Enzyme-mediated reactive crystallization of steviol glycosides

Abstract

Provided herein are methods for processing steviol glycosides. The methods involve enzyme-mediated reactive crystallization. Products are provided that include crystals produced by enzyme-mediated reactive crystallization. Methods are provided for blending enzyme-mediated reactive crystallization products to provide high solubility. Methods are provided for producing highly soluble steviol glycoside products by evaporating and drying an enzyme-mediated reactive crystallization mother liquor. The steviol glycoside crystals of the present disclosure increase the specific sucrose equivalent value of leaves, improving taste, reducing cost, and improving quality.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 419,612, filed October 26, 2022, U.S. Provisional Application No. 63 / 429,342, filed December 1, 2022, and U.S. Provisional Patent Application No. 63 / 443,104, filed February 3, 2023, the contents of each of which are incorporated herein by this reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (ARZE_039_03WO_SeqList_ST26.xml, size: 5,350 bytes, created on October 24, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to methods for enzyme-mediated reactive crystallization of steviol glycosides. [Background technology]

[0004] Excessive sugar consumption in the human diet is associated with poor human health and increased healthcare costs. Replacing sugar with low-calorie, high-intensity sweeteners may address these problems.

[0005] The plant species Stevia rebaudiana is cultivated for its sweet leaves and is traditionally used as a sweetener. Its leaves contain significant amounts of more than 10 different steviol glycosides, most of which exhibit a moderately intense sweetness. In most varieties of Stevia rebaudiana, the primary steviol glycosides in the leaves are stevioside and rebaudioside A. Therefore, stevia leaves contain approximately 10% protein, 4% fat, 8% ash, 5-15% fiber, and 60-70% carbohydrates. These carbohydrates include reducing sugars, fructooligosaccharides, polysaccharides, stevioside, rebaudioside A and C, dulcosides, and other minor species. Additional leaf components include alkaloids, flavonoids, lutein, chlorophyll, and polyphenols. For example, hot water leaf extracts may contain trace amounts of all of these leaf components.

[0006] The present disclosure describes compositions and methods for processing Stevia rebaudiana to create soluble concentrations of stevioside. Summary of the Invention

[0007] The present disclosure provides methods for processing steviol glycosides, e.g., derived from stevia leaf extract or fermentation, wherein the method further comprises an enzymatic reactive crystallization step. In embodiments, the present disclosure further provides compositions prepared according to such methods described herein. In embodiments, the present disclosure further provides co-crystal compositions comprising two or more of rebaudioside M, rebaudioside D, and rebaudioside E.

[0008] In various embodiments, the methods of the present disclosure can be used to make and isolate steviol glycosides from stevia leaves or from some fermentation broths via aqueous solvent systems, optionally without the use of any solvents, resins, or membranes.

[0009] In embodiments, by producing a composition including two or more of rebaudioside M, rebaudioside D, and rebaudioside E, the bitter taste impact commonly associated with steviol glycosides, particularly rebaudioside A and stevioside, is avoided. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a flow diagram of a process for enzyme-responsive crystallization (“ERX”) of a multi-component mixture of steviol glycosides with enhanced solubility, resulting in a crystalline steviol glycoside product. [Figure 2] FIG. 2 is a flow diagram based on the ERX process of FIG. 1, including an additional cooling and / or evaporation step and / or additional recycle of mother liquor and / or seed crystals. [Figure 3] 1 is a flow diagram of the ERX process carried out in two sequential steps of conversion of a feed comprising a multi-component steviol glycoside mixture including at least rebaudioside D and stevioside to produce rebaudioside D-enriched co-crystals and rebaudioside M-enriched co-crystals. [Figure 4] FIG. 4 is a flow diagram of the ERX process described in FIG. 3, including an additional cooling and / or evaporation step. [Figure 5] 1 is a flow diagram of the ERX process implementing a rebaudioside A-enriched recycle stream, where the recycle stream is mixed with a stevioside-enriched stream in a mixer dissolver to create a multi-component steviol glycoside mixture with improved solubility. Rebaudioside A-enriched crystals can then be produced using a β-1,3-glycosylation reaction system. [Figure 6] 1 is a flow diagram of an ERX process implementing a rebaudioside A-enriched recycle stream, where the recycle stream is mixed with a stevioside-enriched stream in a mixer dissolver to produce a multi-component steviol glycoside mixture with improved solubility. Rebaudioside A-enriched crystals can then be produced using a β-1,2-glycosylation and β-1,3-glycosylation reaction system. [Figure 7]1 is an image of rebaudioside D-enriched cocrystals produced by the ERX method described herein, in which RA50 is placed in a 15 liter reactor scale, dried in a vacuum oven, suspended in water, and placed under an optical microscope at 400x magnification. [Figure 8] 1 shows the crystallization kinetics of the ERX process as measured by the volume percent of suspended solids present during a 150 liter scale ERX run. [Figure 9] FIG. 1 is a graphical representation of the reaction rate of the ERX process as measured by stevioside conversion and rebaudioside A conversion in a 150 liter scale ERX run. [Figure 10] This is a graphical representation of the crystallization kinetics from Figure 8 overlaid with the kinetics from Figure 9, showing the time lag or offset between the reaction and crystal formation. The square and triangle tracers in Figure 10 show the conversion of stevioside and Reb A to products as measured by HPLC. The open circles indicate the amount or volume of suspended solids (VSS). [Figure 11] 1 is a graphical representation of a theoretical calculation of sucrose equivalents per kilogram of harvested leaves, illustrating the practical value of effective sweetness obtained by enzymatically upgrading leaf extracts. Steviol glycosides containing rhamnosyl moieties, which are excluded from this graph for visual clarity, have significantly lower sucrose equivalents. [Figure 12] UV-Vis spectrophotometric scan of crystals produced on a laboratory scale by the ERX method, showing excellent purity in terms of the absence of impurities adsorbing at 254 nm, 270 nm, 280 nm, and 315 nm. [Figure 13] 1 shows the enzymatic reactions carried out by beta-1,2-glycosyltransferase and beta-1,3-glycosyltransferase to convert stevioside to rebaudioside M. [Figure 14]The stevioside molecule is shown, indicating which carbon (C13, (G) reaction, or C19 (E) reaction) beta-1,2-glycosyltransferase and beta-1,3-glycosyltransferase can attach an additional glucose unit during conversion to rebaudioside M. [Figure 15] FIG. 1 is a flow diagram of an ERX process in which purification of the ERX product is accomplished by heating the reaction slurry to dissolve the ERX product, form hot break, and remove the hot break, followed by crystalline modification by cooling crystallization. [Figure 16] FIG. 1 is a flow diagram of an ERX process in which purification of the ERX product is accomplished by heating the reaction slurry to dissolve the ERX product, form a hot break, and remove the hot break, followed by crystalline modification by evaporative crystallization. [Figure 17] FIG. 1 is a flow diagram of an ERX process in which purification of the ERX product is accomplished via dissolving recovered ERX crystals in a solvent with or without heating, followed by crystal reformation via either cooling or evaporation or mixed mode crystallization. No hot break or solvent break is removed. [Figure 18] FIG. 1 is a flow diagram of an ERX process that accomplishes purification of the ERX product by dissolving recovered ERX crystals in a solvent with or without heating, providing hot break or solvent break formation and hot or solvent break removal, followed by crystal reformation via either cooling or evaporation or mixed mode crystallization. [Figure 19] FIG. 19 is a flow diagram of an ERX process in which purification of an ERX product is achieved through a sequential combination of the processes shown in FIGS. 15 and 18. [Figure 20] 1 shows a single component feed into ERX where temperature has enhanced the solubility of the feed. [Figure 21]21 is a graphical representation of mother liquor recycling. FIG. 21 shows high-performance liquid chromatography results showing the RXN1 control with 86% Reb M at 3 hours. This can be compared to the RXN2 sample with recycled mother liquor, which has a slower rate of Reb M formation. For the RXN2 sample with recycled mother liquor, Reb M reached 31% at 6 hours, and Reb AM, Reb D, and Reb E reached 47% at 6 hours. For the RXN1 control, Reb AM, Reb D, and Reb E reached 1% after 3 hours. [Figure 22] 1 is a tabular representation of steviol glycoside production at various time points in the presence and / or absence of glycosyltransferases, sucrose synthase, ADP, and / or sucrose. [Figure 23] 1 is a tabular representation of steviol glycoside production at various time points in the presence and / or absence of recycled glycosyltransferases, sucrose synthase, ADP, and / or sucrose. DETAILED DESCRIPTION OF THE INVENTION

[0011] Non-limiting examples of various aspects and variations of the present invention are described herein and illustrated in the accompanying drawings.

[0012] Abbreviation Abbreviations used herein are shown in Table 1.

[0013] [Table 1]

[0014] definition As used herein, the term "a" or "one" refers to one or more of that entity, i.e., it can refer to a plural reference. Thus, the terms "a," "one," "one or more," and "at least one" are used interchangeably herein. In addition, reference to an "element" by the indefinite article "a" or "one" does not exclude the possibility that more than one element is present, unless the context clearly requires that there be only one element.

[0015] Throughout this specification, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method being used to determine the value, or the variation that exists between samples measured. Also, unless expressly stated otherwise or clear from context, the term "approximately" means within 10% of the reported numerical value (except when such number is greater than 100% or less than 0% of the possible values). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of the range or each recited value in the series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalent terms.

[0016] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant across a window of residue, e.g., nucleotide or amino acid, alignment. The "percent identity" of an aligned segment of a test sequence and a reference sequence is the number of identical residues shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a defined small portion of the reference sequence. The "percent identity" is the percent identity multiplied by 100. Comparison of sequences to determine percent identity can be accomplished by several well-known methods, including, for example, using mathematical algorithms such as the BLAST suite of sequence analysis programs (BLOSUM62 matrix, gap opening penalty -1, gap extension penalty -1, GapAlign set to true). Unless otherwise specified, the term "sequence identity" in the claims refers to sequence identity calculated by Clustal Omega® using default parameters. Clustal Omega uses the HHalign algorithm and its default settings as its core alignment engine. The algorithm is described in Soding, J. (2005) 'Protein homology detection by HMM-HMM comparison'. Bioinformatics 21, 951-960. The default transition matrix is ​​Gonnet, the gap opening penalty is 6 bits, and the gap extension is 1 bit.

[0017] As used herein, "biocatalyst" or "biocatalytic" refers to the use of natural catalysts, such as protein enzymes, to carry out chemical transformations on organic compounds. Biocatalysis is also known as biotransformation or biosynthesis. Both isolated biocatalytic methods and whole-cell biocatalytic methods are known in the art. Biocatalytic protein enzymes can be naturally occurring proteins or recombinant proteins.

[0018] As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably, unless otherwise indicated by context, to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, typically DNA.

[0019] As used herein, "expression" refers, depending on the context, to either or both steps in the two-step process by which a polynucleotide is transcribed into mRNA, and the transcribed mRNA is subsequently translated into a polypeptide.

[0020] Regulatory elements, such as enhancers and promoters, can be "homologous" or "heterologous." A "homologous" regulatory element is one that is naturally linked to a particular polynucleotide in the genome; for example, it can be a promoter naturally found in an organism upstream of the encoded polypeptide. A "heterologous" regulatory element is one that is juxtaposed to a polynucleotide by recombinant molecular biology techniques, but is not in a combination found in nature. Often, promoters, enhancers, and other regulatory elements are heterologous to facilitate expression of a polypeptide in a host cell other than the host cell in which the polypeptide naturally occurs. Thus, as used herein, "heterologous expression" refers to producing mRNA and / or a polypeptide in a host cell, such as a microorganism, where the polynucleotide is not naturally found or one or more regulatory elements operably linked to the polynucleotide are not naturally found in the host cell.

[0021] As used herein, the terms "microorganism" or "microorganism" should be interpreted broadly. These terms are used interchangeably and include, but are not limited to, the two prokaryotic domains, bacteria and archaea, and certain eukaryotic fungi and protists. In some embodiments, the present disclosure refers to the "microorganisms" or "microorganisms" in the lists and figures present in this disclosure. This characterization can refer not only to the identified taxonomic genera, but also to the identified taxonomic species, as well as various novel and newly identified or engineered strains of any organism in the tables or figures. The same characterization applies to the listing of these terms elsewhere in this specification, such as in the Examples.

[0022] As used herein, "enzymatic reactivity" refers to the use of natural catalysts, such as protein enzymes, to carry out chemical transformations on organic compounds. Biocatalysis is also known as biotransformation or biosynthesis. Biocatalytic protein enzymes can be naturally occurring proteins or recombinant proteins.

[0023] The term "polypeptide" is used herein to refer to a molecule of two or more subunits of amino acids linked by peptide bonds. Typically, but not always, a polypeptide contains several hundred amino acids, e.g., about 400 to about 900 amino acids.

[0024] As used herein, the term "steviol glycoside(s)" includes, but is not limited to, naturally occurring steviol glycosides (e.g., steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B ("Reb B"), rebaudioside G ("Reb G"), stevioside, rebaudioside C ("Reb C"), rebaudioside F ("Reb F"), rebaudioside ("Reb A"), rebaudioside I ("Reb I"), rebaudioside E ("Reb E"), rebaudioside E2 ("Reb "Reb E2"), rebaudioside AM ("Reb AM"), rebaudioside H ("Reb H"), rebaudioside L ("Reb L"), rebaudioside K ("Reb K"), rebaudioside J ("Reb J"), rebaudioside M ("Reb M"), rebaudioside D ("Reb D"), rebaudioside N ("Reb N"), rebaudioside O ("Reb O"), rebaudioside Q ("Reb Q")), synthetic steviol glycosides (e.g., enzymatically glucosylated steviol glycosides), and combinations thereof.

[0025] As used herein, rebaudioside X may refer to a highly purified variant of rebaudioside M.

[0026] As used herein, a "multi-component mixture of steviol glycosides" refers to a mixture in which at least two different steviol glycosides are present.

[0027] As used herein, "solubility-enhanced" refers to a concentration by weight of a multi-component mixture of a component of interest, e.g., steviol glycoside, that is greater than the solubility of the individual pure components for steviol glycoside, typically in an aqueous solution.

[0028] As used herein, the "specific sucrose equivalent value" of a leaf is the equivalent mass (in kilograms, "kg") of sucrose that can be obtained from 1 kg (dry basis) of leaves at harvest.

[0029] As used herein, "hot break" refers to impurities that become insoluble upon heating. Hot break can also incorporate insoluble impurities that become trapped in the hot break solids.

[0030] As used herein, "solvent break" refers to impurities that become insoluble when dissolved in a solvent other than water, such as an alcohol (e.g., ethanol, methanol, isopropanol). The solvent break step can be followed by heating, which increases the break or accumulation of the impurities.

[0031] As used herein, "recrystallization" refers to a second crystallization step following a first crystallization step. As used herein, "cooling crystallization" refers to crystallization in which the temperature is reduced. As used herein, "evaporative crystallization" refers to crystallization by removal of the solvent, thereby increasing the concentration. The solvent may be water or another solvent such as ethanol.

[0032] As used herein, the term "feedstock composition" refers to any composition (generally an aqueous solution) containing one or more steviol glycosides, which serve as substrates for biotransformation.

[0033] As used herein, "NDP" refers to nucleotide diphosphate. Nucleotide diphosphate can be any form of adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), thymidine diphosphate (TDP), and guanosine diphosphate (GDP). "ADP" refers to any form of adenosine diphosphate, including, but not limited to, anhydrous adenosine 5'-diphosphate, adenosine 5'-diphosphate disodium salt, adenosine 5'-diphosphate monopotassium salt dihydrate, adenosine 5'-diphosphate sodium salt, adenosine-5'-diphosphate disodium salt dihydrate, and adenosine 5'-diphosphate bis(cyclohexylammonium) salt.

[0034] Note that "glycosyl" refers to all sugar residues. Glucosyl is specific for glucose residues.

[0035] A cocrystal is defined as a homogeneous crystalline substance composed of two or more molecules of well-defined stoichiometric amounts held together by noncovalent forces. The FDA further defines that the pKa difference between the coformers must be less than 1, indicating nonionic species and minimal proton sharing. A eutectic melting point is defined as the temperature and molar ratio at which two solids are completely miscible and is recognizable as the minimum coherent point on a phase diagram. Both cocrystals and eutectic melting points exhibit similar short-range order. However, cocrystals differ in that they also exhibit long-range order. Whether a cocrystal or eutectic melting point forms depends on the relative strength of the enthalpic advantage of packing versus the entropic loss due to order.

[0036] In general, the present disclosure provides methods for reacting a mixture of one or more steviol glycosides with one or more glycosyltransferases to create a new mixture from which the steviol glycosides crystallize or precipitate. To this end, the enzyme-mediated reactive crystallization (ERX) method described herein includes a feed composition (or raw material composition) containing one or more steviol glycosides that are soluble at moderate to high concentrations. The feed composition is contacted with an enzyme in a reaction composition that converts at least a portion of the feed composition to create a new, less soluble species (or target steviol glycoside(s)). As a result, at least a portion of the converted target steviol glycoside(s) will crystallize or precipitate. In this way, the target steviol glycoside(s) can be recovered, for example, by simple filtration instead of other intensive methods requiring, for example, more complex and low-throughput resin adsorption / desorption or crystallization. The steviol glycoside component(s) of the feedstock composition serve as substrate(s) for the production of target steviol glycoside(s), as described herein. The target steviol glycoside(s) chemically differ from the corresponding substrate steviol glycoside(s) by one or more additional sugar monomer units. In some embodiments, the target(s) of the target steviol glycoside differ chemically from the corresponding substrate steviol glycoside(s) by one or more additional glucose monomer units.

[0037] In one embodiment, the enzymatic reaction comprises reacting a starting steviol glycoside composition with an NDP-sugar and one or more NDP-glycosyltransferase polypeptides, thereby producing a mixture of precipitated steviol glycosides and, optionally, soluble steviol glycosides. The resulting precipitated steviol glycosides and any soluble steviol glycosides may comprise the target steviol glycoside and, optionally, unreacted steviol glycosides. In one embodiment, the enzymatic reaction comprises reacting a starting steviol glycoside composition with an NDP-glucose and one or more NDP-glycosyltransferase polypeptides, thereby producing a mixture of precipitated steviol glycosides and, optionally, soluble steviol glycosides. The resulting precipitated steviol glycosides and soluble steviol glycosides may consist of the target steviol glycoside and, optionally, unreacted steviol glycosides.

