucose. In some embodiments, the glucose level (e.g., concentration) of the fermentation culture medium comprising the fermentation culture medium supplement described herein (e.g., a soluble fraction of leguminous plant) can be at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower.

[0060] In some embodiments, the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can support growth or activity of microorganisms involvedWSGR Docket No. 69505-701.601 in the agricultural application. For example, in some embodiments, the microorganisms can enhance soil fertility, plant growth promotion, or crop protection. In some embodiments, the microorganisms can be nitrogen-fixing bacteria. In some embodiments, the microorganism can be plant growthpromoting bacteria. In some embodiments, the microorganisms can be any of the genera Bradyrhizobium, Azospirillum, Pseudomonas, Bacillus, Trichoderma, Streptomyces, Actinomyces, Micromonospora, or Metarhizium.

[0061] In some embodiments, the microorganisms can be one of the following species: In the genus Bradyrhizobium, species include, but are not limited to, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium diazoefficiens . In the genus Rhizobium, species can include, but are not limited to, Rhizobium leguminosarum and Rhizobium etli. In the genus, Azospirillum, species can include, but are not limited to, Azospirillum brasilense and Azospirillum lipoferum. The genus Pseudomonas can include, but are not limited to, Pseudomonas fluorescens and Pseudomonas putida. The Bacillus genus can include, but are not limited to, Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus pumilus, Bacillus licheniformis, and Bacillus velezensis. In the genus Trichoderma, species can include, but are not limited to, Trichoderma harzianum and Trichoderma viride. In the Streptomyces genus, species can include, but are not limited to, Streptomyces griseus and Streptomyces lydicus. In the genus Actinomyces, species can include, but are not limited to, Actinomyces israelii, and Micromonospora by Micromonospora lupini. The genus Metarhizium can include, but are not limited to, Metarhizium anisopliae, Metarhizium robertsii, Metarhizium brunneum, Metarhizium flavoviride, and Metarhizium guizhouense.

[0062] In some embodiments, the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can support growth or activity of microorganisms with probiotic benefits for human and animal consumption. For example, in some embodiments, the fermentation culture medium supplement can be utilized for culturing probiotic microorganisms to support efficient fermentation processes. In some embodiments, the fermentation culture medium supplement can be utilized for culturing probiotic microorganisms to support efficient fermentation processes in food products. For example, in some embodiments, the food products can be dairybased fermented products such as yogurt, milk, or fermented cheese varieties. In some embodiments, the food products can be fermented vegetables and condiments (e.g., sauerkraut, miso, pickles, or kimchi). In some embodiments, the food product can be fermented beverages (e.g., kombucha). In some embodiments, the food product can be fermented meats and cured products (e.g., salami). In some embodiments, the fermentation culture medium supplement can support growth and activity of probiotic microorganisms selected to support growth and activity under various fermentationWSGR Docket No. 69505-701.601 conditions. In some embodiments, the microorganisms can be any of the genera Lactobacillus, Bifidobacterium, Enterococcus, Streptococcus, Lactococcus, Bacillus, or Propionibacterium.

[0063] In some embodiments, the microorganisms can be one of the following species: In the genus Lactobacillus, species can include, but are not limited to, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus reuteri, and Lactobacillus bulgaricus. In the genus Bifidobacterium, species can include, but are not limited to, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, and Bifidobacterium bifidum. In the genus Enterococcus, species can include, but are not limited to, Enterococcus faecium. In the genus Streptococcus, species can include, but are not limited to, Streptococcus thermophilus. In the genus Lactococcus, species can include, but are not limited to, Lactococcus lactis subsp. lactis. In the genus Bacillus, species can include, but are not limited to, Bacillus coagulans and Bacillus subtilis. In the genus Propionibacterium, species can include, but are not limited to, Propionibacterium freudenreichii .

[0064] In some embodiments, the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can support growth or activity of industrial fermentation microorganisms. In some embodiments, the fermentation culture medium supplement can be utilized for culturing microorganisms for industrial fermentation processes, including, for example, enzyme production or bioactive compound synthesis. In some embodiments, the fermentation culture medium supplement can enhance microbial growth and metabolic productivity. The microorganisms, for example, can include Escherichia coli (E. coli), or yeast, Saccharomyces, which can include, but are not limited to, Saccharomyces pastorianus and Saccharomyces cerevisiae. In some embodiments, the microorganism can be Aspergillus, which includes, but are not limited to, Aspergillus niger and Aspergillus oryzae. In some embodiments, the microorganisms can be Fusarium venenatum, Bacillus licheniformis, Trichoderma reesei, Pichia pastoris, Corynebacterium glutamicum, Candida antarctica, o Micrococcus luteus.

[0065] In some embodiments, the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can enhance or promote growth or activity of microorganisms. In some embodiments, microorganisms grown in a fermentation culture medium comprising a fermentation culture medium supplement described herein (e.g., a fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant) can significantly enhances the growth rate, metabolic activity, and functional properties as compared to that of microorganisms grown in a control culture medium. For example, in some embodiments, a control culture medium can be a basal culture medium, a basal culture medium comprising one or moreWSGR Docket No. 69505-701.601 additional supplements (e.g., yeast extracts, beef extracts, meat peptone, and / or meat extracts), a fermentation culture medium lacking the fermentation culture medium supplement described herein (e.g., the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant), or a culture medium comprising a fermentation culture medium supplement lacking a soluble fraction derived from a leguminous plant. The fermentation culture medium supplement comprising a solution fraction derived from a leguminous plant can be used in various industrial applications, such as the production of biofuels, pharmaceuticals, food and beverage products, and agricultural products. By incorporating a naturally derived, carbohydrate-rich supplement, the fermentation culture medium supplement can provide a sustainable and efficient solution for optimizing microbial-based fermentation systems.

[0066] In some embodiments, growing microorganisms in the presence of the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can result in an increase in population of microorganisms (e.g., increase in proliferation of the microorganisms). For example, in some embodiments, microorganisms grown in the presence of the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can increase the population of microorganisms by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold or more compared to that of the microorganisms grown in the control culture medium (e.g., a culture medium lacking the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant or a culture medium comprising one or more additional supplements such as beef extracts, yeast extracts, meat peptone, and / or meat extracts). In some embodiments, the growth rate of microorganisms grown in the presence of the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10- fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold or more compared to that of the microorganisms grown in the control culture medium (e.g., a fermentation culture medium lacking the fermentation culture medium supplement comprising a soluble fractionWSGR Docket No. 69505-701.601 derived from a leguminous plant). In some embodiments, the biomass yield of the microorganisms grown in the presence of the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15- fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold or more compared to that of the microorganisms grown in the control culture medium (e.g., a fermentation culture medium lacking the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant). As used herein, “biomass yield” refers to the total amount of microbial mass produced during the growth of a microorganism in a culture medium. It is typically measured as dry weight (e.g., grams per liter) or cell density (e.g., optical density at 600 nm, or colony -forming units per milliliter). In some embodiments, the activity of microorganisms grown in the presence of the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold or more compared to that of the microorganisms grown in the control culture medium (e.g., a fermentation culture medium lacking the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant). As used herein, "activity of microorganisms" refers to the rate and efficiency at which microorganisms perform their metabolic functions or biochemical processes. This can include, but not limited to, the production of enzymes, metabolites or bioactive compounds, nutrient assimilation, and interactions with their environment, such as nitrogen fixation, organic acid production, or pathogen inhibition. In some embodiments, various methods can measure changes in the microbial growth and activity. For example, in some embodiments, the growth rate and biomass yield can be quantified by monitoring optical density (OD) (e.g., at 600 nm in a spectrophotometer), where an increase in OD can reflect a higher cell density. In some embodiments, plate counts (colony -forming units, CFU) can be used to measure viable cell numbers. In some embodiments, microbial activity can be assessed by measuring specific metabolites or enzymes produced by the microorganism. For example, lactic acid bacteria may show increased lactic acid production, measurable by high-performance liquid chromatographyWSGR Docket No. 69505-701.601(HPLC). In some embodiments, enzyme activity can be assessed through spectrophotometric assays, where an increase in enzyme units (U / mL) signifies elevated activity.

