Methods of making carotenoids and products relating thereto

Abstract

The present disclosure provides for a method of making β-carotene, comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and either of a sugar beet molasses or a sorghum syrup to thereby produce the β-carotene; wherein the mineral salt comprises (NH4)2SO4 in an amount of from 0.1 to 0.15 % by weight, KH2PO4 in an amount of from 0.05 to 0.10 % by weight, and MgSO4 in an amount of from 0.4 to 0.5 % by weight. Also provided herein is a personal care product and methods of making the same.

Description

[0001] Attorney Docket No. 11214-012WO1 METHODS OF MAKING CAROTENOIDS AND PRODUCTS RELATING THERETO CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U. S. Provisional Application No.

[0002] 63 / 744,542 filed January 13, 2025, which is hereby incorporated herein by reference in its entirety.

[0003] STATEMENT OF GOVERNMENT SUPPORT

[0004] This invention was made with government support under Grant Nos. 7005999 awarded by the United States Department of Agriculture. The government has certain rights in the invention.

[0005] BACKGROUND

[0006] P-carotene is one of the 1200+ carotenoids found in diverse life forms like plants, algae, fungi, and bacteria. Carotenoids prevent the oxidation of milk, serve as provitamin- A, reduce stress in cells, and are used as food and feed colorants. Animals cannot produce carotenoids but can assimilate carotenoids for improved health and well-being. The global carotenoid market in 2022 was estimated at $2 billion. Carotenoids work by effectively “quenching” reactive oxygen species and offer protective health benefits in the form of antioxidants. Although synthetic carotenoids are widely available and inexpensive, the production of these synthetic carotenoids depends on unsustainable petrochemical processes. Natural carotenoids are also preferred over synthetic carotenoids due to increased chemophobia and conscious health practices among consumers worldwide. Therefore, there is a need for production methods of carotenoids, specifically P-carotene.

[0007] The methods disclosed herein address these and other needs.

[0008] SUMMARY

[0009] In accordance with the purposes of the disclosed materials and methods, as embodied and broadly described herein, the disclosed subject matter, in one aspect, relates to carotenoids and methods of making thereof.

[0010] Thus, in one example, a method of making P-carotene is provided comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and either of a sugar beet molasses or a sorghum syrup to thereby produce the P-carotene; wherein the mineral salt comprises (NH₄)₂SO₄h in an amount of from 0.1 to 0.15 % by weight, KH2PO4 in an amount of from 0.05 to 0.10 % by weight, and MgSCh in an amount of from 0.4 to 0.5 % by weight.

[0011] In a further example, method of making a personal care product is provided comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and either of a sugar beet molassesAttorney Docket No. 11214-012WO1 or a sorghum syrup to thereby produce a yeast cell comprising P-carotene; wherein the mineral salt comprises (NEU^SC in an amount of from 0.1 to 0.15 % by weight, KH2PO4 in an amount of from 0.05 to 0.10 % by weight, and MgSO4 in an amount of from 0.4 to 0.5 % by weight; collecting the yeast cell from the mixture after fermenting; and combining the yeast cell with a personal care product ingredient.

[0012] Additionally, a personal care product is provided, comprising the yeast cell comprising p-carotene disclosed herein, wherein the personal care product comprises sunscreen, lip balm, lotion, or shampoo.

[0013] Additional advantages will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the aspects described below. The advantages described below will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0016] Figure 1 shows a benchtop bioreactor production of beta-carotene from R. glutinis and final freeze-dried yeast cells.

[0017] Figure 2 shows an RSM plot outlining the optimal points for the media components for P-carotene production in R. glutinis (9.18% sorghum syrup, 0.13% (NEU SC, 0.07% KH2PO4, 0.42% MgSCU and 0.96% yeast extract). StatEase-generated RSM interaction plot depicting the interactions / relationship the media components have on cell biomass and P-carotene concentration

[0018] Figure 3A-Figure 3B shows a contour plot illustrating the effects of media components sorghum syrup and yeast extract on (Figure 3A) P-carotene concentration and (Figure 3B) the cell biomass or cell weight of R. glutinis.

[0019] Figure 4 shows a validation experiment using the optimal points obtained from RSM. Samples from two replicates per time point were pooled before analysis. Statistical analyses are not provided since the main objective of the validation study was to quantitatively and qualitatively estimate the highest yield of P-carotene over the 10 days in a larger volume from RSM.

[0020] Figure 5 shows a fermentation of R. glutinis in batch- fed benchtop bioreactors.