[0038] Embodiments of the present disclosure utilize a SuSy NDP-glucose recycling system in combination with one or more NDP-glycosyltransferase polypeptides to convert a raw steviol glycoside composition to a target steviol glycoside composition. The enzymatic reaction can include reacting a raw steviol glycoside composition with an NDP, a source of sugar monomers, one or more NDP-glycosyltransferase polypeptides, and an enzymatic NDP-sugar recycling system, thereby producing a slurry of precipitated steviol glycosides and, optionally, soluble steviol glycosides. The resulting precipitated steviol glycosides and soluble steviol glycosides can consist of the target steviol glycoside and, optionally, unreacted steviol glycosides. The enzymatic reaction can include reacting substrates steviol glycoside, sucrose, and NDP with one or more NDP-glycosyltransferase polypeptides and sucrose synthase, thereby producing a slurry of precipitated and optionally soluble steviol glycosides. The resulting precipitated and soluble steviol glycosides can consist of the target steviol glycoside and, optionally, unreacted steviol glycoside.

[0039] A feedstock composition of Reb A and / or stevioside can be reacted with sucrose, NDP, a B12GT polypeptide, and a SuSy polypeptide to produce Reb D and Reb E, respectively. A feedstock composition of RA50 can be reacted with sucrose, ADP, an engineered B12GT polypeptide, and an engineered SuSy polypeptide to produce precipitated steviol glycosides composed primarily of Reb D and a soluble mother liquor composed primarily of Reb E.

[0040] A feedstock composition of Reb A and / or stevioside can be reacted with sucrose, NDP, a B12GT polypeptide, a B13GT polypeptide, and a SuSy polypeptide to produce Reb M. In another embodiment, a feedstock composition of RA50 is reacted with sucrose, ADP, an engineered B12GT polypeptide, an engineered B13GT, and an engineered SuSy polypeptide to produce precipitated steviol glycosides composed primarily of Reb M and a soluble mother liquor composed primarily of Reb M.

[0041] Regarding rebaudiosides, the major steviol glycosides present in Stevia rebaudiana leaves have a potentially undesirable taste profile due to a lingering bitter aftertaste. To increase the applicable uses of sweeteners, the taste profile needs to be improved. Furthermore, higher molecular weight steviol glycosides, such as rebaudioside D and rebaudioside M, have greater sweetness per mole and reduced lingering bitterness. Enzymatic glycosylation of harvested steviol glycosides can increase the value of stevia crops simply by increasing the sweetness equivalents per harvested unit.

[0042] While many steviol glycosides have only glucosyl residues derived from glucose, some have one or more other sugar residues, such as xylose and rhamnose, among others. For example, rebaudioside F has three glucosyl residues and one xylosyl residue, while rebaudioside C has three glucosyl residues and one rhamnosyl residue.

[0043] The enzymatic reaction can yield linear or branched chains of as many as nine glucosyl or other sugar residues. Steviol glycosides with glucosyl residues are shown in Table 2, where the non-glucosylated core molecule is steviol. In stevia leaves, the level of steviol can be very low and often not detectable by conventional analytical methods. Furthermore, stevia leaf seeds with five or more glucosyl residues may not be present or may be present only at very low concentrations and therefore may not be detectable by conventional measurements.

[0044] [Table 2]

[0045] The value of stevia leaf extract can be increased by improving taste, reducing cost, and improving the purity of steviol glycosides. For example, assume that 1 kilogram (kg) of stevia leaves (dry basis) contains 83.85 grams (g) of stevioside, 45.15 g of rebaudioside A, and smaller amounts of additional steviol glycosides. The total amount of stevioside and rebaudioside A in said 1 kg of leaves contains a total of 0.15 moles, which has approximately 20.6 kg of sucrose equivalents (SE) per kg of leaves. When the same stevia leaves are processed using the method described in this disclosure, the 0.15 moles are upgraded to 0.15 moles of rebaudioside M, which is the theoretical case at a molar yield of 100%. This now has an SE of 53.4 kg per kg of leaves. This is shown in Figure 11, which shows that the measured sucrose equivalent depends on the number of glycosyl or xylosyl moieties present in the steviol glycosides. The process herein increased the effective yield of SE per kg of harvested leaves by 2.6-fold (53.4 / 20.6), which provides a substantial economic benefit.

[0046] Pure steviol glycosides are poorly soluble in water. Solubility measurements can reflect true thermodynamic solubility or short-term kinetic solubility. Kinetic solubility values ​​arise when isomeric forms are prepared that are not thermodynamically stable over long time scales but have higher solubility over short time scales. Some steviol glycoside species have free carboxylic acid groups. These species tend to be more soluble. Nevertheless, steviol glycosides are generally poorly soluble in water, as shown in Table 3 (particular measurements are for species that may not be completely pure).

[0047] [Table 3]

[0048] Typically, steviol glycosides can be recovered from Stevia rebaudiana leaves by hot water extraction, various clarification steps, the use of resin adsorption steps, and / or alcohol desorption steps. This may be followed by additional aqueous and non-aqueous recrystallization steps, as well as additional resin adsorption and desorption steps. Due to the low solubility of pure steviol glycosides in water, these attempts to produce rebaudiosides from leaf extracts are limited to low reaction concentrations, making it difficult to isolate the converted rebaudiosides from the "mother liquor." Therefore, there is a need for improved processes that increase the yield of converted rebaudiosides and improve their isolation and purification. Such improved processes can minimize or avoid the use of resins and solvents, as well as minimize the amount of water processed. Furthermore, such improved processes address the need for more cost-effective processing methods for enzymatic glycosylation by allowing for higher use concentrations (and therefore smaller reactor volumes).

[0049] To this end, the present disclosure provides a method for efficiently processing steviol glycosides by utilizing reactive crystallization to improve the isolation and purification of rebaudioside. The method described herein leverages the enhanced solubility of stevioside along with rebaudioside A to improve reaction concentrations, thereby naturally improving the taste profile of the target composition.

[0050] The present disclosure employs a reactive crystallization step to aid in the processing of steviol glycosides. When supersaturation of a crystalline compound is created by a chemical reaction, the process is known as reactive crystallization. Reactive crystallization, or precipitation, involves a reaction between raw materials to form a solute that crystallizes into a solid product. For example, in the steviol glycoside example herein, a reaction between a steviol glycoside and a glycosyltransferase, or other enzyme capable of adding sugar monomers to the steviol glycoside, produces a new steviol glycoside that is less soluble in solution than the precursor steviol glycoside and therefore precipitates as crystals. This may be the case, for example, when B13GT adds a sugar monomer to Reb D, which is converted to Reb M and precipitates from solution. An example of industrial relevance is the liquid-phase oxidation of paraxylene to terephthalic acid.

[0051] In some variations, the present disclosure describes methods for producing cocrystals as enzyme-mediated reactive crystallization crystalline products. As introduced above, a cocrystal is defined as a homogeneous crystalline substance composed of two or more molecules of well-defined stoichiometric amounts held together by non-covalent forces. The FDA further defines that the pKa difference between the coformers must be less than 1, indicating non-ionic species and minimal proton sharing. If one of the source materials is normally liquid, the combined substance is called a solvate. As described herein, a eutectic melting point is defined as the temperature and molar ratio at which two solids are completely miscible and is recognizable as the point of minimum coincidence on a phase diagram. Both cocrystals and eutectic melting points exhibit similar short-range order. However, cocrystals differ in that they also exhibit long-range order. Whether a cocrystal or eutectic melting point forms depends on the relative strength of the enthalpic advantages of packing versus the entropic losses due to order. A sufficient thermodynamic model may enable predictive calculations through the minimization of the free energy of crystalline phase mixtures. Co-crystal phases can be identified and characterized by thermal analysis and diffraction analysis, including single-crystal X-ray diffraction. The energy of intermolecular interactions within a crystal can be calculated by solid-state DFT and PIXEL methods. Such calculation schemes indicate the total energy of intermolecular interactions. The thermodynamic function of co-crystal formation can be estimated from the solubility of the co-crystal and the solubility of the corresponding pure compound at various temperatures. The driving force of the co-crystal formation process is the Gibbs energy [kJ·mol -1 ], and the endothermic or exothermic enthalpy of formation, as well as the entropy, can be estimated. However, no satisfactory thermodynamic model is available for systems containing steviol glycoside components.

[0052] An ideal, consistent, theory-based approach to the design of reactive crystallization systems would consider enzyme kinetics, diffusion effects, and crystallization kinetics, as well as mass transfer. Appropriate descriptions include reaction, mass transfer, dissolution Dankeler numbers, and nucleation and growth numbers. This allows estimation of the impact of individual steps and their influence on crystal size distribution and crystallizer productivity. To this end, kinetic models of thermal and non-thermal inactivation of the enzyme must also be considered. Secondary crystallizer processes, such as aging, aggregation, and fragmentation, may also occur, complicating reactive crystallizer design.

[0053] Referring now to the drawings, a method for producing and isolating steviol glycoside crystals is described with reference to method 100 in Figure 1. Dashed lines indicate optional steps.

[0054] In optional step 101 of method 100, Stevia rebaudiana leaves and / or stems can be received. In optional steps 102 and 103 of method 100, Stevia rebaudiana leaves and / or stems can be processed to extract steviol glycosides and undergo initial clarification and / or concentration. Extraction can be performed by a batch parabolic tank equipped with a spiral mixer, a batch rotating cylinder extractor, a batch percolation extractor, a horizontal continuous moving belt extractor, a horizontal countercurrent tubular screw extractor, a vertical countercurrent tubular screw extractor, a segmented rotating basket extractor, and a pressurized condensed flow extractor. Clarification can include thermal coagulation of proteins and impurities, diatomaceous earth treatment, ion exchange, and chemical coagulation using calcium carbonate and / or ferric chloride. Concentration can include membrane processing (e.g., nanofiltration), evaporation, and adsorption of one or more steviol glycosides to a solid adsorbent followed by elution and removal with alcohol to obtain a concentrated leaf extract. Fractions may be obtained during this process and reintroduced in step 105 as shown in FIG.

[0055] In step 104 of method 100, a feedstock composition or solubility-enhanced steviol glycoside mixture can be produced from the processed stevia leaf extract. In some embodiments, a concentrated crude stevia leaf hot water extract can be provided as the feedstock composition. In various embodiments, the steviol glycoside mixture can be a single component or a mixture of multiple components of steviol glycosides. In various embodiments, the feedstock composition includes one or more steviol glycosides. In various embodiments, the feedstock composition can include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. The feedstock composition may comprise an RA50 of at least 550 g / L, at least 600 g / L, at least 650 g / L, at least 700 g / L, at least 750 g / L, at least 800 g / L, at least 850 g / L, at least 900 g / L, at least 950 g / L, or at least 1000 g / L. Alternatively, the feedstock composition may be obtained as an already processed version of Stevia rebaudiana. Additionally, the feedstock composition may be synthetic or at least partially purified and commercially available, or prepared by other methods.

[0056] In various embodiments, the feedstock composition may comprise a purified substrate steviol glycoside. For example, the feedstock composition may comprise, on an anhydrous basis, greater than 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99.6% by weight of one or more steviol glycosides. In another embodiment, the feedstock composition comprises a partially purified substrate steviol glycoside composition. For example, the feedstock composition may comprise, on an anhydrous basis, greater than 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50% by weight of one or more substrate steviol glycosides. In another embodiment, the substrate steviol glycoside is purified rebaudioside A or an isomer thereof. In certain embodiments, the substrate steviol glycoside comprises greater than 99% rebaudioside A or an isomer thereof by weight on an anhydrous basis. In another embodiment, the substrate steviol glycoside comprises partially purified rebaudioside A. In certain embodiments, the substrate steviol glycoside comprises greater than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% rebaudioside A by weight on an anhydrous basis. Furthermore, in another embodiment, the substrate steviol glycoside comprises purified stevioside or an isomer thereof. In certain embodiments, the substrate steviol glycoside comprises greater than 99% stevioside or an isomer thereof by weight on an anhydrous basis. In another embodiment, the substrate steviol glycoside comprises partially purified stevioside. In certain embodiments, the substrate steviol glycoside comprises greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% by weight stevioside on an anhydrous basis.

[0057] In step 105 of method 100, the feedstock composition can be reacted with a reactant composition to perform enzymatic reactive crystallization (ERX), which can be carried out batchwise or semi-continuously.

[0058] In various embodiments, the reaction composition comprises at least one enzyme. In some embodiments, crude enzyme mixtures, partially purified enzyme mixtures, and / or purified enzyme mixtures may be used. In various embodiments, at least one enzyme described herein is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces spp., Aspergillus spp., Pichia spp., and Bacillus spp. In various embodiments, at least one enzyme is expressed in E. coli. In various embodiments, at least one enzyme is expressed in Pichia pastoris. In various embodiments, at least one enzyme may be thermotolerant. In various embodiments, at least one enzyme may have enhanced solubility.

[0059] In various embodiments, the at least one enzyme is added to the reaction composition at the beginning of the reaction. For example, if the at least one enzyme includes β12GT and β13GT, the glycosyltransferase may be provided together at the beginning of the reaction. In another embodiment, a first portion of the at least one enzyme may be added at the beginning of the reaction, and one or more additional portions of the at least one enzyme may be added over the course of the reaction. In various embodiments, the aqueous reaction mixture includes one or more of sodium chloride, sodium acetate, potassium chloride, sodium sulfate, and sodium phosphate to help maintain enzyme solubility.

[0060] In various embodiments, at least one enzyme does not produce a significant amount of product having a molecular weight greater than about 1300 g / mol. For example, the enzyme product may have a molecular weight between less than about 1300 g / mol, less than about 1200 g / mol, less than about 1100 g / mol, less than about 1000 g / mol, less than about 900 g / mol, less than about 800 g / mol, less than about 700 g / mol, less than about 600 g / mol, less than about 500 g / mol, less than 400 g / mol, less than about 200 g / mol, and less than about 100 g / mol.

[0061] In various embodiments, the at least one enzyme may be provided in any suitable form, including free, immobilized, or whole cell system. The degree of purity of the at least one enzyme may vary. For example, it may be provided as crude, semi-purified, or purified enzyme preparation(s). In one embodiment, the at least one enzyme is free. In another embodiment, the at least one enzyme is immobilized on a solid support, e.g., an inorganic or organic support. In some embodiments, the solid support is derivatized cellulose, glass, ceramic, methacrylate, styrene, acrylic, metal oxide, or a membrane. In some embodiments, the at least one enzyme is immobilized on the solid support by covalent bonding, adsorption, crosslinking, entrapment, or encapsulation. In some embodiments, the at least one enzyme is provided in the form of a whole cell system, e.g., as live fermenting microbial cells, or as killed and stabilized microbial cells, or in the form of a cell lysate.

[0062] In various embodiments, the at least one enzyme comprises one or more glycosyltransferases. The one or more glycosyltransferases may be wild-type glycosyltransferases, non-naturally occurring engineered glycosyltransferases, polypeptides having glycosyltransferase activity, or combinations thereof. In various embodiments, the one or more glycosyltransferases may comprise β-1,2-glycosyltransferases, β-1,3-glycosyltransferases, and combinations thereof. β-1,2-glycosyltransferase (B12GT) and / or β-1,3-glycosyltransferase (B13GT) may modify steviol glycosides by adding sugars thereto using an NDP-sugar, such as ADP-glucose, as a sugar donor. In embodiments, the NDP-sugar may comprise one of galactose, glucose, xylose, glucosamine, galactosamine, glucuronic acid, galactofuranose, mannose, fucose, rhamnose, acetylneuraminic acid, and mannooctanoic acid.

[0063] Figure 13 is a flow diagram showing the respective roles of B12GT and B13GT in the stevioside to Reb M pathway. In various embodiments, B12GT may add a beta-linked glucose monomer to the C2' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT converts stevioside to Reb E and Reb A to Reb D using an ADP-glucose sugar donor. In various embodiments, B12GT may be a non-naturally occurring engineered B12GT glycosyltransferase described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety. In various embodiments, B13GT may add a beta-linked glucose monomer to the C3' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT uses an ADP-glucose sugar donor to convert Reb E to Reb D and Reb D to Reb M. In embodiments, B13GT can be a non-naturally occurring engineered B13GT glycosyltransferase as described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0064] In various embodiments, when the reaction composition includes at least one glycosyltransferase enzyme, it is added by reacting the feedstock composition with the reaction composition.

[0065] In various embodiments, the at least one enzyme comprises one or more sucrose synthases. The one or more sucrose synthases may be wild-type sucrose synthases, non-naturally occurring engineered sucrose synthases, polypeptides having sucrose synthase activity, or combinations thereof. In various embodiments, the one or more sucrose synthases (SuSy) may convert NDP and sucrose using a sucrose sugar donor to form NDP-glucose and fructose. For example, the one or more SuSy may convert ADP and sucrose to ADP-glucose and fructose. In various embodiments, the SuSy may be a non-naturally occurring engineered SuSy described in International Patent Application No. PCT / US2022 / 016820, the entire contents of which are incorporated herein by reference.

[0066] In embodiments, the at least one enzyme comprises a wild-type, non-naturally occurring, or engineered phosphoglucomutase or a polypeptide having phosphoglucomutase activity. In embodiments, the reaction composition may comprise a pyrophosphate reagent and / or disodium pyrophosphate.

[0067] In various embodiments, the reaction composition used in the reaction of step 105 of method 100 can be an aqueous medium. The reaction composition can include at least one buffer selected from the group including acetate buffer, citrate buffer, HEPES, and phosphate buffer. In various embodiments, the buffer can have a pH of about 4 to about 10, about 4.5 to about 9, about 5 to about 8, about 5.5 to about 7, and about 6 to about 6.5.