[0067] In some embodiments, growing microorganisms in the presence of the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can result in an increase in sporulation yield. For example, in some embodiments, microorganisms grown in the presence of the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can induce sporulation in microorganisms. In some embodiments, sporulating microorganisms include, but are not limited to, the genus bacillus spp. For example, sporulating microorganisms can comprise Bacillus subliHs. Bacillus thuringiensis. Bacillus amyloliquefaciens, Bacillus megalerium. Bacillus pumilus, Bacillus Ucheniformis. and Bacillus velezensis. In some embodiments, microorganisms grown in the presence of the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can result in an increase in sporulation yield of the microorganisms by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.5-fold, at least about 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold, at least about 100-fold or more compared to that of microorganisms grown in the control culture medium (e.g., a culture medium lacking the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant or a culture medium comprising yeast extracts, beef extracts, meat extracts, and / or meat peptone).Method of Making a Fermentation Culture Medium Supplement

[0068] Provided herein are methods of making the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant. In some embodiments, the methods can comprise an extraction process and at least one filtration step to extract the soluble fraction from a leguminous plant. In some embodiments, the methods can further comprise a concentration step. In some embodiments, the methods can further comprise a storing step (e.g., a spontaneous fermentation process or a controlled fermentation process). In some cases, the fermentation step can be spontaneous. In some cases, the fermentation step can be controlled. In some cases, the fermentation step can control or regulate the one or more compositions (e.g., glucose) of the solution fraction derived from a leguminous plant.WSGR Docket No. 69505-701.601

[0069] In some embodiments, a leguminous plant can be yellow pea (Pisum sativum), chickpea (Cicer arietinum), mung bean (Vigna radiala), white bean, pinto bean, red kidney bean (Phaseolus vulgaris), lentil (Vicia lens o Lens culinaris, black bean (Castanospermum ubter), cowpea or caupi bean (Vigna unguiculata), broad bean (Vicia faba), aduki bean (Vigna angularis), soybean (Glycine max), green pea (Pisum sativum var. arvense), lupin (Lupinus albus, Lupinus luteus), pigeon pea (Cajanus cajan), fenugreek (Trigonella foenum-graecum), grass pea (Lathyrus sativus), bambara groundnut Vigna subterranean), velvet bean (Mucuna pruriens), winged bean (Psophocarpus tetragonolobus), or jack bean (Canavalia ensiformis). In some embodiments, a leguminous plant can be a renewable raw material. In some embodiments, the soluble fraction can be derived from a combination of one or more leguminous plants disclosed herein. For example, in some cases, the soluble fraction can be derived by combining at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more leguminous plants disclosed herein.

[0070] In some embodiments, the methods described herein utilize upcycled byproducts (e.g., of a leguminous plant) from the food and agricultural industry. In some embodiments, the method can comprise providing or obtaining a leguminous plant or leguminous plant materials. In some embodiments, the method can comprise providing or obtaining a leguminous plant or leguminous plant materials and extracting a soluble fraction of the leguminous plant. In some embodiments, the providing or obtaining a leguminous plant or leguminous plant materials can comprise providing or obtaining plant flour (e.g, soybean flour or pea flour), dried plant powders, or fresh plant biomass.

[0071] In some embodiments, the method can further comprise mixing the plant materials (e.g, leguminous plant flour) into an aqueous solution to extract a soluble fraction of the leguminous plant. In some embodiments, the aqueous solution can be water. In some embodiments, the aqueous solution can be hot water, cold water, or room-temperature water. In some embodiments, the temperature of aqueous solution (e.g., water) can be adjusted to be at least about 30 °C, at least about 35 °C, at least about 40 °C, at least about 45 °C, at least about 50 °C, at least about 55 °C, at least about 60 °C, at least about 65 °C, at least about 70 °C, at least about 75 °C, at least about 80 °C, at least about 85 °C, at least about 90 °C, at least about 95 °C, or at least about 100 °C. In some embodiments, the aqueous solution (e.g., comprising the plant materials) can undergo alkaline extraction. In some embodiments, the aqueous solution can be further pH adjusted. In some embodiments, pH of the aqueous solution can be adjusted to be at least about pH 7.5, at least about pH 8.0, at least about pH 8.1, at least about pH 8.2, at least about pH 8.3, at least about pH 8.4, at least about pH 8.5, at least about pH 8.6, at least about pH 8.7, at least about pH 8.8, at least about pH 8.9, at least about pH 9.0, at least about pH 9.5, or at least about pH 10.0. In some embodiments,WSGR Docket No. 69505-701.601 the aqueous solution can be an aqueous-solvent extraction solution. In some embodiments, the aqueous-solvent extraction solution can comprise ethanol. In some embodiments, the aqueous- solvent extraction solution can be a solution comprising ethanol and water. In some embodiments, aqueous-solvent extraction solution can comprise a solution, wherein the solution comprises an ethanol to water ratio of 1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9: 1, respectively. In some embodiments, the aqueous-solvent extraction solution comprises the ethanol to water ratio of 6:4, respectively. In some embodiments, pH of the aqueous-solvent extraction solution can be adjusted. In some embodiments, the aqueous-solvent extraction solution can be used without adjusting the pH of the aqueous-solvent extraction solution. In some embodiments, when the aqueous-solvent extraction solution is used, the soluble fraction can be used without an additional filtration or purification steps (e.g., an additional filtration step to remove the insoluble fraction comprising the heavy phase protein slurry).

[0072] In some embodiments, the method further can comprise decanting, purifying, or filtering the aqueous solution comprising a soluble fraction of the leguminous plant. In some embodiments, decanting, purifying, or filtering the aqueous solution can comprise collecting the soluble fraction (in the aqueous solution) in a tank. In some embodiments, insoluble fraction (e.g., wet starch and fibers) can be dried and further processed to obtain dry powders.

[0073] In some embodiments, the pH of the collected soluble fraction (in the aqueous solution) can be further adjusted. In some embodiments, pH of the collected soluble fraction (in the aqueous solution) can be adjusted to at least about pH 2.0, at least about pH 2.5, at least about pH 3.0, at least about pH 3.5, at least about pH 4.0, at least about pH 4.1, at least about pH 4.2, at least about pH 4.3, at least about pH 4.4, at least about pH 4.5, at least about pH 4.6, at least about pH 4.7, at least about pH 4.8, at least about pH 4.9, at least about pH 5.0, at least about pH 5.5, at least about pH 6.0, or at least about pH 6.5. In some embodiments, the collected soluble fraction (in the aqueous solution) can be further decanted, purified, or filtered.

[0074] In some embodiments, the insoluble fraction (e.g., the heavy phase protein slurry) can be collected and further processed to obtain protein concentrate powder. For example, the insoluble fraction can be further processed by evaporation to increase the solid contents at least about 10 %, at least about 15 %, at least about 20 %, at least about 30 %, at least about 40%, at least about 45 %, at least about 45 %, or at least about 50 %. In some embodiments, the protein concentrate powder can be further used as a carrier. In some embodiments, at least one by product (e.g., insoluble fraction) can comprise a protein concentrate (e.g., a pea protein concentrate). In some embodiments, the protein concentrate can comprise globulins. In some embodiments, the protein concentrate can be further processed for food industry applications, cosmetics, or chemicals.WSGR Docket No. 69505-701.601

[0075] In some embodiments, the collected soluble fraction e.g., the liquid phase) disclosed herein can be concentrated. In some embodiments, the collected soluble fraction disclosed herein can undergo one or more concentration steps. In some embodiments, one or more concentration steps can include, but are not limited to, evaporation, ultrafiltration, or precipitation. In some embodiments, following concentration, the soluble fraction can exhibit a total solid content that is at least about 5 %, at least about 10 %, at least about 15 %, at least about 20 %, at least about 25 %, at least about 30 %, at least about 35 %, at least about 40 %, at least about 45 %, at least about 50 % (w / w) or more. In some embodiments, following concentration, the soluble fraction can exhibit a total solid content that is at most about 5 %, at most about 10 %, at most about 15 %, at most about 20 %, at most about 25 %, at most about 30 %, at most about 35 %, at most about 40 %, at most about 45 %, at most about 50 % (w / w,) or less.

[0076] In some embodiments, the collected soluble fraction (e.g., the liquid phase) can be maintained under storage conditions. In some cases, during the storage phase, the soluble fraction may undergo fermentation. In some embodiments, the fermentation can be a spontaneous fermentation process. In some embodiments, the fermentation can be a controlled fermentation process. In some embodiments, the collected soluble fraction can be stored before the concentration step. In some embodiments, the collected soluble fraction can be stored after the concentration step. In some embodiments, the collected soluble fraction can be maintained under storage conditions at ambient temperature. In some cases, the ambient temperature can be about 15 °C, about 20 °C, about 22 °C, about 24 °C, about 26 °C, about 28 °C, or about 30 °C. In some embodiments, the collected soluble fraction may be maintained under storage conditions for at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 1.5 days, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 10 days, at least about 14 days, or more. In some embodiments, the collected soluble fraction may be maintained under storage conditions for at most about 6 hours, at most about 12 hours, at most about 18 hours, at most about 24 hours, at most about 1.5 days, at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 10 days, at most about 14 days, or less.

[0077] In some embodiments, the glucose level of the aqueous solution comprising a soluble fraction of the leguminous plant, upon treatment with one or methods described herein (e.g., enzymatic treatments, chemical treatments, or fermentation process), can be reduced or substantially eliminated. In some embodiments, the amount of glucose level present in the aqueous solution comprising a soluble fraction of the leguminous plant can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at leastWSGR Docket No. 69505-701.601 about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 95%, or about 100% as compared to the glucose level of a starting aqueous solution comprising a soluble fraction of the leguminous plant (e.g., prior to the one or more treatments described herein). In some embodiments, the aqueous solution comprising a soluble fraction of the leguminous plant, upon treatment with one or more methods described herein, can be free or substantially free of glucose (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower). In some embodiments, the aqueous solution comprising a soluble fraction of the leguminous plant, upon treatment with one or more methods described herein, can be free or substantially free of raffinose (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower). In some embodiments, the aqueous solution comprising a soluble fraction of the leguminous plant, upon treatment with one or more methods described herein, can be free or substantially free of fructose (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower).