[0021] Experiments were done in triplicate. Columns represent the average P-carotene production withAttorney Docket No. 11214-012WO1 standard deviation error bars. 1 = day 5, 2 = day 7, and 3 = day 10 of the fermentation time.

[0022] Figure 6 shows the materials and methods used to investigate P-carotene production in R. mucilaginosa in sugar beet molasses substrate.

[0023] Figure 7A - Figure 7C show an RSM-optimized experiment with predicted concentrations that were 8.746 pg / g and 114pg / g of P-carotene and total carotenoids, respectively (Fig. 7A). Three-dimensional contour plots illustrating the interaction of the media components on the concentrations of P-carotene and total carotenoids in R. mucilaginosa (Figs.

[0024] 7B-7C).

[0025] Figure 8 shows a bar graph showing the actual concentration of P- carotene (16.722 pg / g) and total carotenoids (137.249 pg / g). The data on the graph outlines the average yields from three validated samples of the optimized media. Error bars represent the standard error for each validated sample.

[0026] Figure 9 shows a schematic of a method involving feedstock / carbon source acquisition and culture, process optimization and fermentation, quantification of pigment via HPLC, and pigment applications.

[0027] Figure 10 shows a schematic of the materials and methods for fermenting and quantifying yeast.

[0028] Figure HA - Figure HE show contour graphs showing the relationship between sorghum syrup and yeast extract on the production of P-carotene and cell biomass, respectively (Figs. 11 A-l IB). Optimal media composition from RSM to produce beta-carotene in R. glutinis (Fig.

[0029] 11C). Interaction of various media components for beta-carotene and cell biomass of R. glutinis (Fig 1 ID). Bar graph showing the yield of P-carotene after validation experiment (Fig. 1 IE).

[0030] Figure 12 shows a bar graph showing the average yields of P-carotene production in our scale-up experiments. The data on the graph outlines the average yield from three bioreactors at 5 L of the optimized media per bioreactor. 1 = day 5 yields, 2 = day 7 yields, and 3 = day 10 yields.

[0031] Figure 13 A - Figure 13B show cells before and after autoclaving at 121 °C for 30 minutes. Fig. 13A shows cells after autoclaving. Fig. 13B shows control samples that are not autoclaved.

[0032] Figure 14A - Figure 14C show cells after autoclaving at 121 °C for 30 minutes.

[0033] Figure 15 A - Figure 15B show control sample with cells that are not autoclaved.

[0034] Figure 16a- Figure 16c. Contour plots illustrating the effects of media components sorghum syrup and yeast extract on: (Figure 16a) desirability of the interaction (Figure 16b) Cell dry wt. of R. glutinis and (Figure 16c) P-carotene concentration.Attorney Docket No. 11214-012WO1 Figure 17a-Figure 17b. (Figure 17a) RSM plot outlining the optimal points for the media components for |3-carotene production in R. glutinis (9.18% sorghum syrup, 0.13% (NHzfhSCh, 0.07% KH2PO4, 0.42% MgSO4and 0.96% yeast extract). (Figure 17b) StatEase-generated RSM interaction plot depicting

[0035] Figure 18. Validation experiment in media bottles and benchtop bioreactors using the optimal points obtained from RSM. Samples from two replicates per time point were pooled before analysis (x axis-days, y axis-yield pg / g).

[0036] Figure 19. Beta-carotene chromatogram extracted at 455 mm.

[0037] Figure 20. Benchtop bioreactor production of beta-carotene from R. glutinis 32766 (top left to right and bottom left). Final freeze-dried yeast cells from bioreactors (bottom right).

[0038] Figure 21. Predicted and actual values of the optimization beta carotene and dry cell wt. Figure 22. Fermentation profile of three benchtop bioreactors in batch mode for variables like pH, DO, stirring and temperature for fermenter 1.

[0039] Figure 23. Fermentation profile of three benchtop bioreactors in batch mode for variables like pH, DO, stirring and temperature for fermenter 2.

[0040] Figure 24. Fermentation profile of three benchtop bioreactors in batch mode for variables like pH, DO, stirring and temperature for fermenter 3.

[0041] Figure 25. Using sorghum syrup as a low-cost substrate, we optimized P-carotene production by Rhodotorula glutinis via response surface methodology and validated at 7 L scale (up to 1,753 pg / g), while profiling the nutrient-dense biomass (protein, minerals, glucosamine) for food / feed applications.

[0042] Figure 26. Preparation of skin cream incorporating carotenogenic yeast into the process.

[0043] DETAILED DESCRIPTION

[0044] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiments. Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.Attorney Docket No. 11214-012WO1 Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0045] As can be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.

[0046] Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.

[0047] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.

[0048] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It can be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.