[0068] In various embodiments, the reaction in step 105 of method 100 can be carried out at a temperature of about 10° C. to about 80° C., about 10° C. to about 20° C., about 20° C. to about 30° C., about 30° C. to about 40° C., about 40° C. to about 50° C., about 50° C. to about 60° C., about 60° C. to about 70° C., and about 70° C. to about 80° C. In one embodiment, the reaction can be carried out at about 60° C.

[0069] In various embodiments, the reaction in step 105 of method 100 can be carried out for a period of about 0.1 hours to about 10 hours, about 0.1 hours to about 9.5 hours, about 30 minutes to about 9 hours, about 1 hour to about 8.5 hours, about 2 hours to about 8 hours, about 3 hours to about 7.5 hours, about 4 hours to about 7 hours, and about 5 hours to about 6.5 hours. In various embodiments, the reaction can be carried out for a period of about 6 hours.

[0070] In various embodiments, the reaction in step 105 of method 100 is carried out at a pH of about 3 to about 10, about 4 to about 9, about 5 to about 8, and / or about 6 to about 7. In various embodiments, the reaction is carried out at a pH of about 5 to about 8.

[0071] In embodiments, the reaction of step 105 of method 100 is carried out in a vessel designed to support effective separation, efficient washing, and robust scale-up of macromixing (bulk convection), mesomixing (turbulence), and micromixing (including molecular diffusion and momentum diffusion related) to produce crystals large enough for efficient scale-up. In embodiments, the vessel may be a spinner flask, a conical tank, or the like.

[0072] In various embodiments, the reaction composition includes a nucleotide cofactor that can be converted by sucrose synthase to an NDP-sugar (e.g., NDP-glucose). In some embodiments, the nucleotide can be ADP, GDP, UDP, CDP, or TDP. In some embodiments, the nucleotide can be a non-UDP nucleotide (i.e., ADP, GDP, CDP, or TDP). In other embodiments, the nucleotide is ADP. In certain embodiments, the reaction can be carried out with, for example, ADP at a concentration of 0.01 mM to 10 mM, e.g., 0.01 mM to 0.05 mM, 0.05 mM to 0.1 mM, 0.1 mM to 0.5 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, or 5 mM to 10 mM. In certain embodiments, ADP is used at a concentration of 0.5 mM.

[0073] In embodiments, the NDP-sugar or sugar-phosphate is regenerated by transfer of a glycosyl moiety from a sugar dimer or oligomer, hi embodiments, the sugar dimer is sucrose or maltose.

[0074] In various embodiments, the reaction composition comprises sucrose and sucrose synthase, and the sucrose may be present at a sucrose concentration of about 100 mM to about 3 M, about 125 mM to about 2.5 M, about 150 mM to about 2 M, about 175 mM to about 1.5 M, about 200 mM to about 1 M, about 225 mM to about 500 mM, or about 250 mM to about 300 mM. In one example, the reaction can be carried out at a sucrose concentration of 292 mM.

[0075] In embodiments, the reaction of step 105 of method 100 can be monitored by a suitable method, including, but not limited to, high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS), thin layer chromatography (TEC), infrared spectroscopy (IR), or nuclear magnetic resonance (NMR).

[0076] After ERX is performed in step 105, the crystals are isolated by separation and / or washing in step 106 of method 100. In various embodiments, the result of ERX includes crystallized steviol glycosides and a "mother liquor," which includes the buffer and any steviol glycosides that remain soluble. In various embodiments, the precipitated steviol glycosides may be separated from the mother liquor by suitable means, including, but not limited to, vacuum filtration, centrifugation, and a chamber filter press. In various embodiments, the precipitated steviol glycosides may be further processed to increase the purity of the precipitated steviol glycosides. The precipitated steviol glycosides may be washed with water to remove hydrophilic reaction components, such as residual sugars and salts. In various embodiments, the precipitated steviol glycosides may be washed with a hydroalcoholic solvent to remove impurities. In various embodiments, the recovered, processed precipitated steviol glycosides may be further dried to form a powder. The precipitated steviol glycosides can be dried by any standard method, including, but not limited to, freeze drying, vacuum tray drying, spray drying, fluidized bed drying, filter mat drying, or rotary drum drying. In some embodiments, the dried ERX crystals can be milled to produce the desired particle size.

[0077] The resulting crystalline steviol glycoside composition comprises one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. The crystals may, in one example, comprise Reb D, Reb E, and Reb A. Reb D, Reb E, and Reb A may each comprise from about 0% to about 100% of the crystals. Reb D, Reb E, and Reb A may each comprise about 0.1% to about 20%, about 0.2% to about 18%, about 0.3% to about 16%, about 0.4% to about 14%, about 0.5% to about 12%, about 0.6% to about 10%, about 0.7% to about 8%, about 0.8% to about 6%, about 0.9% to about 4%, or about 1% to about 2% of the crystals. Reb D, Reb E, and Reb A may each comprise about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, about 47.5% to about 52.5%, or about 48% to about 52% of the crystals. Reb D, Reb E, and Reb A may each comprise about 70% to about 100%, about 72% to about 98%, about 74% to about 96%, about 76% to about 94%, about 78% to about 92%, about 80% to about 90%, about 82% to about 88%, and about 84% to about 86% of the crystals. In one example, the crystals contain, on an anhydrous basis, 70.9% Reb D, 17.4% Reb E, and 0.4% Reb A. The composition of steviol glycosides in the resulting mother liquor includes one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. In one example, the mother liquor may contain Reb E, Reb D, Reb A, and stevioside in a ratio of 12.2:8.1:1:1.3.However, the ratio between the concentrations of each steviol glycoside is highly variable and depends on the steviol glycoside(s) included in the reaction.

[0078] In embodiments, the mother liquor may be further processed in subprocess 110 of method 100. The output from subprocess 110, described in more detail below, may optionally be provided as a recycle stream input to the feedstock composition.

[0079] Each of the isolation in step 106 and the processing of the mother liquor in subprocess 110 produces a product. Specifically, ERX crystals are produced by step 106. The type of ERX crystals produced depends on the feedstock composition and the at least one enzyme used. In various embodiments, the ERX crystals can define the total suspended solids mass of the reaction, which can be from about 5% to about 50%, from about 10% to about 40%, and from about 15% to about 30%.

[0080] A variation of method 100 will now be described with reference to method 200 of FIG. 2. For the sake of brevity, certain repetitive descriptions will be omitted in favor of the corresponding descriptions above. In optional step 201 of method 200, Stevia rebaudiana leaves and / or stems may be received. In optional steps 202 and 203 of method 200, the Stevia rebaudiana leaves and / or stems may be processed to extract steviol glycosides and undergo initial clarification and / or concentration thereof. Fractions may be obtained during this processing and may be reintroduced in step 205, as shown in FIG. 2.

[0081] In step 204 of method 200, a feedstock composition or solubility-enhanced steviol glycoside mixture can be produced from the processed stevia leaf extract. In some embodiments, a concentrated crude stevia leaf hot water extract can be provided as the feedstock composition. In various embodiments, the steviol glycoside mixture can be a single component or a mixture of multiple components of steviol glycosides. In various embodiments, the feedstock composition includes one or more steviol glycosides. In various embodiments, the feedstock composition can include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. The feedstock composition may comprise an RA50 of at least 550 g / L, at least 600 g / L, at least 650 g / L, at least 700 g / L, at least 750 g / L, at least 800 g / L, at least 850 g / L, at least 900 g / L, at least 950 g / L, or at least 1000 g / L. Alternatively, the feedstock composition may be obtained as an already processed version of Stevia rebaudiana. Additionally, the feedstock composition may be synthetic or at least partially purified and commercially available, or prepared by other methods.

[0082] In step 205 of method 200, the feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In embodiments, the reaction composition includes at least one enzyme. In some embodiments, a crude enzyme mixture, a partially purified enzyme mixture, and / or a purified enzyme mixture can be used. In embodiments, at least one enzyme described herein is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces spp., Aspergillus spp., Pichia spp., and Bacillus spp. In embodiments, at least one enzyme is expressed in E. coli. In embodiments, at least one enzyme is expressed in Pichia pastoris. In embodiments, at least one enzyme can be thermotolerant. In embodiments, at least one enzyme can have enhanced solubility.

[0083] In various embodiments, at least one enzyme is added to the reaction composition at the beginning of the reaction. For example, if the at least one enzyme includes β12GT and β13GT, glycosyltransferase may be provided together at the beginning of the reaction. In another embodiment, a first portion of the at least one enzyme can be added at the beginning of the reaction, and one or more additional portions of the at least one enzyme can be added over the course of the reaction. For example, timing can be adjusted based on the expected conversion product and enzyme activity. In various embodiments, the aqueous reaction mixture includes one or more of sodium chloride, sodium acetate, potassium chloride, sodium sulfate, and sodium phosphate to help maintain enzyme solubility.

[0084] In various embodiments, the at least one enzyme may be provided in any suitable form, including free, immobilized, or whole cell system. The degree of purity of the at least one enzyme may vary. For example, it may be provided as crude, semi-purified, or purified enzyme preparation(s). In one embodiment, the at least one enzyme is free. In another embodiment, the at least one enzyme is immobilized on a solid support, e.g., an inorganic or organic support. In some embodiments, the solid support is derivatized cellulose, glass, ceramic, methacrylate, styrene, acrylic, metal oxide, or a membrane. In some embodiments, the at least one enzyme is immobilized on the solid support by covalent bonding, adsorption, crosslinking, entrapment, or encapsulation. In some embodiments, the at least one enzyme is provided in the form of a whole cell system, e.g., as live fermenting microbial cells, or as killed and stabilized microbial cells, or in the form of a cell lysate.

[0085] In various embodiments, the at least one enzyme comprises one or more glycosyltransferases. The one or more glycosyltransferases may be wild-type glycosyltransferases, non-naturally occurring engineered glycosyltransferases, polypeptides having glycosyltransferase activity, or combinations thereof. In various embodiments, the one or more glycosyltransferases may comprise β-1,2-glycosyltransferases, β-1,3-glycosyltransferases, and combinations thereof. β-1,2-glycosyltransferase (B12GT) and / or β-1,3-glycosyltransferase (B13GT) may convert steviol glycosides using ADP-glucose as a sugar donor.

[0086] Figure 13 is a flow diagram showing the respective roles of B12GT and B13GT in the stevioside to Reb M pathway. In various embodiments, B12GT may add a beta-linked glucose monomer to the C2' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT converts stevioside to Reb E and Reb A to Reb D using an ADP-glucose sugar donor. In various embodiments, B12GT may be a non-naturally occurring engineered B12GT glycosyltransferase described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety. In various embodiments, B13GT may add a beta-linked glucose monomer to the C3' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT uses an ADP-glucose sugar donor to convert Reb E to Reb D and Reb D to Reb M. In embodiments, B13GT can be a non-naturally occurring engineered B13GT glycosyltransferase as described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0087] In various embodiments, the at least one enzyme comprises one or more sucrose synthases. The one or more sucrose synthases may be wild-type sucrose synthases, non-naturally occurring engineered sucrose synthases, polypeptides having sucrose synthase activity, or combinations thereof. In various embodiments, the one or more sucrose synthases (SuSy) may convert NDP and sucrose using a sucrose sugar donor to form NDP-glucose and fructose. For example, the one or more SuSy may convert ADP and sucrose to ADP-glucose and fructose. In various embodiments, the SuSy may be a non-naturally occurring engineered SuSy described in International Patent Application No. PCT / US2022 / 016820, the entire contents of which are incorporated herein by reference.

[0088] In various variations, seed crystals may optionally be added to the reaction in step 205 of method 200. Seed crystals can be added to the reaction to promote crystallization / precipitation of the crystalline steviol glycosides. Seed crystals can be enzymatically generated by reacting a glycosyltransferase with a mixture of steviol glycosides. For example, an initial enzyme dosage of less than 1 / 10 of the final enzyme dosage can be reacted with the feedstock composition to initiate crystal formation. Such initial crystal formation can occur for at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, and / or at least 60 minutes.

[0089] After ERX in step 205, the reacted composition may be further processed in step 206 of method 200 by evaporation and / or cooling and / or other techniques to promote and enhance crystal formation. Evaporation may be referred to as evaporative crystallization. Cooling may be referred to as cooling crystallization and may include cooling by heat transfer surface cooling, evaporative cooling, or a combination thereof. In various embodiments, cooling includes significant evaporation and a reduction in temperature to a temperature below the ERX reaction temperature, thereby recovering product crystals.

[0090] The crystals can then be isolated by separation and / or washing in step 206 of method 200. In various embodiments, the result of the ERX comprises crystallized steviol glycosides and a "mother liquor," which comprises the buffer and any steviol glycosides that remain soluble. In various embodiments, the mother liquor can be provided as a recycle stream to the ERX in step 205 of method 200 and / or can be further processed in subprocess 210 of method 200. The output from subprocess 210, described in more detail below, can optionally be provided as a recycle stream input to the feed composition.

[0091] Each of the isolation in step 206 and the processing of the mother liquor in subprocess 210 produces a product. Specifically, ERX crystals are produced by step 206. The type of ERX crystals produced is based on the feedstock composition and the at least one enzyme used.

[0092] A variation of method 100 will now be described with reference to method 300 of Figure 3, where the products include Red D crystals ("ERX-D crystals"), Reb D-enriched co-crystals, Reb M crystals ("ERX-M crystals"), and rebaudioside M-enriched co-crystals. For the sake of brevity, the corresponding descriptions above will be superseded and repeated.

[0093] In various embodiments, the mother liquor can be further processed in subprocess 310 of method 300. Subprocess 310 is substantially similar to subprocesses 110, 210, and other variations of mother liquor processing described herein. To this end, soluble steviol glycosides from the mother liquor can be purified to produce additional steviol glycoside products. For example, reactive crystallization in which B12GT is reacted with an RA50-based feedstock composition can produce a reaction solid containing primarily Reb D and a soluble mother liquor containing primarily Reb E. The soluble mother liquor can then be subsequently processed to produce a Reb E product. In various embodiments, the soluble reaction mother liquor, which may contain solubilized, non-crystallized Reb D, can be used as a feedstock composition for novel enzymatic and / or ERX reactions.

[0094] In step 311 of sub-process 310, the mother liquor containing Reb E and solubilized, non-crystallized Reb D can be clarified and concentrated.

[0095] In step 312 of subprocess 310, the mother liquor can then be reacted with a reaction composition to perform ERX (or a second ERX). In various embodiments, the reaction composition includes at least one enzyme. For example, the at least one enzyme can include B13GT. The B13GT can be a wild-type glycosyltransferase, a non-naturally occurring engineered glycosyltransferase, a polypeptide having glycosyltransferase activity, or a combination thereof. In various embodiments, the B13GT can be the non-naturally occurring engineered B13GT glycosyltransferase described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0096] After ERX is performed in step 312, the reacted composition can be further processed by separation and / or washing to isolate crystallized Reb M in step 312 of subprocess 310. In various embodiments, the result of ERX in step 312 includes crystallized steviol glycosides and a mother liquor. After isolation in step 313 of subprocess 310, Reb M crystals and Reb M-enriched co-crystals are produced.

[0097] A variation of method 300 will now be described with reference to method 400 of Figure 4, where the products include Red D crystals ("ERX-D crystals"), Reb D-enriched co-crystals, Reb M crystals ("ERX-M crystals"), and rebaudioside M-enriched co-crystals. For the sake of brevity, the corresponding descriptions above will be superseded and repeated.

[0098] In step 404 of method 400, a feedstock composition or a solubility-enhanced steviol glycoside mixture can be provided. The feedstock composition can include the stevia leaf extract described above. In some embodiments, a concentrated crude stevia leaf hot water extract can be provided as the feedstock composition. In various embodiments, the steviol glycoside mixture can be a single component or a mixture of multiple components of steviol glycosides. In various embodiments, the feedstock composition includes one or more steviol glycosides. In various embodiments, the feedstock composition can include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. The feedstock composition may comprise an RA50 of at least 550 g / L, at least 600 g / L, at least 650 g / L, at least 700 g / L, at least 750 g / L, at least 800 g / L, at least 850 g / L, at least 900 g / L, at least 950 g / L, or at least 1000 g / L. Alternatively, the feedstock composition may be obtained as an already processed version of Stevia rebaudiana. Additionally, the feedstock composition may be synthetic or at least partially purified and commercially available, or prepared by other methods.

[0099] In step 405 of method 400, the feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In various embodiments, the reaction composition includes at least one enzyme. For example, the at least one enzyme can include B12GT. The B12GT can be a wild-type glycosyltransferase, a non-natural engineered glycosyltransferase, a polypeptide having glycosyltransferase activity, or a combination thereof. In various embodiments, the B12GT can be the non-natural engineered B12GT glycosyltransferase described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety.

[0100] After ERX is performed in step 405, the reacted composition can be further processed. In step 406 of method 400, evaporation and / or cooling and / or other techniques to promote and enhance crystal formation can be performed. The reaction solids or crystals can then be isolated by separation and / or washing in step 407 of method 400. In various embodiments, the result of ERX includes crystallized steviol glycosides and a "mother liquor," which includes the buffer and any steviol glycosides that remain soluble. As shown in FIG. 4 , the isolated crystals from step 407 of method 400 include Reb D crystals and Reb D-enriched co-crystals. In various embodiments, a Reb D-enriched co-crystal may comprise at least about 50% Reb D, about 55% Reb D, about 60% Reb D, about 65% Reb D, about 70% Reb D, about 75% Reb D, about 80% Reb D, about 85% Reb D, about 90% Reb D, about 91% Reb D, about 92% Reb D, about 93% Reb D, about 94% Reb D, about 95% Reb D, about 96% Reb D, about 97% Reb D, about 98% Reb D, or about 99% Reb D.

[0101] In various embodiments, the mother liquor can be further processed in subprocess 410 of method 400. Subprocess 410 is substantially similar to subprocesses 110, 210, 310, and other variations of mother liquor processing described herein. To this end, soluble steviol glycosides from the mother liquor can be purified to produce additional steviol glycoside products. For example, reactive crystallization in which B12GT is reacted with an RA50-based feedstock composition can produce a reaction solid containing primarily Reb D and a soluble mother liquor containing primarily Reb E. The soluble mother liquor can then be subsequently processed to produce a Reb E product. In various embodiments, the soluble reaction mother liquor, which may contain solubilized, non-crystallized Reb D, can be used as a feedstock composition for novel enzymatic and / or ERX reactions.