[0078] In some embodiments, the method can further comprise a formulation stage. In some embodiments, the formulation stage can comprise pH adjustment, the addition of one or more carriers (e.g., maltodextrin), and / or hydrolysis. In some embodiments, pH of the final product can be adjusted to about pH 7.5, about pH 8.0, or about pH 8.5. In some embodiments, one or more carriers can include protein concentrates obtained from the leguminous plant. In some embodiments, the one or more excipients or binder can include, but is not limited to, silicon dioxide, maltodextrin, calcium silicate, polyvinylpyrrolidone (PVP), magnesium stearate, trehalose, hydroxypropyl methylcellulose (HPMC), or microcrystalline cellulose (MCC).

[0079] In some embodiments, the formulation step of the soluble fraction (or dry powders comprising a soluble fraction) of the leguminous plant described herein can include a hydrolysis step. For example, one or more protease enzymes can be added to the soluble fraction derived from a leguminous plant to hydrolyze or to break down complex carbohydrates, proteins, or other macromolecules into simpler, more bioavailable forms. In some embodiments, hydrolysis can beWSGR Docket No. 69505-701.601 performed enzymatically, chemically, or through a combination of both methods to achieve targeted breakdown of specific components. In some embodiments, the hydrolysis can be performed using specific proteolytic enzymes, such as exoproteases, endoproteases, or a combination thereof. In some embodiments, these enzymes can target proteins within the soluble fraction, breaking them down into smaller peptides or free amino acids, thereby enhancing the fraction’s bioavailability and functional properties. In some embodiments, specific enzymes such as amylases, cellulases, or proteases can be used to selectively break down polysaccharides into monosaccharides or disaccharides, and proteins into peptides or amino acids.

[0080] In some embodiments, the aqueous solution comprising a soluble fraction of the leguminous plant can undergo additional processing (e.g., treatments, purification, or modifications) to reduce or eliminate one or more carbohydrates. For example, in some embodiments, the aqueous solution comprising a soluble fraction of the leguminous plant can undergo additional processing such that the aqueous solution comprising a soluble fraction of the leguminous plant can be free or substantially free of one or more carbohydrates.

[0081] In some embodiments, the methods described herein (e.g., additional processing) comprising modifying, treating, or purifying the aqueous solution comprising a soluble fraction of the leguminous plant can achieve low glucose level. For example, the method can comprise the aqueous solution comprising a soluble fraction of the leguminous plant with one or more enzymatic treatments (e.g., glucose oxidase or catalase), filtrations (e.g., membrane filtration or ultra-filtration), chemical treatments (e.g., chemical treatment that neutralize glucose), activated carbon adsorptions, chromatography (e.g., ion-exchange chromatography or size-exclusion chromatography), or others (e.g., antibody that binds to glucose, chemical precipitation, or adsorption) that selectively reduce glucose level of the aqueous solution comprising a soluble fraction of the leguminous plant. In some embodiments, one or methods described herein (e.g., enzymatic treatments, chemical treatments, or fermentation process), can reduce the amount of glucose level present in the aqueous solution comprising a soluble fraction of the leguminous plant by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 95%, or about 100% as compared to the glucose level of a starting aqueous solution comprising a soluble fraction of the leguminous plant (e.g., prior to the one or more treatments described herein).

[0082] In some embodiments, the aqueous solution comprising a soluble fraction of the leguminous plant can undergo additional processing such that the soluble fraction can be free or substantially free of glucose (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, atWSGR Docket No. 69505-701.601 most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower).

[0083] In some embodiments, the aqueous solution comprising a soluble fraction of the leguminous plant can undergo additional processing such that the soluble fraction is free or substantially free of raffinose (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower).

[0084] In some embodiments, the aqueous solution comprising a soluble fraction of the leguminous plant can undergo additional processing such that the soluble fraction is free or substantially free of fructose (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower).

[0085] In some embodiments, methods described herein (e.g., additional processing) can comprise modifying, treating, or purifying the aqueous solution comprising a soluble fraction of the leguminous plant to achieve low fructose level. For example, the method can comprise treating the aqueous solution comprising a soluble fraction of the leguminous plant with one or more enzymatic treatments (e.g., fructokinase, hexokinase, or fructose isomerase), filtrations (e.g., membrane filtration or ultra-filtration), chemical treatments (e.g., chemical treatment that neutralize fructose), activated carbon adsorptions, chromatography (e.g., ion-exchange chromatography or size-exclusion chromatography), or others (e.g., antibody that binds to fructose, chemical precipitation, or adsorption) that selectively reduce fructose level of the fermentation culture medium or supplement. In some embodiments, the fructose level of the fermentation culture medium or supplement, upon treatment with one or methods described herein (e.g., enzymatic treatments or chemical treatments) can reduce an amount of fructose level present in the fermentation culture medium or supplements by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 95%, or about 100% as compared to the fructose level of a starting fermentation culture medium or supplements (e.g., prior to the one or more treatments described herein).WSGR Docket No. 69505-701.601

[0086] In some embodiments, methods described herein (e.g., additional processing) can comprise modifying, treating, or purifying the aqueous solution comprising a soluble fraction of the leguminous plant to achieve low raffinose level (e.g., at most about 5 g / L, at most about 4 g / L, at most about 3 g / L, at most about 2 g / L, at most about 1 g / L, at most about 0.9 g / L, at most about 0.8 g / L, at most about 0.7 g / L, at most about 0.6 g / L, at most about 0.5 g / L, at most about 0.4 g / L, at most about 0.3 g / L, at most about 0.2 g / L, at most about 0.1 g / L, at most about 0.05 g / L or lower). For example, the method can comprise treating the aqueous solution comprising a soluble fraction of the leguminous plant with one or more enzymatic treatments (e.g., alpha-galactosidase, invertase in combination with alpha-galactosidase), filtrations (e.g., membrane filtration or ultra-filtration), chemical treatments (e.g., chemical treatment that neutralize raffinose), activated carbon adsorptions, chromatography (e.g., ion-exchange chromatography or size-exclusion chromatography), or others (e.g., antibody that binds to raffinose, chemical precipitation, or adsorption) that selectively reduce raffinose level of the fermentation culture medium or supplement. In some embodiments, the raffinose level of the fermentation culture medium or supplements, upon treatment with one or methods described herein (e.g., enzymatic treatment or chemical treatments) can reduce an amount of raffinose level present in the fermentation culture medium or supplements by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 95%, or about 100% as compared to the raffinose level of a starting fermentation culture medium or supplements (e.g., prior to the one or more treatments described herein).

[0087] In some embodiments, the soluble fraction can be further processed to obtain a dry powder. For example, the soluble fraction can be subjected to a drying process, wherein the drying process comprises spray drying process, freeze-drying (e.g., lyophilization) process, drum drying process, fluidized bed drying process, or vacuum drying process. In some embodiments, upon processing by spray drying, freeze-drying, drum drying process, the soluble fraction can form dry powders. In some embodiments, the dry powders of the soluble fraction can be characterized by a moisture content of less than about 50 %, less than about 40 %, less than about 30 %, less than about 20 %, less than about 10 %, less than about 9 %, less than about 8 %, less than about 7 %, less than about 6 %, less than about 5 %, less than about 4 %, less than about 3 %, less than about 2 %, or less than about 1 %. In some embodiments, the soluble fraction or dry powders comprising a soluble fraction of the leguminous plant described herein can further include a powder stabilization step, wherein the powder stability step can comprise adding one or more excipients or binders to improve flowability, reducing hygroscopicity, or increasing thermal stability.WSGR Docket No. 69505-701.601Fermentation cell culture medium

[0088] Provided herein are methods of making fermentation cell culture medium (e.g., a fermentation cell culture medium comprising a soluble fraction derived from a leguminous plant) for supporting growth and activity of microorganisms. In some embodiments, a fermentation cell culture medium can support growth and activity of microorganisms, wherein the fermentation cell culture medium can comprise a basal culture medium and a soluble fraction of leguminous plant. In some embodiments, a fermentation cell culture medium can support growth and activity of microorganisms, wherein the fermentation cell culture medium can comprise a soluble fraction of leguminous plant. In some embodiments, a fermentation cell culture medium can support growth and activity of microorganisms, wherein the cell fermentation culture medium can comprise a hydrolyzed form of the soluble fraction of leguminous plant. In some embodiments, a fermentation cell culture medium can support growth and activity of microorganisms, wherein the cell fermentation culture medium can comprise a mixture of hydrolyzed and unhydrolyzed form of the soluble fraction of leguminous plant.