[0049] Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.Attorney Docket No. 11214-012WO1 Definitions

[0050] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, nonlimiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of and “consisting of.” Similarly, the term “consisting essentially of’ is intended to include examples encompassed by the term “consisting of.”

[0051] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition", or “a disorder”, includes, but is not limited to, two or more such compounds, compositions, or disorders, and the like.

[0052] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other- endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particularvalue. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0053] When a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y', and Tess than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greaterAttorney Docket No. 11214-012WO1 than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0054] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0055] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “substantially free,” when used in the context of a composition or component of a composition that is substantially absent, is intended to refer to an amount that is then about 1 % by weight or less, e.g., less than about 0.5 % by weight, less than about 0.1 % by weight, less than about 0.05 % by weight, or less than about 0.01 % by weight of the stated material, based on the total weight of the composition.

[0056] Method

[0057] Method of Making fi-carotene

[0058] The present disclosure, in one aspect, provides for a method of making P-carotene, comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and cither of a sugarAttorney Docket No. 11214-012WO1 beet molasses or a sorghum syrup to thereby produce the -carotene; wherein the mineral salt comprises (NEL^SC in an amount of from 0.1 to 0.15 % by weight, KH2PO4 in an amount of from 0.05 to 0.10 % by weight, and MgSO₄ in an amount of from 0.4 to 0.5 % by weight In some examples, the mineral salt comprises (NELi SCh in an amount of from 0.10% to 0.11%, 0.11% to 0.12%, 0.12% to 0.13%, 0.13% to 0.14%, or 0.14% to 0.15% by weight. In further examples, the mineral salt comprises (NEUhSC in an amount of from 0.10% to 0.105%, 0.105% to 0.11%, 0.11% to 0.115%, 0.115% to 0.12%, 0.12% to 0.125%, 0.125% to 0.13%, 0.13% to 0.135%, 0.135% to 0.14%, 0.14% to 0.145%, or 0.145% to 0.15% by weight. In some examples, the mineral salt comprises (NEU SCU in an amount of from 0.10% to 0.12%, 0.10% to 0.13%, or 0.10% to 0.14% by weight. In further examples, the mineral salt comprises (NEU SCh in an amount of from 0.10% to 0.115%, 0.10% to 0.125%, 0.10% to 0.135%, or 0.10% to 0.145%, by weight.

[0059] In further examples, the mineral salt comprises KH2PO4 in an amount of from 0.05% to 0.055%, 0.055% to 0.060%, 0.060% to 0.065%. 0.065% to 0.070%, 0.070% to 0.075%, 0.075% to 0.080%, 0.080% to 0.085%, 0.085% to 0.090%, 0.090% to 0.095%, or 0.095% to 0.10% by weight. In some examples, the mineral salt comprises KH2PO4 in an amount of from 0.05% to 0.06%, 0.06% to 0.07%, 0.07% to 0.08%, 0.08% to 0.09%, or 0.09% to 0.10% by weight. In further examples, the mineral salt comprises KH2PO4 in an amount of from 0.05% to 0.060%, 0.05% to 0.065%, 0.05% to 0.070%, 0.05% to 0.075%, 0.05% to 0.080%, 0.05% to 0.085%, 0.05% to 0.090%, or 0.05% to 0.095%, by weight.

[0060] In certain examples, the mineral salt comprises MgSO₄ in an amount of from 0.40% to 0.41%, 0.41% to 0.42%, 0.42% to 0.43%, 0.43% to 0.44%, 0.44% to 0.45%, 0.45% to 0.46%, 0.46% to 0.47%, 0.47% to 0.48%, 0.48% to 0.49%, or 0.49% to 0.50% by weight. In certain examples, the mineral salt comprises MgSCU in an amount of from 0.40% to 0.42%, 0.42% to 0.44%, 0.44% to 0.46%, 0.46% to 0.48%, or 0.48% to 0.50% by weight. In certain examples, the mineral salt comprises MgSO₄ in an amount of from 0.40% to 0.42%, 0.40% to 0.43%, 0.40% to 0.44%, 0.40% to 0.45%, 0.40% to 0.46%, 0.40% to 0.47%, 0.40% to 0.48%, or 0.40% to 0.49% by weight.

[0061] In some examples, the (NEU SC is present in an amount of from 0.12 to 0.14 %, the KH2PO4 is present in an amount of from 0.06 to 0.08 % by weight, and the MgSC is present in an amount of from 0.41 to 0.43% by weight.