[0102] In step 411 of sub-process 410, the mother liquor containing Reb E and solubilized, non-crystallized Reb D can be clarified and concentrated.

[0103] In step 412 of subprocess 410, the mother liquor can then be reacted with a reaction composition to perform ERX. In various embodiments, the reaction composition includes at least one enzyme. For example, the at least one enzyme can include B13GT. The B13GT can be a wild-type glycosyltransferase, a non-naturally occurring engineered glycosyltransferase, a polypeptide having glycosyltransferase activity, or a combination thereof. In various embodiments, the B13GT can be the non-naturally occurring engineered B13GT glycosyltransferase described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0104] After ERX in step 412, the reacted composition may be further processed by evaporation and / or cooling, and / or other techniques to promote and enhance crystal formation in step 413 of subprocess 410. The crystals can then be further processed by separation and / or washing to isolate crystallized Reb M in step 414 of subprocess 410. In various embodiments, the result of ERX in step 412 includes crystallized steviol glycosides and a mother liquor. After isolation in step 414 of subprocess 410, Reb M crystals and Reb M-enriched co-crystals are produced. In various embodiments, a Reb M-enriched co-crystal may comprise at least about 50% Reb M, about 55% Reb M, about 60% Reb M, about 65% Reb M, about 70% Reb M, about 75% Reb M, about 80% Reb M, about 85% Reb M, about 90% Reb M, about 91% Reb M, about 92% Reb M, about 93% Reb M, about 94% Reb M, about 95% Reb M, about 96% Reb M, about 97% Reb M, about 98% Reb M, or about 99% Reb M.

[0105] A variation of the above method will now be described with reference to method 500 of Figure 5, where the product comprises Red A crystals ("ERX-A crystals"). For the sake of brevity, certain repetitive descriptions will be omitted in favor of the corresponding descriptions above.

[0106] After obtaining a stevioside-enriched stevia processing residue (e.g., residue obtained from processing Stevia leaves to obtain a Reb A-enriched stream) from step 501 of method 500, a feedstock composition or solubility-enhanced steviol glycoside mixture can be prepared in a mixer in step 502 of method 500. The mixer can be a dispersion mixer or the like. For example, the mixer can be a rotor-stator high-shear blender. The feedstock composition can also include clarified and concentrated Reb A-enriched mother liquor obtained in step 507 of method 500 (assuming Reb A-enriched mother liquor was previously available). In various embodiments, the feedstock composition can include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. 50, at least 550g / L RA50, at least 600g / L RA50, at least 650g / L RA50, at least 700g / L RA50, at least 750g / L RA50, at least 800g / L RA50, at least 850g / L RA50, at least 900g / L RA50, at least 950g / L RA50, or at least 1000g / L RA50.

[0107] In step 503 of method 500, the feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In various embodiments, the reaction composition includes at least one enzyme. For example, the at least one enzyme can include B13GT. The B13GT can be a wild-type glycosyltransferase, a non-naturally occurring engineered glycosyltransferase, a polypeptide having glycosyltransferase activity, or a combination thereof. In various embodiments, the B13GT can be the non-naturally occurring engineered B13GT glycosyltransferase described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0108] After ERX is performed in step 503, the reacted composition can be further processed. In step 504 of method 500, evaporation and / or cooling and / or other techniques to promote and enhance crystal formation can be performed. The reaction solids or crystals can then be isolated by separation and / or washing in step 505 of method 500. In various embodiments, the result of ERX includes crystallized steviol glycosides and a "mother liquor," which includes the buffer and any steviol glycosides that remain soluble. As shown in FIG. 5, the isolated crystals from step 505 of method 500 include Reb A crystals and a mother liquor enriched in available Reb A. In various embodiments, a Reb A-enriched co-crystal may comprise at least about 50% Reb A, about 55% Reb A, about 60% Reb A, about 65% Reb A, about 70% Reb A, about 75% Reb A, about 80% Reb A, about 85% Reb A, about 90% Reb A, about 91% Reb A, about 92% Reb A, about 93% Reb A, about 94% Reb A, about 95% Reb A, about 96% Reb A, about 97% Reb A, about 98% Reb A, or about 99% Reb A.

[0109] A variation of method 500 will now be described with reference to method 600 of Figure 6, where the product comprises Red M crystals ("ERX-M crystals"). For the sake of brevity, certain repetitive descriptions will be omitted in favor of the corresponding descriptions above.

[0110] After obtaining a stevioside-enriched stevia processing residue (e.g., residue obtained from processing Stevia leaves to obtain a Reb A-enriched stream) from step 601 of method 600, a feedstock composition or solubility-enhanced steviol glycoside mixture can be prepared in a mixer in step 602 of method 600. The mixer can be a dispersion mixer, etc. The feedstock composition can also include the clarified and concentrated mother liquor obtained in step 607 of method 600. The mother liquor can include one or more soluble forms of the steviol glycosides described herein.

[0111] In step 603 of method 600, the feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In various embodiments, the reaction composition includes at least one enzyme. For example, the at least one enzyme can include B12GT and B13GT. B12GT can be a wild-type glycosyltransferase, a non-naturally occurring engineered glycosyltransferase, a polypeptide having glycosyltransferase activity, or a combination thereof. In various embodiments, B12GT can be a non-naturally occurring engineered B12GT glycosyltransferase, as described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety. B13GT can be a wild-type glycosyltransferase, a non-naturally occurring engineered glycosyltransferase, a polypeptide having glycosyltransferase activity, or a combination thereof. In embodiments, the B13GT may be a non-naturally occurring engineered B13GT glycosyltransferase, as described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0112] After ERX is performed in step 603, the reacted composition can be further processed. In step 604 of method 600, evaporation and / or cooling and / or other techniques to promote and enhance crystal formation can be performed. The reaction solids or crystals can then be isolated by separation and / or washing in step 605 of method 600. In various embodiments, the result of ERX includes crystallized steviol glycosides and a "mother liquor," which includes the buffer and any steviol glycosides that remain soluble. As shown in FIG. 6 , the isolated crystals from step 605 of method 600 include Reb M crystals, and the mother liquor is enriched in soluble steviol glycosides.

[0113] As shown in Figures 5 and 6, respectively, in step 502 of method 500 and step 602 of method 600, the soluble ERX mother liquor can be recycled to improve the solubility of the steviol glycoside feedstock composition. For example, as shown in Figure 5, a stevioside feedstock composition, when reacted with B13GT, produces a soluble mother liquor enriched in Reb A. The Reb A can be recycled and mixed with the stevioside-enriched feedstock composition to create a feedstock composition with improved solubility.

[0114] Figure 7 is an image of rebaudioside D-enriched cocrystals produced by the ERX method described herein. The ERX method involves placing RA50 in a 15 liter reactor scale, drying in a vacuum oven, suspending in water, and placing it under an optical microscope at 400x magnification.

[0115] FIG. 8 shows the crystallization kinetics of the ERX process as measured by the volume percent of suspended solids present during a 150 liter scale ERX run.

[0116] FIG. 9 is a graphical representation of the reaction rate of the ERX process as measured by stevioside conversion and rebaudioside A conversion in a 150 liter scale ERX run.

[0117] FIG. 10 is a graph overlaying the crystallization reaction rate of FIG. 8 and the reaction rate of FIG. 9, showing the time lag or offset between the reaction and crystal formation.

[0118] 9 and 10 illustrate how the rates of conversion of stevioside and Reb A to Reb D or Reb M coincide with the formation of crystallized products. Such data can be useful in determining enzyme dosage and other reaction parameters to achieve conversion of raw ingredients to final products in solution and then precipitated solids.

[0119] Figure 11 is a graphical representation of theoretical calculations of sucrose equivalents per kilogram of harvested leaves, illustrating the substantial value of effective sweetness obtained by enzymatically upgrading leaf extracts. Steviol glycosides containing rhamnosyl moieties, which are excluded from this graph for visual clarity, have significantly lower sucrose equivalents.

[0120] FIG. 12 is a UV-Vis spectrophotometric scan of the crystals produced on a laboratory scale by the ERX method, showing excellent purity in terms of the absence of impurities adsorbing at 254 nm, 270 nm, 280 nm, and 315 nm.

[0121] FIG. 13 shows the enzymatic reactions performed by beta-1,2-glycosyltransferase and beta-1,3-glycosyltransferase to convert stevioside to rebaudioside M.

[0122] FIG. 14 shows the stevioside molecule and indicates to which carbon (C13, (G) reaction, or C19 (E) reaction) beta-1,2-glycosyltransferase and beta-1,3-glycosyltransferase can attach an additional glucose unit during conversion to rebaudioside M.

[0123] Referring now to FIG. 15, a method for producing and isolating steviol glycoside crystals is described with reference to method 1500.

[0124] In optional step 1501 of method 1500, Stevia rebaudiana leaves and / or stems can be received. In optional steps 1502 and 1503 of method 1500, the Stevia rebaudiana leaves and / or stems can be processed to extract steviol glycosides and perform an initial clarification and / or concentration thereof. Fractions may be obtained during this processing and can be reintroduced in step 1505, as shown in FIG. 15 .

[0125] In step 1504 of method 1500, the feedstock composition or solubility-enhanced steviol glycoside mixture may be made from a processed stevia leaf extract or may be otherwise provided. In some embodiments, a concentrated crude stevia leaf hot water extract may be provided as the feedstock composition. In various embodiments, the steviol glycoside mixture may be a single component or a mixture of multiple components of steviol glycosides. In various embodiments, the feedstock composition includes one or more steviol glycosides. In various embodiments, the feedstock composition may include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. The feedstock composition may comprise an RA50 of at least 550 g / L, at least 600 g / L, at least 650 g / L, at least 700 g / L, at least 750 g / L, at least 800 g / L, at least 850 g / L, at least 900 g / L, at least 950 g / L, or at least 1000 g / L. Alternatively, the feedstock composition may be obtained as an already processed version of Stevia rebaudiana. Additionally, the feedstock composition may be synthetic or at least partially purified and commercially available, or prepared by other methods.

[0126] In step 1505 of method 1500, the feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In embodiments, the reaction composition includes at least one enzyme. In some embodiments, a crude enzyme mixture, a partially purified enzyme mixture, and / or a purified enzyme mixture can be used. In embodiments, at least one enzyme described herein is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces spp., Aspergillus spp., Pichia spp., and Bacillus spp. In embodiments, at least one enzyme is expressed in E. coli. In embodiments, at least one enzyme is expressed in Pichia pastoris. In embodiments, at least one enzyme can be thermotolerant. In embodiments, at least one enzyme can have enhanced solubility.

[0127] In various embodiments, the at least one enzyme comprises one or more glycosyltransferases. The one or more glycosyltransferases may be wild-type glycosyltransferases, non-naturally occurring engineered glycosyltransferases, polypeptides having glycosyltransferase activity, or combinations thereof. In various embodiments, the one or more glycosyltransferases may comprise β-1,2-glycosyltransferases, β-1,3-glycosyltransferases, and combinations thereof. β-1,2-glycosyltransferase (B12GT) and / or β-1,3-glycosyltransferase (B13GT) may convert steviol glycosides using ADP-glucose as a sugar donor. For example, B12GT converts stevioside to Reb E and Reb A to Reb D using an ADP-glucose sugar donor. In embodiments, the B12GT may be a non-naturally occurring engineered B12GT glycosyltransferase described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety. In embodiments, the B13GT uses an ADP-glucose sugar donor to convert Reb E to Reb D and Reb D to Reb M. In embodiments, the B13GT may be a non-naturally occurring engineered B13GT glycosyltransferase described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0128] After ERX is performed in step 1505, the ERX reaction product can be treated to increase the purity of the steviol glycosides before isolating the ERX reaction product crystals. As shown in step 1506 of method 1500, this can include heating the reaction mixture to redissolve the ERX product, thereby providing the formation of a hot break. In various embodiments, heating may include heating the reaction mixture to a temperature of about 10° C. to about 150° C., about 10° C. to about 20° C., about 20° C. to about 30° C., about 30° C. to about 40° C., about 40° C. to about 50° C., about 50° C. to about 60° C., about 60° C. to about 70° C., about 70° C. to about 80° C., about 80° C. to about 90° C., about 90° C. to about 100° C., about 100° C. to about 110° C., about 110° C. to about 120° C., about 120° C. to about 130° C., about 130° C. to about 140° C., and about 140° C. to about 150° C. In one embodiment, the mixture may be heated to a temperature greater than about 78° C. In embodiments, the mixture may be heated in step 1506 of method 1500 for a period of about 0.1 hours to about 10 hours, about 0.1 hours to about 9.5 hours, about 30 minutes to about 9 hours, about 1 hour to about 8.5 hours, about 2 hours to about 8 hours, about 3 hours to about 7.5 hours, about 4 hours to about 7 hours, and about 5 hours to about 6.5 hours. In embodiments, heating may be carried out for a period of about 6 hours. In embodiments, the heating temperature and heating duration are such that the protein precipitates.

[0129] At step 1507 of method 1500, clarification can be performed to remove hot break (i.e., remove impurities). In embodiments, clarification includes centrifugation, filtration, or a combination thereof. In embodiments, the hot break includes proteins and other cellular debris. In embodiments, clarification can include removing solvent break.

[0130] Crystal reformation may be performed via cooling crystallization in step 1508 of method 1500. In embodiments, cooling crystallization includes cooling by heat transfer surface cooling, evaporative cooling, or a combination thereof. In embodiments, cooling includes significant evaporation and a reduction in temperature to a temperature below the ERX reaction temperature, thereby recovering product crystals.

[0131] The reformed crystals can then be isolated by separation and / or washing in step 1509 of method 1500. In various embodiments, the result of ERX, reheating, and recrystallization includes recrystallized steviol glycosides and a "mother liquor," which includes the buffer and any steviol glycosides that remain soluble. In various embodiments, the precipitated steviol glycosides can be separated from the mother liquor by suitable means, including, but not limited to, vacuum filtration, centrifugation, and a chamber filter press. In various embodiments, the precipitated steviol glycosides can be further processed to increase the purity of the precipitated steviol glycosides. The precipitated steviol glycosides can be washed with water to remove hydrophilic reaction components, such as residual sugars and salts. In various embodiments, the precipitated steviol glycosides can be washed with a hydroalcoholic solvent to remove impurities. In various embodiments, the recovered, processed precipitated steviol glycosides can be further dried to form a powder. The precipitated steviol glycosides can be dried by any standard method, including, but not limited to, freeze drying, vacuum tray drying, spray drying, fluidized bed drying, filter mat drying, or rotary drum drying. In some embodiments, the dried ERX crystals can be milled to produce the desired particle size.

[0132] The resulting crystalline steviol glycoside composition comprises one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. The crystals may, in one example, comprise Reb D, Reb E, and Reb A. Reb D, Reb E, and Reb A may each comprise from about 0% to about 100% of the crystals. Reb D, Reb E, and Reb A may each comprise about 0.1% to about 20%, about 0.2% to about 18%, about 0.3% to about 16%, about 0.4% to about 14%, about 0.5% to about 12%, about 0.6% to about 10%, about 0.7% to about 8%, about 0.8% to about 6%, about 0.9% to about 4%, or about 1% to about 2% of the crystals. Reb D, Reb E, and Reb A may each comprise about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, about 47.5% to about 52.5%, or about 48% to about 52% of the crystals. Reb D, Reb E, and Reb A may each comprise about 70% to about 100%, about 72% to about 98%, about 74% to about 96%, about 76% to about 94%, about 78% to about 92%, about 80% to about 90%, about 82% to about 88%, and about 84% to about 86% of the crystals. In one example, the crystals contain, on an anhydrous basis, 70.9% Reb D, 17.4% Reb E, and 0.4% Reb A. The composition of steviol glycosides in the resulting mother liquor includes one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. In one example, the mother liquor may contain Reb E, Reb D, Reb A, and stevioside in a ratio of 12.2:8.1:1:1.3.However, the ratio between the concentrations of each steviol glycoside is highly variable and depends on the steviol glycoside(s) included in the reaction.

[0133] In embodiments, the mother liquor may be further processed in subprocess 1510 of method 1500. The output from subprocess 1510, described elsewhere herein, may optionally be provided as a recycle stream input to the feedstock composition.

[0134] Each of the isolation in step 1506 and the processing of the mother liquor in subprocess 1510 produces a product. Specifically, ERX crystals are produced by step 1506. The type of ERX crystal produced is based on the feedstock composition and the at least one enzyme used.

[0135] In some embodiments, steps 1506-1507 of method 1500 include mixing ERX crystals with a solvent, heating the slurry to a temperature such that the ERX crystals substantially dissolve, clarifying to remove any particulate or insoluble material, and / or cooling the slurry to an ERX reaction temperature to form the crystals. In some embodiments, the heating temperature may be higher than the ERX reaction temperature. For example, the heating temperature may be greater than about 92°C. In embodiments, the heating temperature may be greater than about 78°C and / or greater than about 110°C and / or may be applied under pressure. In embodiments, the heating temperature may be greater than about 92°C, greater than 110°C, and / or greater than about 130°C and / or may be applied under pressure. In embodiments, the solvent may be water, alcohol, methanol, ethanol, isopropanol, n-propanol, or isoamyl alcohol.

[0136] Referring now to FIG. 16, a method for producing and isolating steviol glycoside crystals is described with reference to method 1600.

[0137] In optional step 1601 of method 1600, Stevia rebaudiana leaves and / or stems can be received. In optional steps 1602 and 1603 of method 1600, the Stevia rebaudiana leaves and / or stems can be processed to extract steviol glycosides and perform an initial clarification and / or concentration thereof. Fractions may be obtained during this processing and can be reintroduced in step 1605, as shown in FIG. 16 .