[0089] In some embodiments, the soluble fraction of the leguminous plant (e.g., hydrolyzed and unhydrolyzed form) can be added to a basal medium at a final concentration of about 0.05 g / L to about 60 g / L. In some embodiments, the fermentation culture medium supplement can be added to a basal medium at a final concentration of about 0.05 g / L to about 1 g / L, about 0.05 g / L to about 5 g / L, about 0.05 g / L to about 10 g / L, about 0.05 g / L to about 12.5 g / L, about 0.05 g / L to about 15 g / L, about 0.05 g / L to about 17.5 g / L, about 0.05 g / L to about 20 g / L, about 0.05 g / L to about 30 g / L, about 0.05 g / L to about 40 g / L, about 0.05 g / L to about 50 g / L, about 0.05 g / L to about 60 g / L, about 1 g / L to about 5 g / L, about 1 g / L to about 10 g / L, about 1 g / L to about 12.5 g / L, about 1 g / L to about 15 g / L, about 1 g / L to about 17.5 g / L, about 1 g / L to about 20 g / L, about 1 g / L to about 30 g / L, about 1 g / L to about 40 g / L, about 1 g / L to about 50 g / L, about 1 g / L to about 60 g / L, about 5 g / L to about 10 g / L, about 5 g / L to about 12.5 g / L, about 5 g / L to about 15 g / L, about 5 g / L to about 17.5 g / L, about 5 g / L to about 20 g / L, about 5 g / L to about 30 g / L, about 5 g / L to about 40 g / L, about 5 g / L to about 50 g / L, about 5 g / L to about 60 g / L, about 10 g / L to about 12.5 g / L, about 10 g / L to about 15 g / L, about 10 g / L to about 17.5 g / L, about 10 g / L to about 20 g / L, about 10 g / L to about 30 g / L, about 10 g / L to about 40 g / L, about 10 g / L to about 50 g / L, about 10 g / L to about 60 g / L, about 12.5 g / L to about 15 g / L, about 12.5 g / L to about 17.5 g / L, about 12.5 g / L to about 20 g / L, about 12.5 g / L to about 30 g / L, about 12.5 g / L to about 40 g / L, about 12.5 g / L to about 50 g / L, about 12.5 g / L to about 60 g / L, about 15 g / L to about 17.5 g / L, about 15 g / L to about 20 g / L, about 15 g / L to about 30 g / L, about 15 g / L to about 40 g / L, about 15 g / L to about 50 g / L, about 15 g / L to about 60 g / L, about 17.5 g / L to about 20 g / L, about 17.5 g / L to about 30 g / L, about 17.5 g / L to about 40 g / L, about 17.5WSGR Docket No. 69505-701.601 g / L to about 50 g / L, about 17.5 g / L to about 60 g / L, about 20 g / L to about 30 g / L, about 20 g / L to about 40 g / L, about 20 g / L to about 50 g / L, about 20 g / L to about 60 g / L, about 30 g / L to about 40 g / L, about 30 g / L to about 50 g / L, about 30 g / L to about 60 g / L, about 40 g / L to about 50 g / L, about 40 g / L to about 60 g / L, or about 50 g / L to about 60 g / L. In some embodiments, the fermentation culture medium supplement can be added to a basal medium at a final concentration of about 0.05 g / L, about 1 g / L, about 5 g / L, about 10 g / L, about 12.5 g / L, about 15 g / L, about 17.5 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, or about 60 g / L. In some embodiments, the fermentation culture medium supplement can be added to a basal medium at a final concentration of at least about 0.05 g / L, about 1 g / L, about 5 g / L, about 10 g / L, about 12.5 g / L, about 15 g / L, about 17.5 g / L, about 20 g / L, about 30 g / L, about 40 g / L, or about 50 g / L. In some embodiments, the fermentation culture medium supplement can be added to a basal medium at a final concentration of at most about 1 g / L, about 5 g / L, about 10 g / L, about 12.5 g / L, about 15 g / L, about 17.5 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, or about 60 g / L.

[0090] In some embodiments, the fermentation culture medium can comprise both a hydrolyzed and unhydrolyzed soluble fractions of the leguminous plant. In some embodiments, the ratio between the unhydrolyzed soluble fraction and the hydrolyzed form of the soluble fraction of the fermentation culture medium supplement added to a basal medium can be 1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9:2, respectively. In some embodiments, the fermentation culture medium can further comprise one or more carriers. In some embodiments, the fermentation culture medium comprising a soluble fraction of the leguminous plant (e.g., a hydrolyzed or unhydrolyzed) may be free or substantially free of glucose.

[0091] In some embodiments, the fermentation culture medium can be a mixed fermentation culture media, wherein the mixed fermentation culture medium can comprise both a soluble fraction of a leguminous plant as described herein (e.g., hydrolyzed or unhydrolyzed soluble fractions of the leguminous plant) with a second fermentation culture medium supplement (e.g., yeast extracts or non-leguminous plant extracts). The ratio of a soluble fraction of a leguminous plant and a second fermentation culture medium supplement can be 1 :9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, or 9: 1, respectively. In some embodiments, the mixed fermentation culture media can exhibit synergistic effects, wherein the combined impact on the growth and activity of the microorganism is greater than the additive effects of individual components alone. For example, in some embodiments, the mixed fermentation culture media can enhance growth and activity of the microorganism by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1.2-fold, at least about 1.4-fold, at least about 1.6-fold, at least about 1.8-fold, at least about 2.0-fold,WSGR Docket No. 69505-701.601 at least about 2.5-fold, at least about 3.0-fold, at least about 3.5-fold, at least about 4.5-fold, at least about 5.0-fold, at least about 6.0-fold, at least about 7.0-fold, at least about 8.0-fold, at least about 9.0-fold, or at least about 10.0-fold higher than that of individual components alone or a combined effects of the components.

[0092] In some embodiments, the fermentation culture media can further comprise salts (such as sodium chloride, calcium, magnesium, and phosphate), amino acids, vitamins, buffers, nucleotides, antibiotics, antioxidants, or carbohydrates or equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture condition, such as pH, and the like, will be apparent to the ordinary skilled artisan.

[0093] In some embodiments, the methods comprise growing or inoculating microorganisms in the fermentation culture medium. In some embodiments, the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can support growth or activity of microorganisms involved in the agricultural application. For example, in some embodiments, the microorganisms can enhance soil fertility, plant growth promotion, or crop protection. In some embodiments, the microorganisms can be nitrogen-fixing bacteria. In some embodiments, the microorganism can be plant growth-promoting bacteria. In some embodiments, the microorganisms can be any of the genera Bradyrhizobium, Azospirillum, Pseudomonas, Bacillus, Trichoderma, Streptomyces, Actinomyces, Micromonospora, or Metarhizium.

[0094] In some embodiments, the microorganisms can be one of the following species: In the genus Bradyrhizobium, species include, but are not limited to, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium diazoefficiens . In the genus Rhizobium, species can include, but are not limited to, Rhizobium leguminosarum and Rhizobium etli. In the genus, Azospirillum, species can include, but are not limited to, Azospirillum brasilense and Azospirillum lipoferum. The genus Pseudomonas can comprise, but are not limited to, Pseudomonas fluorescens and Pseudomonas putida. The diverse Bacillus genus can include, but are not limited to, Bacillus subtilis, Bacillus thuringiensis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus pumilus, Bacillus licheniformis, and Bacillus velezensis. In the genus Trichoderma, species can include, but are not limited to Trichoderma harzianum and Trichoderma viride. In the Streptomyces genus, species can include, but are not limited to Streptomyces griseus and Streptomyces lydicus. In the genus Actinomyces, species can include, but are not limited to, Actinomyces israelii, and Micromonospora by Micromonospora lupini. The genus Metarhizium can include, but are not limited to, Metarhizium anisopliae, Metarhizium robertsii, Metarhizium brunneum, Metarhizium flavoviride, and Metarhizium guizhouense.WSGR Docket No. 69505-701.601

[0095] In some embodiments, the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can support growth or activity of microorganisms with probiotic benefits for human and animal consumption. For example, in some embodiments, the fermentation culture medium supplement can be utilized for culturing probiotic microorganisms to support efficient fermentation processes. In some embodiments, the fermentation culture medium supplement can be utilized for culturing probiotic microorganisms to support efficient fermentation processes in food products. For example, in some embodiments, the food products can be dairybased fermented products such as yogurt, milk, or fermented cheese varieties. In some embodiments, the food products can be fermented vegetables and condiments (e.g., sauerkraut, miso, pickles, kimchi). In some embodiments, the food product can be fermented beverages (e.g., kombucha). In some embodiments, the food product can be fermented meats and cured products (e.g., salami). In some embodiments, the fermentation culture medium supplement can support growth and activity of probiotic microorganisms specifically selected to support growth and activity under various fermentation conditions. In some embodiments, the microorganisms can be any of the genera Lactobacillus, Bifidobacterium, Enterococcus, Streptococcus, Lactococcus, Bacillus, or Propionibacterium.

[0096] In some embodiments, the microorganisms can be one of the following species: In the genus Lactobacillus, species can include, but are not limited to, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus reuteri, and Lactobacillus bulgaricus. In the genus Bifidobacterium, species can include, but are not limited to, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, and Bifidobacterium bifidum. In the genus Enterococcus, species can include, but are not limited to, Enterococcus faecium. In the genus Streptococcus, species can include, but are not limited to, Streptococcus thermophilus. In the genus Lactococcus, the species can include, but are not limited to, Lactococcus lactis subsp. lactis. In the genus Bacillus, species can include, but are not limited to, Bacillus coagulans and Bacillus subtilis. In the genus Propionibacterium, species can include, but are not limited to, Propionibacterium freudenreichii .

[0097] In some embodiments, the fermentation culture medium supplement comprising a soluble fraction derived from a leguminous plant can support growth or activity of industrial fermentation microorganisms. In some embodiments, the fermentation culture medium supplement can be utilized for culturing microorganisms for industrial fermentation processes, including, for example, enzyme production and bioactive compound synthesis. In some embodiments, the fermentation culture medium supplement can be utilized for culturing the microorganisms to enhance microbial growthWSGR Docket No. 69505-701.601 and metabolic productivity. The microorganisms, for example, can include Escherichia coli (E. coli), or yeast, Saccharomyces, which can include Saccharomyces pastorianus and Saccharomyces cerevisiae. In some embodiments, the microorganism can be Aspergillus, which includes, but are not limited to, Aspergillus niger and Aspergillus oryzae. In some embodiments, the microorganisms can be Fusarium venenatum, Bacillus licheniformis, Trichoderma reesei, Pichia pastoris, Corynebacterium glutamicum, Candida antarctica, o Micrococcus luteus.Kits

[0098] In some embodiments, provided herein relate to a kit that includes the composition described herein with instructions for use. For convenience, the kit-of-parts may comprise reagents (e.g., a soluble fraction derived from a leguminous plant) in predetermined amounts with instructions for use. In some embodiments, disclosed herein are kits comprising a composition comprising a fermentation culture medium supplement, a basal medium, or a soluble fraction derived from a leguminous plant.