[0138] In step 1604 of method 1600, the feedstock composition or solubility-enhanced steviol glycoside mixture may be made from a processed stevia leaf extract or may be otherwise provided. In some embodiments, a concentrated crude stevia leaf hot water extract may be provided as the feedstock composition. In various embodiments, the steviol glycoside mixture may be a single component or a mixture of multiple components of steviol glycosides. In various embodiments, the feedstock composition includes one or more steviol glycosides. In various embodiments, the feedstock composition may include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. The feedstock composition may comprise an RA50 of at least 550 g / L, at least 600 g / L, at least 650 g / L, at least 700 g / L, at least 750 g / L, at least 800 g / L, at least 850 g / L, at least 900 g / L, at least 950 g / L, or at least 1000 g / L. Alternatively, the feedstock composition may be obtained as an already processed version of Stevia rebaudiana. Additionally, the feedstock composition may be synthetic or at least partially purified and commercially available, or prepared by other methods.

[0139] In step 1605 of method 1600, the feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In embodiments, the reaction composition includes at least one enzyme. In some embodiments, a crude enzyme mixture, a partially purified enzyme mixture, and / or a purified enzyme mixture can be used. In embodiments, at least one enzyme described herein is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces spp., Aspergillus spp., Pichia spp., and Bacillus spp. In embodiments, at least one enzyme is expressed in E. coli. In embodiments, at least one enzyme is expressed in Pichia pastoris. In embodiments, at least one enzyme can be thermotolerant. In embodiments, at least one enzyme can have enhanced solubility.

[0140] In various embodiments, the at least one enzyme comprises one or more glycosyltransferases. The one or more glycosyltransferases may be wild-type glycosyltransferases, non-naturally occurring engineered glycosyltransferases, polypeptides having glycosyltransferase activity, or combinations thereof. In various embodiments, the one or more glycosyltransferases may comprise β-1,2-glycosyltransferases, β-1,3-glycosyltransferases, and combinations thereof. β-1,2-glycosyltransferase (B12GT) and / or β-1,3-glycosyltransferase (B13GT) may convert steviol glycosides using ADP-glucose as a sugar donor. For example, B12GT converts stevioside to Reb E and Reb A to Reb D using an ADP-glucose sugar donor. In embodiments, the B12GT may be a non-naturally occurring engineered B12GT glycosyltransferase described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety. In embodiments, the B13GT uses an ADP-glucose sugar donor to convert Reb E to Reb D and Reb D to Reb M. In embodiments, the B13GT may be a non-naturally occurring engineered B13GT glycosyltransferase described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0141] After ERX is performed in step 1605, the ERX reaction product can be treated to increase the purity of the steviol glycosides before isolating the ERX reaction product crystals. As shown in step 1606 of method 1600, this can include heating the reaction mixture to redissolve the ERX product, thereby providing the formation of a hot break. In various embodiments, heating may include heating the reaction mixture to a temperature of about 10° C. to about 150° C., about 10° C. to about 20° C., about 20° C. to about 30° C., about 30° C. to about 40° C., about 40° C. to about 50° C., about 50° C. to about 60° C., about 60° C. to about 70° C., about 70° C. to about 80° C., about 80° C. to about 90° C., about 90° C. to about 100° C., about 100° C. to about 110° C., about 110° C. to about 120° C., about 120° C. to about 130° C., about 130° C. to about 140° C., and about 140° C. to about 150° C. In one embodiment, the mixture may be heated to a temperature greater than about 78° C. In embodiments, the mixture may be heated in step 1506 of method 1500 for a period of about 0.1 hours to about 10 hours, about 0.1 hours to about 9.5 hours, about 30 minutes to about 9 hours, about 1 hour to about 8.5 hours, about 2 hours to about 8 hours, about 3 hours to about 7.5 hours, about 4 hours to about 7 hours, and about 5 hours to about 6.5 hours. In embodiments, heating may be carried out for a period of about 6 hours. In embodiments, the heating temperature and heating duration are such that the protein precipitates.

[0142] In step 1607 of method 1600, clarification can be performed to remove the hot break (i.e., remove impurities). In various embodiments, clarification includes centrifugation, filtration, or a combination thereof. In various embodiments, the hot break includes proteins and other cellular debris. In various embodiments, clarification can include removing the solvent break. Crystal reformation can be performed via evaporative crystallization in step 1608 of method 1600. The reformed crystals can then be isolated by separation and / or washing in step 1609 of method 1600. In various embodiments, the result of ERX, reheating, and recrystallization includes recrystallized steviol glycosides and a "mother liquor," which includes the buffer and any steviol glycosides that remain soluble. In various embodiments, the precipitated steviol glycosides can be separated from the mother liquor by suitable means, including, but not limited to, vacuum filtration, centrifugation, and a chamber filter press. In various embodiments, the precipitated steviol glycosides can be further processed to increase their purity. The precipitated steviol glycosides can be washed with water to remove hydrophilic reaction components, such as residual sugars and salts. In various embodiments, the precipitated steviol glycosides can be washed with an aqueous alcoholic solvent to remove impurities. In various embodiments, the recovered and processed precipitated steviol glycosides can be further dried to form a powder. The precipitated steviol glycosides can be dried by any standard method, including, but not limited to, freeze drying, vacuum tray drying, spray drying, fluidized bed drying, filter mat drying, or rotary drum drying. In some embodiments, the dried ERX crystals can be milled to produce a desired particle size.

[0143] The resulting crystalline steviol glycoside composition comprises one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. The crystals may, in one example, comprise Reb D, Reb E, and Reb A. Reb D, Reb E, and Reb A may each comprise from about 0% to about 100% of the crystals. Reb D, Reb E, and Reb A may each comprise about 0.1% to about 20%, about 0.2% to about 18%, about 0.3% to about 16%, about 0.4% to about 14%, about 0.5% to about 12%, about 0.6% to about 10%, about 0.7% to about 8%, about 0.8% to about 6%, about 0.9% to about 4%, or about 1% to about 2% of the crystals. Reb D, Reb E, and Reb A may each comprise about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, about 47.5% to about 52.5%, or about 48% to about 52% of the crystals. Reb D, Reb E, and Reb A may each comprise about 70% to about 100%, about 72% to about 98%, about 74% to about 96%, about 76% to about 94%, about 78% to about 92%, about 80% to about 90%, about 82% to about 88%, and about 84% to about 86% of the crystals. In one example, the crystals contain, on an anhydrous basis, 70.9% Reb D, 17.4% Reb E, and 0.4% Reb A. The composition of steviol glycosides in the resulting mother liquor includes one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. In one example, the mother liquor may contain Reb E, Reb D, Reb A, and stevioside in a ratio of 12.2:8.1:1:1.3.However, the ratio between the concentrations of each steviol glycoside is highly variable and depends on the steviol glycoside(s) included in the reaction.

[0144] In embodiments, the mother liquor may be further processed in subprocess 1610 of method 1600. The output from subprocess 1610, described elsewhere herein, may optionally be provided as a recycle stream input to the feedstock composition.

[0145] Each of the isolation in step 1606 and the processing of the mother liquor in subprocess 1610 produces a product. Specifically, ERX crystals are produced by step 1606. The type of ERX crystals produced is based on the feedstock composition and the at least one enzyme used.

[0146] In some embodiments, steps 1606-1607 of method 1600 include mixing ERX crystals with a solvent, heating the mixture to a temperature such that the ERX crystals substantially dissolve, clarifying to remove any particulate or insoluble material, and / or cooling the mixture to an ERX reaction temperature to form the crystals. In various embodiments, the mixture may be heated to a temperature of about 10°C to about 150°C, about 10°C to about 20°C, about 20°C to about 30°C, about 30°C to about 40°C, about 40°C to about 50°C, about 50°C to about 60°C, about 60°C to about 70°C, about 70°C to about 80°C, about 80°C to about 90°C, about 90°C to about 100°C, about 100°C to about 110°C, about 110°C to about 120°C, about 120°C to about 130°C, about 130°C to about 140°C, and about 140°C to about 150°C. In one embodiment, the mixture may be heated to a temperature greater than about 78°C. In some embodiments, the heating temperature may be higher than the ERX reaction temperature. For example, the heating temperature may be greater than about 92°C. In embodiments, the heating temperature may be greater than about 78° C. and / or greater than about 110° C. and / or may be applied under pressure. In embodiments, the heating temperature may be greater than about 92° C., greater than 110° C. and / or greater than about 130° C. and / or may be applied under pressure. In embodiments, the solvent may be water, alcohol, methanol, ethanol, isopropanol, n-propanol, or isoamyl alcohol.

[0147] Referring now to Figure 17, a method is described that includes, after isolation of the ERX reaction product crystals, further processing the ERX reaction product to increase the purity of the steviol glycosides. Certain steps of method 1700 of Figure 17 are substantially similar to those of method 100 of Figure 1, and therefore, certain steps have been omitted or shortened for the sake of brevity.

[0148] In optional step 1701 of method 1700, Stevia rebaudiana leaves and / or stems can be received. In optional steps 1702 and 1703 of method 1700, the Stevia rebaudiana leaves and / or stems can be processed to extract steviol glycosides and perform an initial clarification and / or concentration thereof. Fractions may be obtained during this processing and can be reintroduced in step 1705, as shown in FIG. 17 .

[0149] In step 1704 of method 1700, a feedstock composition or solubility-enhanced steviol glycoside mixture can be produced from the processed stevia leaf extract. In some embodiments, a concentrated crude stevia leaf hot water extract can be provided as the feedstock composition. In various embodiments, the steviol glycoside mixture can be a single component or a mixture of multiple components of steviol glycosides. In various embodiments, the feedstock composition includes one or more steviol glycosides. In various embodiments, the feedstock composition can include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. The feedstock composition may comprise an RA50 of at least 550 g / L, at least 600 g / L, at least 650 g / L, at least 700 g / L, at least 750 g / L, at least 800 g / L, at least 850 g / L, at least 900 g / L, at least 950 g / L, or at least 1000 g / L. Alternatively, the feedstock composition may be obtained as an already processed version of Stevia rebaudiana. Additionally, the feedstock composition may be synthetic or at least partially purified and commercially available, or prepared by other methods.

[0150] In step 1705 of method 1700, the feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In embodiments, the reaction composition includes at least one enzyme. In some embodiments, a crude enzyme mixture, a partially purified enzyme mixture, and / or a purified enzyme mixture can be used. In embodiments, at least one enzyme described herein is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces spp., Aspergillus spp., Pichia spp., and Bacillus spp. In embodiments, at least one enzyme is expressed in E. coli. In embodiments, at least one enzyme is expressed in Pichia pastoris. In embodiments, at least one enzyme can be thermotolerant. In embodiments, at least one enzyme can have enhanced solubility.

[0151] In various embodiments, the at least one enzyme comprises one or more glycosyltransferases. The one or more glycosyltransferases may be wild-type glycosyltransferases, non-naturally occurring engineered glycosyltransferases, polypeptides having glycosyltransferase activity, or combinations thereof. In various embodiments, the one or more glycosyltransferases may comprise β-1,2-glycosyltransferases, β-1,3-glycosyltransferases, and combinations thereof. β-1,2-glycosyltransferase (B12GT) and / or β-1,3-glycosyltransferase (B13GT) may convert steviol glycosides using ADP-glucose as a sugar donor.

[0152] Figure 13 is a flow diagram showing the respective roles of B12GT and B13GT in the stevioside to Reb M pathway. In various embodiments, B12GT may add a beta-linked glucose monomer to the C2' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT converts stevioside to Reb E and Reb A to Reb D using an ADP-glucose sugar donor. In various embodiments, B12GT may be a non-naturally occurring engineered B12GT glycosyltransferase described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety. In various embodiments, B13GT may add a beta-linked glucose monomer to the C3' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT uses an ADP-glucose sugar donor to convert Reb E to Reb D and Reb D to Reb M. In embodiments, B13GT can be a non-naturally occurring engineered B13GT glycosyltransferase as described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0153] After ERX is performed in step 1705, the crystals are isolated by separation and / or washing in step 1706 of method 1700. In various embodiments, the result of ERX includes crystallized steviol glycosides and a "mother liquor," which includes the buffer and any steviol glycosides that remain soluble. In various embodiments, the precipitated steviol glycosides may be separated from the mother liquor by suitable means, including, but not limited to, vacuum filtration, centrifugation, and a chamber filter press. In various embodiments, the precipitated steviol glycosides may be further processed to increase the purity of the precipitated steviol glycosides. The precipitated steviol glycosides may be washed with water to remove hydrophilic reaction components, such as residual sugars and salts. In various embodiments, the precipitated steviol glycosides may be washed with a hydroalcoholic solvent to remove impurities. In various embodiments, the recovered, processed precipitated steviol glycosides may be further dried to form a powder. The precipitated steviol glycosides can be dried by any standard method, including, but not limited to, freeze drying, vacuum tray drying, spray drying, fluidized bed drying, filter mat drying, or rotary drum drying. In some embodiments, the dried ERX crystals can be milled to produce the desired particle size.

[0154] The resulting crystalline steviol glycoside composition comprises one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. The crystals may, in one example, comprise Reb D, Reb E, and Reb A. Reb D, Reb E, and Reb A may each comprise from about 0% to about 100% of the crystals. Reb D, Reb E, and Reb A may each comprise about 0.1% to about 20%, about 0.2% to about 18%, about 0.3% to about 16%, about 0.4% to about 14%, about 0.5% to about 12%, about 0.6% to about 10%, about 0.7% to about 8%, about 0.8% to about 6%, about 0.9% to about 4%, or about 1% to about 2% of the crystals. Reb D, Reb E, and Reb A may each comprise about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, about 47.5% to about 52.5%, or about 48% to about 52% of the crystals. Reb D, Reb E, and Reb A may each comprise about 70% to about 100%, about 72% to about 98%, about 74% to about 96%, about 76% to about 94%, about 78% to about 92%, about 80% to about 90%, about 82% to about 88%, and about 84% to about 86% of the crystals. In one example, the crystals contain, on an anhydrous basis, 70.9% Reb D, 17.4% Reb E, and 0.4% Reb A. The composition of steviol glycosides in the resulting mother liquor includes one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. In one example, the mother liquor may contain Reb E, Reb D, Reb A, and stevioside in a ratio of 12.2:8.1:1:1.3.However, the ratio between the concentrations of each steviol glycoside is highly variable and depends on the steviol glycoside(s) included in the reaction.

[0155] In embodiments, the mother liquor may be further processed in subprocess 1710 of method 1700. The output from subprocess 1710, described elsewhere herein, may optionally be provided as a recycle stream input to the feedstock composition.

[0156] Each of the isolation in step 1706 and the processing of the mother liquor in subprocess 1710 produces a product. Specifically, ERX crystals are produced by step 1706. The type of ERX crystal produced is based on the feedstock composition and the at least one enzyme used.

[0157] The ERX crystals produced in step 1706 can be further processed in steps 1707-1709 of method 1700. In various embodiments, prior to isolating the ERX reaction product crystals, the ERX reaction product can be treated to increase the purity of the steviol glycosides. As shown in step 1707 of method 1700, this can include heating the reaction mixture to redissolve the ERX product, thereby providing the formation of a hot break. In various embodiments, heating may include heating the reaction mixture to a temperature of about 10° C. to about 150° C., about 10° C. to about 20° C., about 20° C. to about 30° C., about 30° C. to about 40° C., about 40° C. to about 50° C., about 50° C. to about 60° C., about 60° C. to about 70° C., about 70° C. to about 80° C., about 80° C. to about 90° C., about 90° C. to about 100° C., about 100° C. to about 110° C., about 110° C. to about 120° C., about 120° C. to about 130° C., about 130° C. to about 140° C., and about 140° C. to about 150° C. In one embodiment, the mixture may be heated to a temperature greater than about 78° C. In embodiments, the mixture may be heated in step 1506 of method 1500 for a period of about 0.1 hours to about 10 hours, about 0.1 hours to about 9.5 hours, about 30 minutes to about 9 hours, about 1 hour to about 8.5 hours, about 2 hours to about 8 hours, about 3 hours to about 7.5 hours, about 4 hours to about 7 hours, and about 5 hours to about 6.5 hours. In embodiments, heating may be carried out for a period of about 6 hours. In embodiments, the heating temperature and heating duration are such that the protein precipitates.

[0158] Crystal reformation can be performed via cooling crystallization and / or evaporative crystallization in step 1708 of method 1700. Mixing may be applied, if desired. In embodiments, cooling crystallization includes cooling by heat transfer surface cooling, evaporative cooling, or a combination thereof. In embodiments, cooling includes significant evaporation and reducing the temperature to a temperature below the ERX reaction temperature, thereby recovering the product crystals. The reformed crystals can then be isolated as purified ERX-crystals in step 1709 of method 1700 by separating and / or washing them.

[0159] A variation of the method 1700 will now be described with reference to method 1800 of FIG.

[0160] In optional step 1801 of method 1800, Stevia rebaudiana leaves and / or stems can be received. In optional steps 1802 and 1803 of method 1800, the Stevia rebaudiana leaves and / or stems can be processed to extract steviol glycosides and perform an initial clarification and / or concentration thereof. Fractions may be obtained during this processing and can be reintroduced in step 1805, as shown in FIG. 18 .

[0161] In step 1804 of method 1800, a feedstock composition or solubility-enhanced steviol glycoside mixture can be produced from the processed stevia leaf extract. In some embodiments, a concentrated crude stevia leaf hot water extract can be provided as the feedstock composition. In various embodiments, the steviol glycoside mixture can be a single component or a mixture of multiple components of steviol glycosides. In various embodiments, the feedstock composition includes one or more steviol glycosides. In various embodiments, the feedstock composition can include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. The feedstock composition may comprise an RA50 of at least 550 g / L, at least 600 g / L, at least 650 g / L, at least 700 g / L, at least 750 g / L, at least 800 g / L, at least 850 g / L, at least 900 g / L, at least 950 g / L, or at least 1000 g / L. Alternatively, the feedstock composition may be obtained as an already processed version of Stevia rebaudiana. Additionally, the feedstock composition may be synthetic or at least partially purified and commercially available, or prepared by other methods.

[0162] In step 1805 of method 1800, the feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In embodiments, the reaction composition includes at least one enzyme. In some embodiments, a crude enzyme mixture, a partially purified enzyme mixture, and / or a purified enzyme mixture can be used. In embodiments, at least one enzyme described herein is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces spp., Aspergillus spp., Pichia spp., and Bacillus spp. In embodiments, at least one enzyme is expressed in E. coli. In embodiments, at least one enzyme is expressed in Pichia pastoris. In embodiments, at least one enzyme can be thermotolerant. In embodiments, at least one enzyme can have enhanced solubility.

[0163] In various embodiments, the at least one enzyme comprises one or more glycosyltransferases. The one or more glycosyltransferases may be wild-type glycosyltransferases, non-naturally occurring engineered glycosyltransferases, polypeptides having glycosyltransferase activity, or combinations thereof. In various embodiments, the one or more glycosyltransferases may comprise β-1,2-glycosyltransferases, β-1,3-glycosyltransferases, and combinations thereof. β-1,2-glycosyltransferase (B12GT) and / or β-1,3-glycosyltransferase (B13GT) may convert steviol glycosides using ADP-glucose as a sugar donor.

[0164] Figure 13 is a flow diagram showing the respective roles of B12GT and B13GT in the stevioside to Reb M pathway. In various embodiments, B12GT may add a beta-linked glucose monomer to the C2' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT converts stevioside to Reb E and Reb A to Reb D using an ADP-glucose sugar donor. In various embodiments, B12GT may be a non-naturally occurring engineered B12GT glycosyltransferase described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety. In various embodiments, B13GT may add a beta-linked glucose monomer to the C3' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT uses an ADP-glucose sugar donor to convert Reb E to Reb D and Reb D to Reb M. In embodiments, B13GT can be a non-naturally occurring engineered B13GT glycosyltransferase as described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0165] After ERX is performed in step 1805, the crystals are isolated by separation and / or washing in step 1806 of method 1800. In various embodiments, the result of ERX includes crystallized steviol glycosides and a "mother liquor," which includes the buffer and any steviol glycosides that remain soluble. In various embodiments, the precipitated steviol glycosides may be separated from the mother liquor by suitable means, including, but not limited to, vacuum filtration, centrifugation, and a chamber filter press. In various embodiments, the precipitated steviol glycosides may be further processed to increase the purity of the precipitated steviol glycosides. The precipitated steviol glycosides may be washed with water to remove hydrophilic reaction components, such as residual sugars and salts. In various embodiments, the precipitated steviol glycosides may be washed with a hydroalcoholic solvent to remove impurities. In various embodiments, the recovered, processed precipitated steviol glycosides may be further dried to form a powder. The precipitated steviol glycosides can be dried by any standard method, including, but not limited to, freeze drying, vacuum tray drying, spray drying, fluidized bed drying, filter mat drying, or rotary drum drying. In some embodiments, the dried ERX crystals can be milled to produce the desired particle size.

[0166] The resulting crystalline steviol glycoside composition comprises one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. The crystals may, in one example, comprise Reb D, Reb E, and Reb A. Reb D, Reb E, and Reb A may each comprise from about 0% to about 100% of the crystals. Reb D, Reb E, and Reb A may each comprise about 0.1% to about 20%, about 0.2% to about 18%, about 0.3% to about 16%, about 0.4% to about 14%, about 0.5% to about 12%, about 0.6% to about 10%, about 0.7% to about 8%, about 0.8% to about 6%, about 0.9% to about 4%, or about 1% to about 2% of the crystals. Reb D, Reb E, and Reb A may each comprise about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, about 47.5% to about 52.5%, or about 48% to about 52% of the crystals. Reb D, Reb E, and Reb A may each comprise about 70% to about 100%, about 72% to about 98%, about 74% to about 96%, about 76% to about 94%, about 78% to about 92%, about 80% to about 90%, about 82% to about 88%, and about 84% to about 86% of the crystals. In one example, the crystals contain, on an anhydrous basis, 70.9% Reb D, 17.4% Reb E, and 0.4% Reb A. The composition of steviol glycosides in the resulting mother liquor includes one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. In one example, the mother liquor may contain Reb E, Reb D, Reb A, and stevioside in a ratio of 12.2:8.1:1:1.3.However, the ratio between the concentrations of each steviol glycoside is highly variable and depends on the steviol glycoside(s) included in the reaction.

[0167] In embodiments, the mother liquor may be further processed in subprocess 1810 of method 1800. The output from subprocess 1810, described elsewhere herein, may optionally be provided as a recycle stream input to the feedstock composition.

[0168] Each of the isolation in step 1806 and the processing of the mother liquor in subprocess 1810 produces a product. Specifically, ERX crystals are produced by step 1806. The type of ERX crystals produced is based on the feedstock composition and the at least one enzyme used.

[0169] The ERX crystals produced in step 1806 can be further processed in steps 1807-1809 and 1821 of method 1800. In various embodiments, prior to isolating the ERX reaction product crystals, the ERX reaction product can be treated to increase the purity of the steviol glycosides. As shown in step 1807 of method 1800, this can include heating the reaction mixture to redissolve the ERX product, thereby providing the formation of a hot break. In various embodiments, heating may include heating the reaction mixture to a temperature of about 10° C. to about 150° C., about 10° C. to about 20° C., about 20° C. to about 30° C., about 30° C. to about 40° C., about 40° C. to about 50° C., about 50° C. to about 60° C., about 60° C. to about 70° C., about 70° C. to about 80° C., about 80° C. to about 90° C., about 90° C. to about 100° C., about 100° C. to about 110° C., about 110° C. to about 120° C., about 120° C. to about 130° C., about 130° C. to about 140° C., and about 140° C. to about 150° C. In one embodiment, the mixture may be heated to a temperature greater than about 78° C. In embodiments, the mixture may be heated in step 1807 of method 1800 for a period of about 0.1 hours to about 10 hours, about 0.1 hours to about 9.5 hours, about 30 minutes to about 9 hours, about 1 hour to about 8.5 hours, about 2 hours to about 8 hours, about 3 hours to about 7.5 hours, about 4 hours to about 7 hours, and about 5 hours to about 6.5 hours. In embodiments, heating may be carried out for a period of about 6 hours. In embodiments, the heating temperature and heating duration are such that the protein precipitates.

[0170] In step 1808 of method 1800, clarification can be performed to remove hot break (i.e., remove impurities). In embodiments, clarification includes centrifugation, filtration, or a combination thereof. In embodiments, the hot break includes proteins and other cellular debris. In embodiments, clarification can include removing solvent break. Crystal reformation can be performed in step 1809 of method 1800 via cooling crystallization and / or evaporative crystallization. Mixing can be applied as needed. In embodiments, cooling crystallization includes cooling by heat transfer surface cooling, evaporative cooling, or a combination thereof. In embodiments, cooling includes significant evaporation and reducing the temperature to a temperature below the ERX reaction temperature, thereby recovering the product crystals. The reformed crystals can then be isolated as purified ERX-crystals in step 1821 of method 1800 by separating and / or washing.

[0171] A variation of method 1800 in which purification occurs before and after ERX will now be described with reference to method 1900 in FIG.

[0172] In step 1901 of method 1900, a feedstock composition or solubility-enhanced steviol glycoside mixture can be provided from a processed stevia leaf extract. In some embodiments, a concentrated crude stevia leaf hot water extract can be provided as the feedstock composition. In various embodiments, the steviol glycoside mixture can be a single component or a mixture of multiple components of steviol glycosides. In various embodiments, the feedstock composition includes one or more steviol glycosides. In various embodiments, the feedstock composition can include one of RA20, RA40, RA50, and / or RA60. In one embodiment, the feed composition has an RA50 of at least 50g / L, at least 100g / L, at least 150g / L, at least 200g / L, at least 250g / L, at least 300g / L, at least 350g / L, at least 400g / L, at least 450g / L, at least 500g / L. The feedstock composition may comprise an RA50 of at least 550 g / L, at least 600 g / L, at least 650 g / L, at least 700 g / L, at least 750 g / L, at least 800 g / L, at least 850 g / L, at least 900 g / L, at least 950 g / L, or at least 1000 g / L. Alternatively, the feedstock composition may be obtained as an already processed version of Stevia rebaudiana. Additionally, the feedstock composition may be synthetic or at least partially purified and commercially available, or prepared by other methods.

[0173] In step 1902 of method 1900, a feedstock composition can be reacted with a reaction composition to perform enzymatic reactive crystallization (ERX). In embodiments, the reaction composition includes at least one enzyme. In some embodiments, a crude enzyme mixture, a partially purified enzyme mixture, and / or a purified enzyme mixture can be used. In embodiments, at least one enzyme described herein is prepared by expression in a host microorganism. Suitable host microorganisms include, but are not limited to, E. coli, Saccharomyces spp., Aspergillus spp., Pichia spp., and Bacillus spp. In embodiments, at least one enzyme is expressed in E. coli. In embodiments, at least one enzyme is expressed in Pichia pastoris. In embodiments, at least one enzyme can be thermotolerant. In embodiments, at least one enzyme can have enhanced solubility.

[0174] In various embodiments, the at least one enzyme comprises one or more glycosyltransferases. The one or more glycosyltransferases may be wild-type glycosyltransferases, non-naturally occurring engineered glycosyltransferases, polypeptides having glycosyltransferase activity, or combinations thereof. In various embodiments, the one or more glycosyltransferases may comprise β-1,2-glycosyltransferases, β-1,3-glycosyltransferases, and combinations thereof. β-1,2-glycosyltransferase (B12GT) and / or β-1,3-glycosyltransferase (B13GT) may convert steviol glycosides using ADP-glucose as a sugar donor.

[0175] Figure 13 is a flow diagram showing the respective roles of B12GT and B13GT in the stevioside to Reb M pathway. In various embodiments, B12GT may add a beta-linked glucose monomer to the C2' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT converts stevioside to Reb E and Reb A to Reb D using an ADP-glucose sugar donor. In various embodiments, B12GT may be a non-naturally occurring engineered B12GT glycosyltransferase described in International Patent Application No. PCT / US2022 / 016820, which is incorporated herein by reference in its entirety. In various embodiments, B13GT may add a beta-linked glucose monomer to the C3' of the 13-O-glucose and / or 19-O-glucose of a steviol glycoside substrate. For example, B13GT uses an ADP-glucose sugar donor to convert Reb E to Reb D and Reb D to Reb M. In embodiments, B13GT can be a non-naturally occurring engineered B13GT glycosyltransferase as described in International Patent Application No. PCT / US2023 / 073344, which is incorporated herein by reference in its entirety.

[0176] The ERX crystals produced in step 1902 can be further processed in steps 1903-1906 of method 1900. In various embodiments, prior to isolating the ERX reaction product crystals, the ERX reaction product can be treated to increase the purity of the steviol glycosides. As shown in step 1903 of method 1900, this can include heating the reaction mixture to redissolve the ERX product, thereby providing the formation of a hot break. In various embodiments, heating may include heating the reaction mixture to a temperature of about 10° C. to about 150° C., about 10° C. to about 20° C., about 20° C. to about 30° C., about 30° C. to about 40° C., about 40° C. to about 50° C., about 50° C. to about 60° C., about 60° C. to about 70° C., about 70° C. to about 80° C., about 80° C. to about 90° C., about 90° C. to about 100° C., about 100° C. to about 110° C., about 110° C. to about 120° C., about 120° C. to about 130° C., about 130° C. to about 140° C., and about 140° C. to about 150° C. In one embodiment, the mixture may be heated to a temperature greater than about 78° C. In embodiments, the mixture may be heated in step 1903 of method 1900 for a period of about 0.1 hours to about 10 hours, about 0.1 hours to about 9.5 hours, about 30 minutes to about 9 hours, about 1 hour to about 8.5 hours, about 2 hours to about 8 hours, about 3 hours to about 7.5 hours, about 4 hours to about 7 hours, and about 5 hours to about 6.5 hours. In embodiments, heating may be carried out for a period of about 6 hours. In embodiments, the heating temperature and heating duration are such that the protein precipitates.

[0177] In step 1904 of method 1900, clarification can be performed to remove hot break (i.e., remove impurities). In embodiments, clarification includes centrifugation, filtration, or a combination thereof. In embodiments, the hot break includes proteins and other cellular debris. In embodiments, clarification can include removing solvent break. Crystal reformation can be performed in step 1905 of method 1900 via cooling crystallization and / or evaporative crystallization. In embodiments, cooling crystallization includes cooling by heat transfer surface cooling, evaporative cooling, or a combination thereof. In embodiments, cooling includes significant evaporation and reducing the temperature to a temperature below the ERX reaction temperature, thereby recovering the product crystals. The reformed crystals can then be isolated as purified ERX-crystals in step 1906 of method 1900 by separating and / or washing.

[0178] After ERX is performed and an initial purification is performed in steps 1902-1906, the ERX crystals can be further processed in steps 1907-1910 of method 1900 to perform a second purification. In various embodiments, prior to isolating the ERX reaction product crystals, the ERX reaction product can be treated to increase the purity of the steviol glycosides. As shown in step 1907 of method 1900, this can include heating the reaction mixture to redissolve the ERX product, thereby providing the formation of a hot break. In various embodiments, heating may include heating the reaction mixture to a temperature of about 10° C. to about 150° C., about 10° C. to about 20° C., about 20° C. to about 30° C., about 30° C. to about 40° C., about 40° C. to about 50° C., about 50° C. to about 60° C., about 60° C. to about 70° C., about 70° C. to about 80° C., about 80° C. to about 90° C., about 90° C. to about 100° C., about 100° C. to about 110° C., about 110° C. to about 120° C., about 120° C. to about 130° C., about 130° C. to about 140° C., and about 140° C. to about 150° C. In one embodiment, the mixture may be heated to a temperature greater than about 78° C. In embodiments, the mixture may be heated in step 1907 of method 1900 for a period of about 0.1 hours to about 10 hours, about 0.1 hours to about 9.5 hours, about 30 minutes to about 9 hours, about 1 hour to about 8.5 hours, about 2 hours to about 8 hours, about 3 hours to about 7.5 hours, about 4 hours to about 7 hours, and about 5 hours to about 6.5 hours. In embodiments, heating may be carried out for a period of about 6 hours. In embodiments, the heating temperature and heating duration are such that the protein precipitates.

[0179] In step 1908 of method 1900, clarification can be performed to remove hot break (i.e., remove impurities). In embodiments, clarification includes centrifugation, filtration, or a combination thereof. In embodiments, the hot break includes proteins and other cellular debris. In embodiments, clarification can include removing solvent break. Crystal reformation can be performed in step 1909 of method 1900 via cooling crystallization and / or evaporative crystallization. Mixing can be applied as needed. In embodiments, cooling crystallization includes cooling by heat transfer surface cooling, evaporative cooling, or a combination thereof. In embodiments, cooling includes significant evaporation and reducing the temperature to a temperature below the ERX reaction temperature, thereby recovering product crystals. The reformed crystals can then be isolated as purified ERX-crystals in step 1921 of method 1900 by separation and / or washing.

[0180] The resulting crystalline steviol glycoside composition comprises one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. The crystals may, in one example, comprise Reb D, Reb E, and Reb A. Reb D, Reb E, and Reb A may each comprise from about 0% to about 100% of the crystals. Reb D, Reb E, and Reb A may each comprise about 0.1% to about 20%, about 0.2% to about 18%, about 0.3% to about 16%, about 0.4% to about 14%, about 0.5% to about 12%, about 0.6% to about 10%, about 0.7% to about 8%, about 0.8% to about 6%, about 0.9% to about 4%, or about 1% to about 2% of the crystals. Reb D, Reb E, and Reb A may each comprise about 10% to about 90%, about 15% to about 85%, about 20% to about 80%, about 25% to about 75%, about 30% to about 70%, about 35% to about 65%, about 40% to about 60%, about 45% to about 55%, about 47.5% to about 52.5%, or about 48% to about 52% of the crystals. Reb D, Reb E, and Reb A may each comprise about 70% to about 100%, about 72% to about 98%, about 74% to about 96%, about 76% to about 94%, about 78% to about 92%, about 80% to about 90%, about 82% to about 88%, and about 84% to about 86% of the crystals. In one example, the crystals contain, on an anhydrous basis, 70.9% Reb D, 17.4% Reb E, and 0.4% Reb A. The composition of steviol glycosides in the resulting mother liquor includes one or more of the steviol glycosides described herein, including stevioside, Reb B, Reb G, Reb C, Reb F, Reb A, Reb I, Reb E, Reb E2, Reb AM, Reb H, Reb L, Reb K, Reb J, Reb M, Reb D, Reb N, Reb O, Reb Q, and synthetic steviol glycosides. In one example, the mother liquor may contain Reb E, Reb D, Reb A, and stevioside in a ratio of 12.2:8.1:1:1.3.However, the ratio between the concentrations of each steviol glycoside is highly variable and depends on the steviol glycoside(s) included in the reaction.

[0181] 20, when the feedstock composition contains only a single steviol glycoside, such as stevioside, other means for enhancing solubility can be used. As in step 2001 of method 2000, the feedstock composition can be heated to a temperature greater than about 40° C., about 50° C., about 60° C., about 70° C., about 80° C., about 90° C., and / or about 100° C. ERX can then be performed in step 2002 of method 2000 according to the methods described herein. [Example]

[0182] Example 1 - ERX Reaction to Produce Rebaudioside D-Enriched Product "ERX-D" A 1-liter spinner flask was used as a reactive crystallizer. To express engineered SuSy (SEQ ID NO: 1), a Pichia strain was grown in a 1-liter fermentor. To express engineered B12GT (SEQ ID NO: 2), a second Pichia strain was grown in a separate 1-liter fermentor. In each case, cells were harvested and mechanically lysed using a French press. The expressed proteins from the lysates were partially purified by heat treatment, centrifuged to remove clotted proteins and cell debris, and decolorized by treatment with a hydrophobic resin. Engineered SuSy and B12GT were reacted with a feedstock composition containing 100 g / L RA50 (a stevia leaf extract containing >95% by weight steviol glycosides, >50% by weight Reb A, and >30% by weight stevioside), 100 g / L sucrose, and 0.5 mM ADP in 50 mM phosphate buffer (pH 6) and 250 mM NaCl. The reaction was carried out at 60°C for 6 hours. Enzyme-mediated reactive crystallization yielded a precipitate and mother liquor. The reaction was stored and cooled overnight (2-8°C). The precipitate was collected by centrifugation, washed three times with water, and dried in a vacuum oven. The dried reaction precipitate contained 75.5% Reb D, 18.2% Reb E, and 0.4% Reb A on an anhydrous basis. The ratio of the major steviol glycosides in the mother liquor was determined to be 12.2:8.1:1:1.3 Reb E:Reb D:Reb A:Steviol.