[0099] In some embodiments, the kit is a ready -to-use kit, wherein the composition described herein included in the kit can be ready to use by the users without further alterations (e.g., by having a predetermined concentration). In some embodiments, the composition described herein is provided in the kit in a container for application to the cell culture system. In some embodiments, the kit comprises a powder form of the fermentation culture medium supplement. In some embodiments, the kit comprises a solution of the fermentation culture medium supplement. In some embodiments, the kit may comprise additional components, such as a basal culture medium, additional supplements, or minerals.

[0100] In some embodiments, the cell culture medium is packaged in a container with a label affixed to the container or included in the package that describes use of the compositions for use in vitro, in vivo, or ex vivo. Exemplary containers include, but are not limited to, a vessel, vial, tube, ampoule, bottle, flask, and the like. In some embodiments, the container is adapted for packaging the media. In some embodiments, the container is made from material well-known in the art, including, but is not limited to, glass, polypropylene, polystyrene, and other plastics.

[0101] In some embodiments, the compositions are packaged in a unit dosage form. For example, in some embodiments, the soluble fraction derived from a leguminous plant can be packaged in a volume of 10 mL, 50 mL, 100 mL, 500 mL, or 1 L. In some embodiments, the soluble fraction derived from a leguminous plant can be packaged in a dry powder form. In some embodiments, the soluble fraction derived from leguminous plants can be packaged in 10 mg, 50 mg, 100 mg, 500 mg, 1g, 50g, 100g, or 1000 g of dry powders.WSGR Docket No. 69505-701.601

[0102] The kit optionally includes a device suitable for combining a basal medium, fermentation culture medium supplement, and alternatively combining the media with additional supplements (e.g., yeast extracts). In various aspects, the kit contains a label and / or instructions that describes use of the media for cell culture.

[0103] In some embodiments, instructions for use may be provided in a kit. These instructions may be presented in the kit in a variety of forms, such as printed information on a suitable medium or substrate (e.g., a piece or pieces of paper on which the information is printed), in the packaging of the kit, in a package insert, etc. In some embodiments, instructions for use can be provided on a computer readable medium (e.g., jump / thumb drive, CD, etc.), for which the information has been recorded, or at a website address which may be used via the internet to access the information at a website.Definition

[0104] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0105] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0106] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0107] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can includeWSGR Docket No. 69505-701.601 quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute.

[0108] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0109] As used herein, the term “agriculture” has its ordinary meaning as understood in light of the specification and refers to plant cultivation. Agriculture can include production of food, medicines, or products from plants. The type of plants that may be cultivated is not particularly limiting, and can include any tree, crop that has industrial, commercial, medical, recreational, ornamental, or aesthetic value.

[0110] As used herein, the term a “supplement” or “supplements” refers to an additional nutrient composition or component that is introduced to a basal culture medium to enhance or optimize its nutrient profile for improved growth, metabolic activity, or productivity of microorganisms. The supplement may comprise one or more nutrients, such as carbohydrates, amino acids, vitamins, minerals, or other growth factors, that may be limited or lacking in the basal medium.EXAMPLES

[0111] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Methods of making a fermentation culture medium supplement from pea soluble

[0112] As outlined in FIG. 1, the pea flour was loaded into the extraction tanks. The pea flour was dissolved in hot water (temperature: 50-60°C), and then adjusted pH to 8.0 - 8.5. Once the extraction time was reached, the mixture (soluble and insoluble fraction) was separated by a decantation process. In the separation process, the liquid phase (e.g., comprising a soluble fraction of the pea flour) was collected in an acid tank, while the heavy phase (e.g., comprising wet starch and fibers) was moved to a drying stage to form dry powders. The pH of the liquid phase collected in the acid tank was then adjusted to 4.5 and passed through a second separation process. This step resulted in separation of the heavy phase comprising protein slurry, and the liquid phase (e.g., referred to as pea soluble).

[0113] The protein slurry (e.g., an insoluble fraction) was then further processed to obtain the pea protein concentrate.

[0114] Pea soluble fraction (e.g., the liquid phase) was further processed to concentrate the pea soluble. For example, the concentration steps were carried out by evaporation of the liquid toWSGR Docket No. 69505-701.601 increase the solids content up to 35% to obtain the concentrated pea soluble. The concentrated pea soluble (batch 1 : day 1) was stored in a storage tank at an ambient temperature. Subsequently, the pea soluble from batch 2 and batch 3 were further collected from the following two days of production.

[0115] On the 4th day, the pea soluble was further formulated. The formulation stage may include pH adjustment, carrier treatments, and / or hydrolysis, depending on the final product desired. Finally, the drying process was carried out in a spray dryer, resulting in dry powders of the soluble fraction.Example of Manufacturing Pea Soluble with Carriers;

[0116] 50% of the pea soluble (Supplement A) and 50% maltodextrin, which functions as a carrier, were mixed to obtain Supplement B. The concentrated pea soluble obtained during batches 1, 2, and 3 (Supplement A) were mixed in a stirred tank while maltodextrin is added to achieve a carrier content of 50% dry basis (d.b.) in the final product. Then, the pH was adjusted from an initial value of 4-5 to a final pH of 7.5-8.5.Example of Manufacturing Hydrolyzed Pea Soluble

[0117] The concentrated pea soluble was dissolved in water at an amount between 20 to 30%. pH of the solution was adjusted to pH 7-8, and the temperature was adjusted to 50-60°C. The enzyme blend was added to the solution. The enzyme blend comprised an exoprotease and endoprotease. In the tank, while stirring, the enzyme reaction (e.g., hydrolysis) was continued for about 1 hour. A thermal treatment process was carried out to deactivate the enzymes. Once the hydrolyzed concentrated pea soluble was obtained, the hydrolyzed concentrated pea soluble was further treated with or without carrier (Supplement C and Supplement D, respectively). The steps further included a drying step to obtain powders.Example 2: Composition of Pea Soluble with or Without Carriers (Supplement A & Supplement B) Supplement A

[0118] The composition of pea soluble is shown in Tables 1 - 4. Table 1 describes typical composition of pea soluble. Table 2 describes carbohydrates composition. Table 3 describes Mineral composition. Table 4 describes an amino acid profile based on nitrogen content. It was surprising that the carbohydrates composition of pea soluble is free or substantially free of glucose, fructose, and raffinose.WSGR Docket No. 69505-701.601Table 1. Typical Composition of Pea Soluble (Supplement A)Table 2: Carbohydrates Composition DetailTable 3: Mineral Composition DetailTable 4: Amino Acid Profile Based on Nitrogen Content (Supplement A and Supplement B)WSGR Docket No. 69505-701.601Supplement B

[0119] The composition of pea soluble with carriers is shown in Tables 5 - 8. Supplement B comprises 50% pea soluble (of Supplement A) and 50% of maltodextrin as carriers. Table 5 describes typical composition of pea soluble with carriers. Table 6 describes carbohydrates composition. Table 7 describes Mineral composition. Table 8 describes an amino acid profile based on nitrogen content. Although the glucose level was increased as compared to Supplement A due to the presence of maltodextrin, it can be seen that the composition is substantially free or is free of glucose (e.g., less than 1% w / w dry basis).Table 5: Typical compositionTable 6: Carbohydrates composition detailWSGR Docket No. 69505-701.601Table 7: Mineral composition detailTable 8: Amino Acid Profile Based on Nitrogen Content (Supplement A and Supplement B)WSGR Docket No. 69505-701.601Example 3: Performance of Pea Soluble for Bacillus subtilis growth and activity

[0120] Different pea soluble fermentation culture medium (e.g., comprising supplements A-D as shown in Table 9) were used to assess growth and activity of Bacillus subtilis.Table 9: Pea Soluble MediumInoculum Preparation

[0121] 100 ml Erlenmeyer flasks containing 10 ml of the following culture media: Luria Broth(LB), Supplement A, Supplement B, Supplement C, and Supplement D. The media were inoculated with stocks of a strain of Bacillus subtilis. The Erlenmeyer flasks were incubated in a shaker at 200 RPM and 37°C for 16 hours (Overnight).B. subtilis Cultures

[0122] From the overnight grown cultures (inoculum cultures), 100 ml Erlenmeyer flasks were inoculated with 20 ml of the following culture media: LB and Supplements A-D. Each culture medium was evaluated in duplicate. The Erlenmeyer flasks were incubated in a shaker at 37°C and 200 RPM. The fermentation was monitored for 96 hours, and cell growth was determined with OD at 650 nm.Spores determination

[0123] The samples to determine the spores were taken after 24 hours of fermentation. Spores CFU were quantified by plating cultures which had been boiled for 30 minutes at 80°C to kill vegetative cells.Mediums:Luria broth: 20 g / 1Supplement A: 15 g / 1Supplement B: 15 g / 1 Supplement C: 15 g / 1 Supplement D: 15 g / 1 ResultsWSGR Docket No. 69505-701.601

[0124] As shown in Table 10 and FIG. 2, the fermentation culture medium comprising Supplement A (e.g., 100% pea soluble) demonstrated an increase in B. subtilis growth compared to the control medium with LB, as measured by optical density. While other culture conditions (e.g., Supplements B-D) did not show a significant difference in optical density, they still achieved higher optical density values than the control medium containing LB. It should be noted that optical density is an indirect indicator of cell counts and is efficient for monitoring growth during fermentation.

[0125] As shown in Table 11, Table 12, and Table 13, bacteria and spore count after 24 hours of incubation in culture medium comprising Supplement A (Table 11), Supplement C (Table 12), Supplement B (Table 13), or Supplement D (Table 13) further demonstrate that pea soluble, regardless whether the pea soluble is hydrolyzed or not, both CFU and spore yield are significant increased as compared to culture medium comprising LB. More specifically, it’s surprising that culture medium comprising Supplement A and culture medium comprising Supplement C led to an increase in sporulation yield of over 80-120 - fold in a culture medium that is free or substantially free of glucose.Table 10: Average optical density after 24 hours of incubationTable 11: Bacteria and spores count after 24 hours of incubation in Supplement ATable 12: Bacteria and spores count after 24 hours of incubation in Supplement CWSGR Docket No. 69505-701.601Table 13: Bacteria and spores count after 24 hours of incubation in Supplements B and D

[0126] It’s further noted that there are no significant differences between Supplement A and Supplement B regarding growth. Furthermore, there were no significant differences in microorganism growth count and growth rate between the culture media comprising 100% pea soluble or hydrolyzed pea soluble (Supplement A and Supplement C). Also Supplements A-D can replace all or a portion of yeast extracts (e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 70%, at least about 80%, or at least about 90). For example, using Supplement A or Supplement B to adjust the nitrogen content of a medium as a substitute for yeast extract in Bradyrhizobium culture resulted in no significant differences in growth, growth rate, or activity of microorganism (data not shown).Example 4: Replacement of animal-derived nitrogen source with pea soluble for Bacillus subtilis growth and activity

[0127] The ability of Supplement B (comprising 50% Pea soluble: 50% maltodextrin) to replace conventional animal-derived nitrogen source in bacterial culture media was evaluated. Growth and sporulation rates of Bacillus subtilis were measured in different culture conditions, where Supplement B replaced peptone from meat and / or meat extract

[0128] Four different culture media were tested:Control: Typical optimized medium for Bacillus subtilis sporulation.- Medium 1 : 100% replacement of meat peptone with Supplement B.- Medium 2: 100% replacement of meat extract with Supplement B.- Medium 3 : 100% replacement of both meat peptone and meat extract with Supplement B.Table 14: Media compositions (g / 1), Experiment 1WSGR Docket No. 69505-701.601

[0129] Bacteria and sporulation efficiency were assessed by colony-forming units (CFU) and spore counts (Table 15).Table 15: CFU and Sporulation Rate in Tested Media experiment 1

[0130] The results indicate that Supplement B can effectively replace conventional animal-based nitrogen sources in bacterial culture media. The replacement of meat peptone with Supplement B (Medium 1) resulted in a higher sporulation rate compared to the control. This suggests that Supplement B provides suitable nutrients for bacterial growth and differentiation.

[0131] Based on the results, a second set of trials were performed, focusing on the final nitrogen content in the culture media formulation. Supplement B was used at the same dosage as meat peptone and also at a higher concentration, to make the nitrogen level comparable with the control media.

[0132] Typical total nitrogen content on ingredients used:- Meat peptone: 12 %- Meat extract: 10 %Supplement s: 2.2 %Table 16: Media compositions (g / 1)WSGR Docket No. 69505-701.601

[0133] Bacteria growth and sporulation efficiency were assessed by colony -forming units (CFU) and spore counts (Table 17).Table 17: CFU and Sporulation Rate in Tested Media

[0134] In this second experiment, the evaluation focused on replacing meat peptone in the culture medium with Supplement B, since meat peptone is an expensive ingredient and Supplement B contains peptone-like nitrogen compounds in its composition. Two formulations from the first experiment (control and Medium 1) were repeated, and a third one (Medium 4) was included, in which the total nitrogen content was adjusted to match the control medium (Table 16). As observed, both Medium 1 and Medium 4 showed higher sporulation rates compared to the control, with very similar performance between them. Importantly, despite Medium 1 containing a lower total nitrogen level, its sporulation was comparable to Medium 4 and superior to the control.

[0135] The data suggest that the stimulatory effect of Supplement B on sporulation is not solely attributable to its nitrogen contribution, but may involve specific compositional features that differentiate it from conventional peptone sources.Example 5: Sugar profile of the pea solubles

[0136] After the alkaline extraction - isoelectric precipitation process, concentrated pea solubles can be treated, purified, or modified for various purposes, including the modification of their sugar profile.

[0137] Multiple strategies exist for this purpose, including techniques such as tangential flow filtration, chromatography, enzymatic treatments, or other physical, chemical, biological methods or their combinations. Fermentation can be applied to transform specific sugars or modify their overall content.

[0138] Due to the high solids content, low pH, and mineral-rich composition of soluble legume extracts, these substrates are known to favor spontaneous lactic acid fermentation, as they pose a barrier to the growth of many microorganisms. In this context, spontaneous fermentation on the concentrated soluble extract represents an advantageous alternative, owing to its operationalWSGR Docket No. 69505-701.601 simplicity and low technological requirements compared to more complex treatments. However, a controlled fermentation would allow for more consistent product characteristics over time.

[0139] To demonstrate the modification of the sugar profile and content, particularly glucose (Glu), galactose (Gal), and sucrose (Sac), in the concentrated soluble extract of yellow pea, through spontaneous fermentation during storage.

[0140] A sample of concentrated yellow pea solubles was used, obtained as described in Example 1 : Methods of making a fermentation culture medium supplement from pea soluble. Through the alkaline extraction - isoelectric precipitation process of yellow pea protein, a concentrated yellow pea soluble extract was obtained.

[0141] Three consecutive batches were produced. The concentrate from Batch 1 (Day 1) was stored in a tank at room temperature under aerobic conditions. On Days 2 and 3, the concentrated solubles from Batches 2 and 3 were collected into the same tank.

[0142] The presence of glucose (Glu), galactose (Gal), and sucrose (Sac) was quantified in two samples of the concentrate:Sample 1 : concentrated soluble extract from Day 1 (storage time = 0 days) Sample 2: mixture of the three batches, after 3 days of storage under aerobic conditions at room temperature.

[0143] The sugar determination was performed by high-performance liquid chromatography (HPLC) using a refractive index detector (RID) with the following setup:Column: Phenomenex Luna Omega Sugar, 3 pm, 100 A, 150 x 4.6 mm- Detector: Refractive Index (RID)- Detection limit: 0.03 g / 100 mL for each sugar analyzed

[0144] The concentrations of glucose (Glu), galactose (Gal), and sucrose (Sac) detected in the two analyzed samples are presented below (Table 18). The sugar content is expressed on a dry basis (% w / w d.b.).Table 18: Results obtained for the determination of dry mass and the concentration of glucose (Glu), galactose (Gal), and sucrose (Sac) in both analyzed samples.WSGR Docket No. 69505-701.601

[0145] The results obtained show that the storage of concentrated pea solubles under the described conditions enables spontaneous fermentation, which reduces the content of various sugars, leading to a modification in the sugar profile of the sample.

[0146] In this case, galactose and sucrose were reduced by 23% and 33.9%, respectively. This change occurred without accumulation of glucose as a product of sucrose digestion, suggesting that as sucrose is consumed, any glucose generated is immediately metabolized, despite the presence of galactose, another simple sugar in the medium.

[0147] As a result, the process contributes to an effective reduction of reducing sugars, which could potentially mitigate Maillard reactions during further processing or storage of the final product. Taken together, these findings demonstrate that the applied method naturally modifies the sugar profile of the concentrate without the need for enzymatic or physical treatments, and offers functional benefits in terms of stability, chemical reactivity, and industrial application potential.