[0183] Example 2 - Growth and Characterization of ERX Crystals in a 150 Liter Reactor Example 2a - Preparation of Reactive Enzyme Two 150 liter scale fermentation runs were conducted using two different engineered Pichia strains. One strain expressed an engineered SuSy enzyme (SEQ ID NO: 1). The other Pichia strain expressed an engineered B12GT enzyme (SEQ ID NO: 2). After fermentation was completed, the fermentation broths were combined, diluted, and centrifuged to isolate the cells from the fermentation broth, resulting in 185 kg and diluted to a total of 500 L. The cells were then separated using a disc centrifuge, yielding 115.46 kg of cell cake.

[0184] The cell cake was resuspended 2:1 in buffer, and the cells were lysed by passing them through a homogenizer five times. The resulting lysed cell stream was pasteurized in a flow system and then rapidly cooled. The liquid stream was further diluted and clarified using a disc centrifuge to remove coagulated proteins and unwanted cell debris. The clear liquid was purified using a suitable dead-end filtration system and then stored overnight in a cooling tank. The liquid enzyme solution was then purified using a fixed-bed resin process. This was subsequently ultrafiltered to concentrate the enzyme mixture and diafiltered to exchange the buffer from the lysis buffer to the reaction buffer.

[0185] Example 2b - Enzyme-Mediated Reactive Crystallization The enzyme-mediated reactive crystallization process used a hot-water jacketed conical tank with a maximum working volume of 200 liters. Agitation was provided by a single overhead-driven stirring shaft with a small-diameter screw impeller at the bottom, in addition to two simple flat-blade turbines with two blades each oriented at a 30-degree angle. The clarified enzyme from Example 2a was reacted with a feedstock composition containing 100 g / L of RA50 (a stevia leaf extract containing >95% steviol glycosides, >50% rebaudioside A, and >30% stevioside by weight), 100 g / L of sucrose, and 0.5 mM ADP in 50 mM potassium phosphate buffer (pH 6) and 250 mM sodium acetate. Notably, the mixed steviol glycoside feedstock composition fed to the enzyme-mediated reactive crystallization process has a higher solubility than either of the individual steviol glycosides alone.

[0186] The reaction was carried out at 60°C for 6 hours. The system rapidly reached the target reaction and primary crystallization temperature setpoints through the mixing process. Gentle agitation was maintained throughout the reaction time course. After 6 hours of reaction, the system reached a suspended solids content of 18% crystals by volume. Figure 8 shows the kinetics of crystal formation, and Figure 9 shows the kinetics of steviol glycoside conversion.

[0187] The system was then cooled with cold water and stored for 20 hours to allow further crystal growth. The crystals were collected using a chamber filter press and air-dried. A total of 19 kg of air-dried crystals was produced. These crystals were vacuum tray dried at 55°C using a vacuum pump operating at 50 mbar absolute pressure. The final weight of the dried crystals was 9.06 kg. The dried crystals consisted of 70.9% Reb D, 17.4% Reb E, and 0.4% Reb A on an anhydrous basis.

[0188] Example 2c - Treatment of Mother Liquor from Enzyme-Mediated Reactive Crystallization The crystals from the enzyme-mediated reactive crystallization of Example 2b were collected using a filter press, resulting in a clarified mother liquor that may be unique in that it may contain a particularly soluble steviol glycoside composition.

[0189] The mother liquor was clarified by ultrafiltration and diafiltration to remove the enzymes and collect any residual steviol glycosides present, yielding 204 liters of combined ultrafiltration filtrate containing a Reb E to Reb D ratio of 3.56:1.

[0190] The soluble steviol glycoside solution was loaded onto the non-ionic resin by adsorption. The column was then washed with water to remove hydrophilic reaction components. Rebaudioside was then eluted using 70% isopropyl alcohol in water at room temperature. The first 1.5 bed volumes contained 77.14% by weight of steviol glycosides, the next bed volume contained approximately 1%, and the final 1.5 bed volumes contained 0.03%.

[0191] The 348 liters of eluate was continuously evaporated down to 20 liters. At this point, water was added as a solvent chase to facilitate final alcohol stripping. The elution mixture was then concentrated to 57% dissolved solids, and the concentrate was transferred to trays in preparation for vacuum tray drying. Upon cooling, immediate crystallization occurred at the surface, indicating that crystals can be produced from the mother liquor using evaporation and / or cooling crystallization, but relatively high concentrations may be required. The steviol glycoside concentrate was vacuum tray dried to yield 4 kg of final dried crystals. The crystals consisted primarily of Reb D and Reb E in a 1:3.4 ratio. The crystals readily dissolved in water.

[0192] Example 3 - Low Solubility of Mixed Rebaudioside A and Rebaudioside D A composition representative of some point in enzyme-mediated reactive crystallization was prepared as follows: 100 grams of deionized water was added to 4 grams of sucrose and 2.17 grams of fructose, and the mixture was heated to 60°C with stirring. Rebaudioside D (0.50 grams) and rebaudioside A (0.513 grams) were added. The final mixture had a composition of 0.48% by weight rebaudioside A and 0.45% by weight rebaudioside D. A white solid remained present, and the combined rebaudiosides were not soluble, even at a total of 0.93% by weight.

[0193] Example 4 - High Solubility of Mixed Rebaudioside A and Stevioside A rebaudioside mixture representative of a partially purified leaf extract was prepared as follows: 200 grams of RA50 (a Stevia leaf extract containing >95% steviol glycosides, >50% rebaudioside A, and >30% stevioside by weight) was dissolved in 800 ml of water, heated to 80° C., and mixed. At 60° C., the mixture was clear, indicating high solubility of the Rebaudioside A and stevioside mixture.

[0194] Example 5 - Concentration of ERX Mother Liquor to Obtain Additional Crystals Example 5 demonstrates the principle of treating ERX mother liquor by concentration to obtain additional crystals. The Reb D and Reb E-enriched reaction mother liquor from Example 1 was purified via ultrafiltration, adsorption, desorption, and desolvation to remove enzymes, sugars, and ADP. The resulting material contained substantially all of the steviol glycosides present in the ERX mother liquor. This material was dried to allow for experiments with precise levels of dissolved solids (DS). This material was dissolved in water at a ratio of 1 gram of dissolved material per 10 grams of water. Complete solubility was observed, which is consistent with an ERX mother liquor containing highly soluble mixed rebaudiosides. Analysis showed that the ERX mother liquor was enriched in both rebaudioside E and rebaudioside D. Further evaporation and cooling of this aqueous mixture resulted in the formation of new crystals.

[0195] Example 6 - ERX Preparation of Rebaudioside M, Hot Break Clarification, Recrystallization, Redissolution, and Recrystallization Example 6 shows the preparation of the enzyme, the formation of Reb M by ERX, the formation of the hot break, the removal of the hot break, and the re-formation of crystals by cooling crystallization.

[0196] Example 6a - Preparation of Reactive Enzyme Three 1-liter scale fermentation runs were performed using three different engineered Pichia strains. One strain expressed an engineered SuSy enzyme (SEQ ID NO: 1). Another Pichia strain expressed an engineered B12GT enzyme (SEQ ID NO: 2). Another Pichia strain expressed an engineered B13GT enzyme (SEQ ID NO: 3). After the fermentations were completed, the fermentation broths were individually centrifuged to isolate the cells from the fermentation broth. Each fermentation produced a significant amount of wet cell cake.

[0197] The cell cakes from above were combined at a 1:1:2 ratio of SuSyPichia:B12GTPichia:B13GTPichia wet cell weight. This mixture was resuspended 2:1 in sodium acetate buffer, and the cells were lysed by passing five times through a high-pressure homogenizer. The resulting lysed cell stream was pasteurized and rapidly cooled in a continuous-flow pasteurizer. The liquid stream was further diluted and clarified using centrifugation to remove coagulated proteins and unwanted cell debris. The clear liquid was stored overnight at 4°C. The clear liquid was concentrated and the buffer exchanged by ultrafiltration. A total of 1370 ml of concentrate was produced. A sample of this was diluted 570 / 730 times, and 400 grams of this enzyme mixture was taken and used here.

[0198] Example 6b - ERX Reaction 400 g of the enzyme mixture from Example 6a was combined with 400 ml of a steviol glycoside-based composition. The resulting 800 gram mixture contained 10 g / L of RA50 (a stevia leaf extract containing >95% steviol glycosides, >50% rebaudioside A, and >30% stevioside by weight) in 50 mM phosphate buffer (pH 6) containing 250 mM sodium acetate, 50 g / L of sucrose, and 1 mM ADP. The reaction mixture was preheated for at least 1 hour and reacted at 56.7°C for 5 hours, yielding a precipitate and mother liquor.

[0199] Example 6c - Preparation of Hot Break The above reaction mixture was heated in a reaction flask at 86°C for 30 minutes. It was then cooled to 75°C and filtered to remove the hot break. The clarified filtrate weighed 802.61 grams. The hot break mass was 1.73 grams. The filtered ERX product was cooled at 4°C for 12 hours to reform the ERX crystals. The ERX crystals were collected on a filter and washed with three aliquots of ice-cold water using a total wash mass of 68.3 grams. The initial mass of the recovered dried ERX product crystals was 3.67 grams. The rate of crystal growth was moderate at 4°C, and the addition time allowed for an additional 3.96 grams of dried crystals to be collected after washing. The combined crystal analysis was as follows:

[0200] [Table 4]

[0201] Example 6d - Recrystallization The combined crystals were dissolved in 323 grams of purified water at 70°C, then cooled and recrystallized overnight. After washing, a dry crystal mass of 2.43 grams was recovered. The crystal analysis was as follows:

[0202] [Table 5]

[0203] Example 7 - Mother Liquor Treatment and Steviol Glycoside Recycling Example 7 demonstrates mother liquor processing and recycling of steviol glycosides within an ERX reaction system.

[0204] Example 7a - Preparation of Reactive Enzyme SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 from Example 6a were combined with 60 g / L RA60 (>95% steviol glycosides, >60% rebaudioside A, and >20% stevioside by weight), 250 g / L sucrose, and 1 mM ADP at 56°C at an enzyme dosage of 6.1 mg enzyme per gram of lyophilized material. Three sequential reactions were performed. First, 100 ml of the composition was placed in a rotary evaporator. An immersion blender was used to break down the custard for 2, 2.5, and 3 hours. Crystals comprised approximately 25%-30% of the volume. The resulting estimated mother liquor volume was 70 ml. Next, 600 g / L RA60 7.8 was combined with 70 ml of the mother liquor in the rotary evaporator. The immersion blender was used as described above. The estimated mother liquor volume obtained was 56 ml. Finally, 5.6 ml of 600 g / L RA60 was combined with 50 ml of treated mother liquor in a rotary evaporator. An immersion blender was used as described above. Total protein concentration was monitored throughout the experiment. Samples were collected at time intervals for HPLC analysis of crystal formation and composition.

[0205] This data is shown in Figure 21, which is a graphical representation of mother liquor recycle. Figure 21 shows high performance liquid chromatography results showing the RXN1 control with 86% Reb M at 3 hours. This can be compared to the RXN2 sample with recycled mother liquor, which has a slower rate of Reb M formation. For the RXN2 sample with recycled mother liquor, Reb M reached 31% at 6 hours, and Reb AM, Reb D, and Reb E reached 47% at 6 hours. For the RXN1 control, Reb AM, Reb D, and Reb E, they reached 1% by 3 hours.

[0206] Example 7b - Preparation of steviol glycosides SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 from Example 6a were combined with 60 g / L RA60 (>95% steviol glycosides, >60% rebaudioside A, and >20% stevioside by weight), 250 g / L sucrose, and 1 mM ADP at 56°C at an enzyme dosage of 6.1 mg enzyme per gram of lyophilized material. Three sequential reactions were performed. First, a 150 mL volume of the composition was placed in the first rotary evaporator, and an additional 50 mL volume of the composition was placed in the second rotary evaporator as a control. The first rotary evaporator contained the enzyme and reactants. The custard was broken down using an immersion blender for 2, 2.5, and 3 hours. Crystals comprised approximately 25%-30% of the volume. The resulting estimated mother liquor volume was 70 mL. The second rotary evaporator contained the reconstituted enzyme without the addition of reactants or an immersion blender. The reconstituted enzyme was further heated and gently sheared on the rotary evaporator. Next, 75 mL of the mother liquor was passed through a Sephadex column to remove excess fructose (a fructose assay was used to confirm removal). The protein concentration was tested. Each solution (and the control solution) was then diluted with additional buffer to the same concentration. Five 9 mL aliquots were then prepared from the reserved untreated mother liquor. Each aliquot was supplemented with additional reagents as follows: (1) another 9 mL aliquot was prepared from the Sephadex-filtered solution; (2) 12.5 g of sucrose and 2 mM ADP were added to the solution from the above reaction; and (3) a 9 mL aliquot of the aforementioned solution was prepared. All 9 mL aliquots were then reacted using a 10 mL bioconversion QC setup. The reaction was initiated by adding 1 mL of 600 g / L RA60. Samples for HPLC were obtained every hour for 4 hours to test for rebaudioside.

[0207] Figure 22 shows the amount of rebaudioside produced at each time point for the numerous conditions identified under the "Injection Name" column. Samples marked 1A correspond to the auxiliary reagent ADP, samples marked 1B2 correspond to the auxiliary reagent β12GT, samples marked 1B3 correspond to the auxiliary reagent β13GT, and samples marked 1S correspond to the auxiliary reagent sucrose. Additionally, 1C refers to a control in which a first reaction was performed under standard conditions and then used in a second reaction without any treatment. 1A refers to an ADP test in which a first reaction was performed under standard conditions and then supplemented with additional ADP before use in a second reaction. 1F refers to a fructose test in which a first reaction was performed under standard conditions and then treated to remove excess fructose before use in a second reaction. 1B2 refers to a β12GT test in which a first reaction was performed under standard conditions and then supplemented with additional β12GT before use in a second reaction. 1BB refers to a β13GT test in which the first reaction was performed under standard conditions and then supplemented with additional β13GT before use in the second reaction. 1S refers to a sucrose test in which the first reaction was performed under standard conditions and then supplemented with additional sucrose before use in the second reaction. 2 refers to an absolute control that was subjected to the same temperature conditions as 1C, 1A, 1F, 1B2, 1B3, and 1S, but did not perform the first reaction and was not subjected to high shear.

[0208] Example 7c - Residue-Based Enzymatic Production of Steviol Glycosides The β12GT, β13GT, and SuSy remaining in the mother liquor were used as recycled enzymes for an additional 10 ml of bioconversion.

[0209] Specifically, the reactions used two steviol glycoside feedstocks, namely, RA60 powder and concentrated RA60 syrup at approximately 60 g / L. Additionally, two levels of Na-ADP dosage with an additional 1 mM Na-ADP were used. The reactions were carried out overnight without heating. After the reactions, the vials were warmed to 60°C and sampled every two hours thereafter. The vials were finally sampled after three time points.

[0210] Referring to Figure 23, the production of steviol glycosides from these reactions is shown. RXN1 was reacted with 60 g / L of RA60 powder but without ADP. RXN2 was reacted with 60 g / L of RA60 powder and ADP. RXN3 was reacted with approximately 75 g / L of RA60 syrup but without ADP. RXN4 was reacted with approximately 75 g / L of RA60 syrup and ADP.

[0211] The foregoing description, for purposes of explanation, used specific nomenclature to provide a thorough understanding of the invention. However, it will be apparent to those skilled in the art that specific details are not required to practice the present invention. The foregoing descriptions of specific embodiments of the subject matter of the present disclosure have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in light of the above teachings. The embodiments were chosen and described in order to explain the principles of the invention and its practical application, so that others skilled in the art can utilize the invention and its various embodiments with various modifications suited to the particular use intended. It is the following claims and their equivalents that define the scope of the invention.

[0212] Numbered Embodiments of the Invention Without limiting the scope of any appended claims, the present disclosure describes the following numbered embodiments of the disclosure:

[0213] (1) A method for producing and isolating steviol glycoside crystals, the method comprising: performing enzyme-mediated reactive crystallization by reacting a feedstock composition comprising at least one soluble steviol glycoside with an enzyme composition comprising at least one enzyme configured to add one or more sugar monomers to the at least one soluble steviol glycoside; and isolating at least one steviol glycoside crystal produced by the reaction, wherein the isolated steviol glycoside crystal comprises at least one additional sugar monomer compared to the at least one soluble steviol glycoside.

[0214] (2) The method of (1), wherein the reacting comprises converting the at least one soluble steviol glycoside into a different steviol glycoside having reduced solubility relative to the at least one steviol glycoside, thereby causing crystallization of the different steviol glycoside.

[0215] (3) The method according to either (1) or (2), wherein the one or more sugar monomers include galactose, glucose, xylose, glucosamine, galactosamine, glucuronic acid, galactofuranose, mannose, fucose, rhamnose, acetylneuraminic acid, and mannooctanoic acid.

[0216] (4) The method according to any one of (1) to (3), wherein the raw material composition contains two or more soluble steviol glycosides.

[0217] (5) The method described in (4), wherein the two or more soluble steviol glycosides are present in the raw material composition at a concentration of at least 100 grams per liter.

[0218] (6) The two or more soluble steviol glycosides are steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside E, The method of either (4) or (5), comprising a naturally occurring steviol glycoside selected from the group consisting of rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside X, rebaudioside N, rebaudioside O, rebaudioside Q, and enzymatically glycosylated steviol glycosides.

[0219] (7) The method described in any one of (4) to (6), wherein the two or more soluble steviol glycosides have improved solubility.