[0148] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0149] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby

Claims

WSGR Docket No. 69505-701.601CLAIMSWHAT IS CLAIMED IS:

1. A fermentation culture medium supplement comprising: a soluble fraction derived from a leguminous plant, wherein the soluble fraction comprises carbohydrates that are free or substantially free of glucose, wherein the carbohydrates are present in the soluble fraction at a concentration of at least about 50% w / w, and wherein the fermentation culture medium supplement provides nutrient substrates for microbial metabolism.2 The fermentation culture medium supplement of claim 1, wherein the soluble fraction further comprises proteins.3 The fermentation culture medium supplement of claim 2, wherein the proteins are present in the soluble fraction at a concentration of at least about 10% w / w.4 The fermentation culture medium supplement of any one of claims 1-3, wherein the proteins are present in the soluble fraction at a concentration of from about 10.0% to about 40% w / w.5 The fermentation culture medium supplement of any one of claims 1-4, wherein the carbohydrates are present in the soluble fraction at a concentration of from about 50% to about 90% w / w.6 The fermentation culture medium supplement of any one of claims 1-5, wherein the carbohydrates comprise galactose, sucrose, stachyose, or a combination thereof.7 The fermentation culture medium supplement of claim 6, wherein the galactose is present in the soluble fraction at a concentration of at least about 10% w / w.8 The fermentation culture medium supplement of claim 6, wherein the sucrose is present in the soluble fraction at a concentration of at least about 13% w / w.9 The fermentation culture medium supplement of any one of claims 1-8, wherein the soluble fraction further comprises nitrogen, ashes, mineral(s), or a combination thereof.10 The fermentation culture medium supplement of claim 9, wherein the mineral comprises potassium, sodium, magnesium, calcium, phosphorus, sulfur, iron, zinc, copper, manganese, molybdenum, or a combination thereof.11 The fermentation culture medium supplement of claim 9, wherein the nitrogen is present in the soluble fraction at a concentration of at least about 1.5 % w / w.WSGR Docket No. 69505-701.60112. The fermentation culture medium supplement of any one of claims 1-11, wherein the microbial metabolism supports growth or activity of microorganisms.

13. The fermentation culture medium supplement of claim 12, wherein the microorganisms are selected from the group consisting of: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense , Bradyrhizobium diazoefficiens, Rhizobium leguminosarum. Rhizobium etli , Azospirillum brasilense, Azospirillum lipoferum, Pseudomonas fhiorescens. Pseudomonas putida, Bacillus subtilis, Bacillus ihuringiensis. Bacillus amyloliquefaciens, Bacillus megalerium. Bacillus pumilus,, Bacillus licheniformis Bacillus velezensis, Trichoderma harzianum. Trichoderma viride. Streptomyces griseus. Streptomyces lydicus. Actinomyces israelii, Micromonospora lupini, Metarhizium anisopliae, Metarhizium robertsii, Metarhizium brunneum, Metarhizium flavoviride, Metarhizium guizhouense. Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus reuteri, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Bacillus coagulans, Bacillus subtilis, Lactobacillus bulgaricus, Propionibacterium freudenreichii, E.coli, Saccharomyces pastorianus, Saccharomyces cerevisiae, Aspergillus niger, Fusarium venenatum, Bacillus licheniformis, Trichoderma reesei, Pichia pastoris, Corynebacterium glutamicum, Aspergillus oryzae, Candida antarctica, and Micrococcus luteus.

14. The fermentation culture medium supplement of claim 12 or 13, wherein the growth or the activity of the microorganisms is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant.

15. The fermentation culture medium supplement of claim 14, wherein the growth or the activity of the microorganisms is measured by an optical density.

16. The fermentation culture medium supplement of any one of claims 12-15, wherein the soluble fraction induces sporulation of the microorganisms.

17. The fermentation culture medium supplement of any one of claims 12-16, wherein the soluble fraction enhances sporulation of the microorganisms.WSGR Docket No. 69505-701.60118. The fermentation culture medium supplement of claim 16 or 17, wherein the sporulation is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5 -fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant.

19. The fermentation culture medium supplement of any one of claims 1-18, wherein the leguminous plant is selected from the group consisting of yellow pea, chickpea, mung bean, white bean, pinto bean, red kidney bean, lentil, black bean, cowpea, caupi bean, broad bean, and aduki bean.

20. The fermentation culture medium supplement of any one of claims 1-19, wherein the carbohydrates are present in the soluble fraction at an amount of at most 5% glucose.

21. The fermentation culture medium supplement of any one of claims 1-17, wherein the carbohydrates are present in the soluble fraction at an amount of at most 1% glucose.

22. The fermentation culture medium supplement of any one of claims 1-21, wherein the soluble fraction further comprises a carrier.

23. The fermentation culture medium supplement of claim 22, wherein the carrier comprises protein concentrates obtained from the leguminous plant.

24. The fermentation culture medium supplement of claim 22, wherein the carrier comprises silicon dioxides, salts, sugars, or a combination thereof.

25. The fermentation culture medium supplement of claim 22, wherein the carrier comprises a biopolymer.

26. The fermentation culture medium supplement of claim 25, wherein the biopolymer is selected from the group consisting of a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, and maltodextrin.

27. The fermentation culture medium supplement of any one of claims 22-26, wherein the carrier is added to the soluble fraction at a dose of from about 0.1% to about 50%.

28. The fermentation culture medium supplement of any one of claims 1-27, wherein the soluble fraction is not liquefied or treated with a proteolytic enzyme.WSGR Docket No. 69505-701.60129. The fermentation culture medium supplement of any one of claims 1-27, wherein the soluble fraction is liquefied or treated with a proteolytic enzyme.

30. The fermentation culture medium supplement of claim 29, wherein the proteolytic enzyme comprises an endoprotease, an exoprotease, or a combination thereof.

31. The fermentation culture medium supplement of claim 29 or 30, wherein the proteolytic enzyme treatment or liquefaction results in a hydrolyzed soluble fraction.

32. The fermentation culture medium supplement of claim 31, wherein the hydrolyzed soluble fraction further comprises the carrier.

33. The fermentation culture medium supplement of any one of claims 1-32, wherein the soluble fraction replaces a yeast extract.

34. The fermentation culture medium supplement of any one of claims 1-33, wherein the soluble fraction replaces meat peptones or meat extracts.

35. The fermentation culture medium supplement of any one of claims 1-34, wherein the soluble fraction is processed by spray drying to produce a dry powder.

36. The fermentation culture medium supplement of claim 35, wherein the soluble fraction is characterized by a moisture content of less than 50%.

37. A fermentation culture medium supplement comprising: a soluble fraction derived from a leguminous plant, wherein the soluble fraction comprises at least one carbon source and at least one nitrogen source for microbial metabolism, wherein the at least one carbon source is free or substantially free of glucose.

38. The fermentation culture medium supplement of claim 37, wherein the at least one nitrogen source is derived from a unhydrolyzed protein of the soluble fraction derived from the leguminous plant.

39. The fermentation culture medium supplement of claim 37 or 38, wherein the at least one nitrogen source is present in an amount of at least about 10% by weight of the soluble fraction.

40. The fermentation culture medium supplement of any one of claims 37-39, wherein the at least one carbon source is carbohydrate present at a concentration of at least about 50% by weight of the soluble fraction.

41. The fermentation culture medium supplement of claim 40, wherein the carbohydrate comprises galactose, stachyose, sucrose, or a combination thereof.WSGR Docket No. 69505-701.60142. The fermentation culture medium supplement of claim 40, wherein the carbohydrate is free or substantially free of glucose.

43. The fermentation culture medium supplement of any one of claims 37-42, wherein the soluble fraction derived from the leguminous plant further comprises at least one mineral.

44. The fermentation culture medium supplement of claim 43, wherein the at least one mineral comprises potassium, sodium, magnesium, calcium, phosphorus, sulfur, iron, zinc, copper, manganese, molybdenum, or a combination thereof.

45. The fermentation culture medium supplement of any one of claims 37-44, wherein the microbial metabolism supports growth or activity of microorganisms.

46. The fermentation culture medium supplement of claim 45, wherein the microorganisms are selected from the group consisting of: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense , Bradyrhizobium diazoefficiens, Rhizobium leguminosarum.Rhizobium etli , Azospirillum brasilense, Azospirillum lipoferum, Pseudomonas fhiorescens. Pseudomonas putida, Bacillus subtilis, Bacillus ihuringiensis. Bacillus amyloliquefaciens, Bacillus megalerium. Bacillus pumilus,, Bacillus licheniformis Bacillus velezensis, Trichoderma harzianum. Trichoderma viride. Streptomyces griseus. Streptomyces lydicus. Actinomyces israelii, Micromonospora lupini, Metarhizium anisopliae, Metarhizium robertsii, Metarhizium brunneum, Metarhizium flavoviride, Metarhizium guizhouense. Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus reuteri, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Bacillus coagulans, Bacillus subtilis, Lactobacillus bulgaricus, Propionibacterium freudenreichii, E.coli, Saccharomyces pastorianus, Saccharomyces cerevisiae, Aspergillus niger, Fusarium venenatum, Bacillus licheniformis, Trichoderma reesei, Pichia pastoris, Corynebacterium glutamicum, Aspergillus oryzae, Candida antarctica, and Micrococcus luteus.

47. The fermentation culture medium supplement of claim 45 or 46, wherein the growth or the activity of the microorganisms is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, atWSGR Docket No. 69505-701.601 least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant.

48. The fermentation culture medium supplement of claim 47, wherein the growth or the activity of the microorganisms is measured by an optical density.

49. The fermentation culture medium supplement of any one of claims 43-48, wherein the soluble fraction induces sporulation of the microorganisms.

50. The fermentation culture medium supplement of any one of claims 43-49, wherein the soluble fraction enhances sporulation of the microorganisms.

51. The fermentation culture medium supplement of claim 49 or 50, wherein the sporulation is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5 -fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant.

52. The fermentation culture medium supplement of any one of claims 37-51, wherein the leguminous plant is selected from the group consisting of yellow pea, chickpea, mung bean, white bean, pinto bean, red kidney bean, lentil, black bean, cowpea, caupi bean, broad bean, and aduki bean.

53. The fermentation culture medium supplement of any one of claims 37-52, wherein the at least one carbon source comprises at most 5% glucose.