[0220] (8) The method according to any one of (1) to (7), wherein the raw material composition contains a stevia leaf extract.

[0221] (9) The method according to any one of (1) to (8), wherein the raw material composition contains stevioside and rebaudioside A.

[0222] (10) The method according to any one of (1) to (9), wherein the raw material composition contains at least 30 g / L of stevioside and at least 50 g / L of rebaudioside A.

[0223] (11) The method according to any one of (1) to (10), wherein the isolated steviol glycoside crystals comprise one or more of rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside X, rebaudioside D, and enzymatically glycosylated steviol glycosides.

[0224] (12) The method according to any one of (1) to (11), wherein the at least one enzyme configured to add the one or more sugar monomers to the at least one soluble steviol glycoside comprises one or more of a β-1,2-glycosyltransferase (B12GT) and a β-1,3-glycosyltransferase (B13GT).

[0225] (13) The method according to (12), wherein the B12GT and / or the B13GT are engineered enzymes.

[0226] (14) The method according to any one of (1) to (13), wherein the at least one enzyme configured to add the one or more sugar monomers to the at least one soluble steviol glycoside comprises a nucleotide-sugar (NDP-sugar) as a substrate.

[0227] (15) The method according to (14), wherein the NDP comprises one of adenosine diphosphate (ADP), cytidine diphosphate (CDP), thymidine diphosphate (TDP), and guanosine diphosphate (GDP).

[0228] (16) The method according to any one of (1) to (15), wherein the enzyme composition further contains sucrose synthase.

[0229] (17) The method according to (16), wherein the sucrose synthase is capable of producing NDP-glucose.

[0230] (18) The method according to (17), wherein the sucrose synthase is capable of producing ADP-glucose.

[0231] (19) The method according to any one of (1) to (18), further comprising heating a reaction composition containing the raw material composition and the enzyme composition.

[0232] (20) The method according to (19), wherein the reaction composition is heated to a predetermined reaction temperature of about 10°C, about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, and about 80°C or higher.

[0233] (21) The method according to either (19) or (20), wherein the reaction composition comprises a pH of about 4 to about 10, about 5 to about 9, and about 6 to about 8.

[0234] (22) The method according to any one of (19) to (21), wherein the reaction composition further contains NDP.

[0235] (23) The method according to (22), wherein the NDP includes ADP, CDP, TDP, and / or GDP.

[0236] (24) The method according to any one of (19) to (22), wherein the reaction composition further contains a sugar.

[0237] (25) The method according to (24), wherein the sugar comprises one or more of galactose, glucose, xylose, glucosamine, galactosamine, glucuronic acid, galactofuranose, mannose, fucose, rhamnose, acetylneuraminic acid, and mannooctanoic acid.

[0238] (26) The method according to any one of (1) to (25), wherein the reaction of the raw material composition with the enzyme composition is carried out for a predetermined duration.

[0239] (27) The method of (26), wherein the predetermined duration is at least about 6 minutes, about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, and about 6 hours.

[0240] (28) The method according to any one of (1) to (27), further comprising adding a secondary enzyme composition to the reaction composition during the enzyme-mediated reactive crystallization.

[0241] (29) The method according to any one of (1) to (28), further comprising performing evaporation crystallization and / or cooling crystallization.

[0242] (30) The method according to any one of (1) to (29), wherein isolating the at least one steviol glycoside crystal comprises recovering a mother liquor from the reaction, the mother liquor containing residual soluble steviol glycoside.

[0243] (31) The method of (30), further comprising treating the mother liquor by clarifying and concentrating the remaining soluble steviol glycosides and performing a second enzyme-mediated reactive crystallization on the remaining soluble steviol glycosides to produce second steviol glycoside crystals.

[0244] (32) The method according to (31), further comprising carrying out evaporation crystallization and / or cooling crystallization.

[0245] (33) The method according to (30) or (31), further comprising recycling the mother liquor into a subsequent raw material composition.

[0246] (34) The method according to any one of (1) to (33), further comprising reheating the reaction composition after the reacting to form a hot break containing impurities.

[0247] (35) The method of (34), further comprising clarifying the reaction composition by removing the hot break.

[0248] (36) The method according to (35), further comprising performing evaporation crystallization and / or cooling crystallization on the clarified reaction composition.

[0249] (37) The method according to any one of (1) to (36), further comprising heating the raw material composition to a predetermined temperature.

[0250] (38) The method according to (37), wherein the predetermined temperature is at least about 40°C, about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, and about 100°C.

[0251] (39) The method according to any one of (1) to (38), wherein the isolating step further comprises separating and washing a reaction composition containing the raw material composition and the enzyme composition.

[0252] (40) A crystalline composition comprising steviol glycoside cocrystals having a ratio of rebaudioside E to rebaudioside D of 1:3 to 1:5 on an anhydrous basis.

[0253] (41) A crystalline composition comprising, on an anhydrous basis, steviol glycoside cocrystals having at least 65% rebaudioside D and at least 10% rebaudioside E.

[0254] (42) A crystalline composition comprising, on an anhydrous basis, steviol glycoside co-crystals having at least 10% rebaudioside D and at least 70% rebaudioside M.

[0255] (43) A crystalline composition comprising rebaudioside E to rebaudioside D in a ratio of 2:1 to 5:1 on an anhydrous basis.

[0256] (44) A crystalline composition comprising crystalline steviol glycoside species.

[0257] (45) A crystalline composition comprising, on an anhydrous basis, at least 65% rebaudioside D and at least 10% rebaudioside E.

[0258] (46) A crystalline composition comprising, on an anhydrous basis, at least about 90% Reb M, about 91% Reb M, about 92% Reb M, about 93% Reb M, about 94% Reb M, about 95% Reb M, about 96% Reb M, about 97% Reb M, about 98% Reb M, or about 99% Reb M.

[0259] (47) The crystalline composition according to any one of claims 40 to 46, produced according to the method according to any one of (1) to (46).

[0260] (48) A method comprising spray-crushing the crystalline composition according to (47).

[0261] (49) A spray-crushed composition comprising the crystalline composition according to any one of (40) to (46).

[0262] (50) A steviol glycoside composition comprising a mother liquor, wherein the mother liquor has high solubility of steviol glycoside.

[0263] (51) The steviol glycoside composition according to claim 48, produced according to any one of the methods described in (1) to (50).

[0264] (52) A beverage composition prepared from an ingredient composition containing 10 ppm to 1000 ppm of any one of the crystalline compositions according to any one of (40) to (46).

[0265] (53) A baked food product prepared from a raw material composition containing 10 ppm to 1000 ppm of any one of the crystalline compositions according to any one of (40) to (46).

[0266] (54) The method of either (30) or (50), wherein the steviol glycosides in the mother liquor contain, on average, at least 0.5, at least 1, at least 2, and / or at least 3 units of glucose per steviol glycoside than the at least one soluble steviol glycoside in the raw material composition.

[0267] (55) The method according to any one of (1) to (54), wherein the at least one steviol glycoside crystal produced by the reaction contains, on average, at least 0.5, at least 1, at least 2, and / or at least 3 units of glucose per steviol glycoside than the at least one soluble steviol glycoside in the raw material composition.

[0268] (56) The method of either (30) or (50), wherein the at least one soluble steviol glycoside has an average molecular weight lower than the average molecular weight of the steviol glycoside in the mother liquor.

[0269] (57) The method of any one of (1) to (56), wherein the at least one enzyme comprises an engineered beta-1,3-glycosyltransferase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3, wherein the polypeptide comprises H at position 24, D at position 123, D at position 379, and Q at position 380.

[0270] (58) The method of any one of (1) to (57), wherein the at least one enzyme comprises an engineered beta-1,2-glycosyltransferase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2.

[0271] (59) The method according to any one of (1) to (58), wherein the at least one enzyme comprises an engineered sucrose synthase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:1.

[0272] (60) A method for making and isolating steviol glycoside crystals, the method comprising: performing enzyme-mediated reactive crystallization by reacting a feedstock composition comprising at least one soluble steviol glycoside with an enzyme composition comprising at least one enzyme configured to add one or more sugar monomers to the at least one soluble steviol glycoside; isolating at least one steviol glycoside crystal produced by the reaction, wherein the isolated steviol glycoside crystal comprises at least one additional sugar monomer compared to the at least one soluble steviol glycoside; and recovering a mother liquor produced from the reaction.

Claims

1. 1. A method for making and isolating steviol glycoside crystals, comprising: performing enzyme-mediated reactive crystallization by reacting a feedstock composition comprising at least one soluble steviol glycoside with an enzyme composition comprising at least one enzyme configured to add one or more sugar monomers to the at least one soluble steviol glycoside; and isolating at least one steviol glycoside crystal produced by the reaction, wherein the isolated steviol glycoside crystal comprises at least one additional sugar monomer compared to the at least one soluble steviol glycoside.

2. 2. The method of claim 1, wherein the reacting comprises converting the at least one soluble steviol glycoside to a different steviol glycoside having reduced solubility relative to the at least one steviol glycoside, thereby causing crystallization of the different steviol glycoside.

3. 10. The method of claim 1, wherein the one or more sugar monomers comprise galactose, glucose, xylose, glucosamine, galactosamine, glucuronic acid, galactofuranose, mannose, fucose, rhamnose, acetylneuraminic acid, and mannooctanoic acid.

4. 10. The method of claim 1, wherein the raw material composition comprises two or more soluble steviol glycosides.

5. 5. The method of claim 4, wherein the two or more soluble steviol glycosides are present in the feedstock composition at a concentration of at least 100 grams per liter.

6. The two or more soluble steviol glycosides may be steviol-13-O-glucoside, steviol-19-O-glucoside, rubusoside, steviol-1,2-bioside, steviol-1,3-bioside, rubusoside, dulcoside B, dulcoside A, rebaudioside B, rebaudioside G, stevioside, rebaudioside C, rebaudioside F, rebaudioside A, rebaudioside I, rebaudioside B, rebaudioside C, rebaudioside F, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside G, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside I, rebaudioside B, rebaudioside I ...

5. The method of claim 4, wherein the steviol glycoside comprises a naturally occurring steviol glycoside selected from the group consisting of rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside D, rebaudioside X, rebaudioside N, rebaudioside O, rebaudioside Q, and enzymatically glycosylated steviol glycosides.

7. 5. The method of claim 4, wherein the two or more soluble steviol glycosides have enhanced solubility.

8. 10. The method of claim 1, wherein the raw material composition comprises a stevia leaf extract.

9. 10. The method of claim 1, wherein the feed composition comprises stevioside and rebaudioside A.

10. 10. The method of claim 1, wherein the feed composition comprises at least 30 g / L of stevioside and at least 50 g / L of rebaudioside A.

11. 2. The method of claim 1, wherein the isolated steviol glycoside crystals comprise one or more of rebaudioside A, rebaudioside I, rebaudioside E, rebaudioside E2, rebaudioside AM, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside X, rebaudioside D, and enzymatically glycosylated steviol glycosides.

12. 2. The method of claim 1, wherein the at least one enzyme configured to add the one or more sugar monomers to the at least one soluble steviol glycoside comprises one or more of a β-1,2-glycosyltransferase (B12GT) and a β-1,3-glycosyltransferase (B13GT).

13. 13. The method of claim 12, wherein the B12GT and / or the B13GT are engineered enzymes.

14. 10. The method of claim 1, wherein the at least one enzyme configured to add the one or more sugar monomers to the at least one soluble steviol glycoside includes a nucleotide-sugar (NDP-sugar) as a substrate.

15. 15. The method of claim 14, wherein the NDP comprises one of adenosine diphosphate (ADP), cytidine diphosphate (CDP), thymidine diphosphate (TDP), and guanosine diphosphate (GDP).

16. The method of claim 1 , wherein the enzyme composition further comprises a sucrose synthase.

17. 17. The method of claim 16, wherein the sucrose synthase is capable of making NDP-glucose.

18. 18. The method of claim 17, wherein the sucrose synthase is capable of producing ADP-glucose.

19. 10. The method of claim 1, further comprising heating a reaction composition comprising the raw material composition and the enzyme composition.

20. 20. The method of claim 19, wherein the reaction composition is heated to a predetermined reaction temperature of about 10°C, about 20°C, about 30°C, about 40°C, about 50°C, about 60°C, about 70°C, and about 80°C or greater.

21. 20. The method of claim 19, wherein the reaction composition comprises a pH of about 4 to about 10, about 5 to about 9, and about 6 to about 8.

22. 20. The method of claim 19, wherein the reaction composition further comprises an NDP.

23. 23. The method of claim 22, wherein the NDP comprises ADP, CDP, TDP, and / or GDP.

24. 20. The method of claim 19, wherein the reaction composition further comprises a sugar.

25. 25. The method of claim 24, wherein the sugar comprises one or more of galactose, glucose, xylose, glucosamine, galactosamine, glucuronic acid, galactofuranose, mannose, fucose, rhamnose, acetylneuraminic acid, and mannooctanoic acid.

26. 10. The method of claim 1, wherein reacting the feedstock composition and the enzyme composition occurs for a predetermined duration.

27. 27. The method of claim 26, wherein the predetermined duration is at least about 6 minutes, about 30 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, and about 6 hours.

28. 10. The method of claim 1, further comprising adding a secondary enzyme composition to the reaction composition during the enzyme-mediated reactive crystallization.

29. The method of claim 1 further comprising performing evaporative crystallization and / or cooling crystallization.

30. 10. The method of claim 1, wherein isolating the at least one steviol glycoside crystals comprises recovering a mother liquor from the reaction, the mother liquor comprising residual soluble steviol glycoside.

31. treating the mother liquor by clarifying and concentrating the remaining soluble steviol glycosides; 31. The method of claim 30, further comprising performing a second enzyme-mediated reactive crystallization on the remaining soluble steviol glycoside to produce second steviol glycoside crystals.

32. 32. The method of claim 31, further comprising performing evaporative crystallization and / or cooling crystallization.

33. 31. The method of claim 30, further comprising recycling the mother liquor to a subsequent feed composition.

34. 10. The method of claim 1, further comprising reheating the reaction composition after said reacting to form a hot break containing impurities.

35. 35. The method of claim 34, further comprising clarifying the reaction composition by removing the hot break.

36. 36. The method of claim 35, further comprising performing evaporative crystallization and / or cooling crystallization on the clarified reaction composition.

37. 10. The method of claim 1, further comprising heating the feed composition to a predetermined temperature.

38. 38. The method of claim 37, wherein the predetermined temperature is at least about 40°C, about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, and about 100°C.

39. 10. The method of claim 1, wherein said isolating further comprises separating and washing a reaction composition comprising said raw material composition and said enzyme composition.

40. 1. A crystalline composition comprising steviol glycoside co-crystals having a ratio of rebaudioside E to rebaudioside D of 1:3 to 1:5 on an anhydrous basis.

41. 1. A crystalline composition comprising steviol glycoside co-crystals having at least 65% rebaudioside D and at least 10% rebaudioside E on an anhydrous basis.

42. 1. A crystalline composition comprising steviol glycoside co-crystals having at least 10% rebaudioside D and at least 70% rebaudioside M on an anhydrous basis.

43. A crystalline composition comprising rebaudioside E to rebaudioside D in a ratio of 2:1 to 5:1 on an anhydrous basis.

44. A crystalline composition comprising crystalline steviol glycoside species.

45. 1. A crystalline composition comprising, on an anhydrous basis, at least 65% rebaudioside D and at least 10% rebaudioside E.

46. A crystalline composition comprising, on an anhydrous basis, at least about 90% Reb M, about 91% Reb M, about 92% Reb M, about 93% Reb M, about 94% Reb M, about 95% Reb M, about 96% Reb M, about 97% Reb M, about 98% Reb M, or about 99% Reb M.

47. 47. The crystalline composition of any one of claims 40 to 46, produced according to the method of claim 1.

48. 48. A method comprising spray-milling the crystalline composition of claim 47.

49. A spray-milled composition comprising the crystalline composition of any one of claims 40 to 46.

50. A steviol glycoside composition comprising a mother liquor, wherein the mother liquor has high solubility of steviol glycoside.

51. 49. The steviol glycoside composition of claim 48 produced according to the method of claim 1.

52. A beverage composition prepared from an ingredient composition containing 10 ppm to 1000 ppm of any one of the crystalline compositions according to claims 40 to 46.

53. A baked food product prepared from an ingredient composition comprising 10 ppm to 1000 ppm of any of the crystalline compositions of claims 40 to 46.

54. 51. The method of claim 30, wherein the steviol glycosides in the mother liquor contain, on average, at least 0.5, at least 1, at least 2, and / or at least 3 units of glucose per steviol glycoside than the at least one soluble steviol glycoside in the feedstock composition.

55. 10. The method of claim 1, wherein the at least one steviol glycoside crystals produced by the reaction contain, on average, at least 0.5, at least 1, at least 2, and / or at least 3 units of glucose per steviol glycoside than the at least one soluble steviol glycoside of the feedstock composition.

56. 51. The method of claim 30, wherein the at least one soluble steviol glycoside comprises an average molecular weight that is lower than the average molecular weight of the steviol glycoside in the mother liquor.

57. 2. The method of claim 1, wherein the at least one enzyme comprises an engineered beta-1,3-glycosyltransferase polypeptide comprising an amino acid sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:3, wherein the polypeptide comprises an H at position 24, a D at position 123, a D at position 379, and a Q at position 380.

58. 2. The method of claim 1, wherein the at least one enzyme comprises an engineered beta-1,2-glycosyltransferase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

2.

59. 2. The method of claim 1, wherein the at least one enzyme comprises an engineered sucrose synthase polypeptide comprising an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

1.

60. 1. A method for making and isolating steviol glycoside crystals, comprising: performing enzyme-mediated reactive crystallization by reacting a feedstock composition comprising at least one soluble steviol glycoside with an enzyme composition comprising at least one enzyme configured to add one or more sugar monomers to the at least one soluble steviol glycoside; isolating at least one steviol glycoside crystal produced by the reaction, wherein the isolated steviol glycoside crystal comprises at least one additional sugar monomer compared to the at least one soluble steviol glycoside; and recovering a mother liquor produced by the reaction.

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