54. The fermentation culture medium supplement of any one of claims 37-53, wherein the at least one carbon source comprises at most 1% glucose.

55. The fermentation culture medium supplement of any one of claims 37-54, wherein the soluble fraction further comprises a carrier.

56. The fermentation culture medium supplement of claim 55, wherein the carrier comprises a protein concentrate obtained from the leguminous plant.

57. The fermentation culture medium supplement of claim 55, wherein the carrier comprises silicon dioxides, salts, sugars, or a combination thereof.WSGR Docket No. 69505-701.60158. The fermentation culture medium supplement of claim 55, wherein the carrier comprises a biopolymer.

59. The fermentation culture medium supplement of claim 58, wherein the biopolymer is selected from the group consisting of a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, and maltodextrin.

60. The fermentation culture medium supplement of any one of claims 55-59, wherein the carrier is added at a dose of from about 0.1% to about 50% of the soluble fraction.

61. The fermentation culture medium supplement of any one of claims 37-60, wherein the soluble fraction is not liquefied or treated with a proteolytic enzyme.

62. The fermentation culture medium supplement of any one of claims 37-60, wherein the soluble fraction is liquefied or treated with a proteolytic enzyme.

63. The fermentation culture medium supplement of claim 62, wherein the proteolytic enzyme comprises an endoprotease, an exoprotease, or a combination thereof.

64. The fermentation culture medium supplement of claim 62 or 63, wherein the proteolytic enzyme treatment or liquefaction results in a hydrolyzed soluble fraction.

65. A fermentation culture medium comprising: a basal culture medium, a fermentation culture medium supplement of any one of claims 1-64, wherein the fermentation culture medium supplement is added at a final concentration of from 0.05 g / L to about 30 g / L in the basal culture medium.

66. The fermentation culture medium of claim 65, wherein the fermentation culture medium supplement is added at a final concentration of about 14 g / L in the basal culture medium.

67. The fermentation culture medium of claim 65 or 66, wherein the fermentation culture medium lacks a yeast extract.

68. The fermentation culture medium of any one of claims 65-67, wherein the fermentation culture medium is free or substantially free of glucose.

69. A method of making a fermentation culture medium, comprising: a) providing or obtaining a leguminous plant or a leguminous plant material; b) extracting a soluble fraction of the leguminous plant or the leguminous plant material,WSGR Docket No. 69505-701.601 wherein the soluble fraction comprises carbohydrates, wherein the carbohydrates are free or substantially free of glucose; and c) drying the soluble fraction of the leguminous plant or the leguminous plant material.

70. The method of claim 69, wherein the leguminous plant or the leguminous plant material comprises plant flours, dried plant powders, or fresh plant biomass.

71. The method of claim 69 or 70, wherein the leguminous plant or the leguminous plant material is selected from the group consisting of yellow pea, chickpea, mung bean, white bean, pinto bean, red kidney bean, lentil, black bean, cowpea, caupi bean, broad bean, and aduki bean.

72. The method of any one of claims 69-71, wherein the extracting the soluble fraction comprises mixing the leguminous plant or the leguminous plant material into an aqueous solution.

73. The method of claim 72, wherein the extracting the soluble fraction further comprises decanting, purifying, or filtering the aqueous solution, thereby obtaining the soluble fraction.

74. The method of any one of claims 69-73, after the extracting, the method further comprises concentrating the soluble fraction.

75. The method of claim 74, wherein the concentration of the soluble fraction produces a soluble fraction having a total solid content of at least about 20 %, at least about 30%, or at least about 40% (w / w).

76. The method of any one of claims 69-75, further comprising, before the drying the soluble fraction, adding one or more carriers into the soluble fraction.

77. The method of any one of claims 69-75, further comprising, after the drying the soluble fraction, adding one or more carriers into the soluble fraction.

78. The method of claim 76 or 77, wherein the one or more carriers comprises a protein concentrate obtained from the leguminous plant.

79. The method of claim 76 or 77, wherein the one or more carriers comprises silicon dioxides, salts, sugars, or a combination thereof.

80. The method of claim 76 or 77, wherein the one or more carriers comprises a biopolymer.

81. The method of claim 80, wherein the biopolymer is selected from the group consisting of a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, and maltodextrin.

82. The method of any one of claims 69-81, the extracting further comprises treating the soluble fraction with a proteolytic enzyme, thereby producing a hydrolyzed soluble fraction.WSGR Docket No. 69505-701.60183. The method of claim 82, wherein the proteolytic enzyme comprises an endoprotease, an exoprotease, or a combination thereof.

84. A method for growing microorganisms, comprising: a) providing a fermentation culture medium comprising: a basal culture medium and a soluble fraction derived from a leguminous plant, wherein the soluble fraction comprises carbohydrates that are free or substantially free of glucose, and wherein the carbohydrates are present in the soluble fraction at a concentration of at least about 50% w / w; and b) inoculating the fermentation culture medium with the microorganisms to promote microbial growth or microbial activity.

85. The method of claim 84, wherein the soluble fraction further comprises proteins.

86. The method of claim 85, wherein the proteins are present in the soluble fraction at a concentration of at least about 10% w / w.

87. The method of claim 85 or 86, wherein the proteins are present in the soluble fraction at a concentration of from about 10.0% to about 40% w / w.

88. The method of any one of claims 84-87, wherein the carbohydrates are present in the soluble fraction at a concentration of from about 50% to about 90% w / w.

89. The method of any one of claims 84-88, wherein the carbohydrates comprise galactose, sucrose, stachyose, or a combination thereof.

90. The method of claim 89, wherein the galactose is present in the soluble fraction at a concentration of at least about 10% w / w.

91. The method of claim 89, wherein the sucrose is present in the soluble fraction at a concentration of at least about 13% w / w.

92. The method of any one of claims 84-91, wherein the soluble fraction further comprises nitrogen, ashes, mineral, or a combination thereof.

93. The method of claim 92, wherein the mineral comprises potassium, sodium, magnesium, calcium, phosphorus, sulfur, iron, zinc, copper, manganese, molybdenum, or a combination thereof.

94. The method of any one of claims 84-93, wherein the carbohydrates have at most 5% glucose.

95. The method of any one of claims 84-94, wherein the carbohydrates have at most 1% glucose.WSGR Docket No. 69505-701.60196. The method of any one of claims 84-95, wherein the microbial metabolism supports growth or activity of the microorganisms.

97. The method of claim 96, wherein the microorganisms are selected from the group consisting of: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium liaoningense, Bradyrhizobium diazoefficiens, Rhizobium leguminosarum, Rhizobium etli, Azospirdlum brasilense, Azospirdlum lipoferum, Pseudomonas fhiorescens. Pseudomonas putida, Bacdlus subtilis, Bacdlus ihuringiensis. Bacdlus amyloliquefaciens, Bacdlus megalerium. Bacdlus pumilus,, Bacdlus licheniformis Bacdlus velezensis, Trichoderma harzianum. Trichoderma viride, Streptomyces griseus. Streptomyces lydicus. Actinomyces israelii, Micromonospora lupini, Metarhizium anisopliae, Metarhizium robertsii, Metarhizium brunneum, Metarhizium flavoviride, Metarhizium guizhouense. Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus brevis, Lactobacillus sakei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus helveticus, Bifidobacterium animalis subsp. lactis, Bifidobacterium breve, Bifidobacterium longum subsp. infantis, Bifidobacterium bifidum, Enterococcus faecium, Lactobacillus reuteri, Streptococcus thermophilus, Lactococcus lactis subsp. lactis, Bacillus coagulans, Bacillus subtilis, Lactobacillus bulgaricus, Propionibacterium freudenreichii, E.coli, Saccharomyces pastorianus, Saccharomyces cerevisiae, Aspergillus niger, Fusarium venenatum, Bacillus licheniformis, Trichoderma reesei, Pichia astoris, Corynebacterium glutamicum, Aspergillus oryzae, Candida antarctica, and Micrococcus luteus.

98. The method of claim 96 or 97, wherein the growth or the activity of the microorganisms is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5 -fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40- fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant.

99. The method of claim 98, wherein the growth or the activity of the microorganisms is measured by an optical density.

100. The method of any one of claims 96-99, wherein the soluble fraction induces sporulation of the microorganisms.

101. The method of any one of claims 96-100, wherein the soluble fraction enhances sporulation of the microorganisms.WSGR Docket No. 69505-701.601102. The method of claim 100 or 101, wherein the sporulation is enhanced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30, at least about 35%, at least about 40%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold compared to that of the microorganisms cultured with a culture medium supplement lacking the soluble fraction derived from the leguminous plant.

103. The method of any one of claims 84-102, wherein the leguminous plant is selected from the group consisting of yellow pea, chickpea, mung bean, white bean, pinto bean, red kidney bean, lentil, black bean, cowpea, caupi bean, broad bean, and aduki bean.

104. The method of any one of claims 84-103, wherein the soluble fraction further comprises a carrier.

105. The method of claim 104, wherein the carrier comprises a protein concentrate obtained from the leguminous plant.

106. The method of claim 104, wherein the carrier comprises silicon dioxides, salts, sugars, or a combination thereof.

107. The method of claim 104, wherein the carrier comprises a biopolymer.

108. The method of claim 107, wherein the biopolymer is selected from the group consisting of a native starch, a modified starch, dextrin, cyclodextrin, inulin, arabic gum, pectin, and maltodextrin.

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