[0062] In some examples, the mineral salt comprises (NELO2SO4 in an amount of from 0.120% to 0.122%, 0.122% to 0.124%, 0.124% to 0.126%, 0.126% to 0.128%, 0.128% to 0.130%, 0.130% to 0.132%, 0.132% to 0.134%, 0.134% to 0.136%, or 0.136% to 0.140% by weight. InAttorney Docket No. 11214-012WO1 further examples, the mineral salt comprises (NI hhSCh in an amount of from 0.120% to 0.122%, 0.120% to 0.124%, 0.120% to 0.126%, 0.120% to 0.128%, 0.120% to 0.130%, 0.120% to 0.132%, 0.120% to 0.134%, 0.120% to 0.136%, 0.120% to 0.138%, or 0.120% to 0.140% by weight. In some examples, the mineral salt comprises (NHr SCri in an amount of from 0.120% to 0.125%, 0.125% to 0.130%, 0.130% to 0.135%, and 0.135% to 0.140% by weight. In further examples, the mineral salt comprises (NFU SC in an amount of from 0.120% to 0.125%, 0.120% to 0.130%, 0.120% to 0.135%, and 0.120% to 0.140% by weight.

[0063] In some examples, the mineral salt comprises KH2PO4 in an amount from 0.060% to 0.062%, 0.062% to 0.064%, 0.064% to 0.066%, 0.066% to 0.068%, 0.068% to 0.070%, 0.070% to 0.072%, 0.072% to 0.074%, 0.074% to 0.076%, 0.076% to 0.078%, or 0.078% to 0.080% by weight. In some examples, the mineral salt comprises KH2PO4 in an amount from 0.060% to 0.062%, 0.060% to 0.064%, 0.060% to 0.066%, 0.060% to 0.068%, 0.060% to 0.070%, 0.060% to 0.072%, 0.060% to 0.074%, 0.060% to 0.076%, 0.060% to 0.078%, or 0.060% to 0.080% by weight. In some examples, the mineral salt comprises KH2PO4 in an amount from 0.060% to 0.065%, 0.060% to 0.070%, 0.060% to 0.075%, 0.060% to 0.080% by weight. In some examples, the mineral salt comprises KH2PO4 in an amount from 0.060% to 0.065%, 0.065% to 0.070%, 0.070% to 0.075%, 0.075% to 0.080% by weight.

[0064] In some examples, the mineral salt comprises MgSCU in an amount from 0.410% to 0.411%, 0.411% to 0.412%, 0.412% to 0.413%, 0.413% to 0.414%, 0.414% to 0.415%, 0.415% to 0.416%, 0.416% to 0.417%, 0.417% to 0.418%, 0.418% to 0.419%, 0.419% to 0.420%, 0.420% to 0.421%, 0.421% to 0.422%, 0.422% to 0.423%, 0.423% to 0.424%, 0.424% to 0.425%, 0.425% to 0.426%, 0.426% to 0.427%, 0.427% to 0.428%, 0.428% to 0.429%, 0.429% to 0.430% by weight. In some examples, the mineral salt comprises MgSCh in an amount from 0.410% to 0.415%, 0.415% to 0.420%, 0.420% to 0.425%, 0.425% to 0.430% by weight. In some examples, the mineral salt comprises MgSCh in an amount from 0.410% to 0.415%, 0.410% to 0.420%, 0.410% to 0.425%, and 0.410% to 0.430% by weight. In some examples, the mineral salt comprises MgSCh in an amount from 0.410% to 0.411%, 0.410% to 0.412%, 0.410% to 0.413%, 0.410% to 0.414%, 0.410% to 0.415%, 0.410% to 0.416%, 0.410% to 0.417%, 0.410% to 0.418%, 0.410% to 0.419%, 0.410% to 0.420%, 0.410% to 0.421%, 0.410% to 0.422%, 0.410% to 0.423%, 0.410% to 0.424%, 0.410% to 0.425%, 0.410% to 0.426%, 0.410% to 0.427%, 0.410% to 0.428%, and 0.410% to 0.430% by weight.

[0065] In some examples, the Rhodotorula yeast is Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula rubra, or any combination thereof.

[0066] In further examples, the Rhodotorula yeast is Rhodotorula mucilaginosa.Attorney Docket No. 11214-012WO1 In certain examples, the Rhodotorula mucilaginosa is deposited under ATCC 32763 (Rhodotorula mucilaginosa (Jorgensen) Harrison (ATCC 32763)). Incorporated by reference herein is the Rhodotorula mucilaginosa (Jorgenson) Harrison ATCC 32763 Product Sheet.

[0067] In specific examples, the Rhodotorula yeast is Rhodotorula glutinis.

[0068] In some examples, the Rhodotorula glutinis is deposited under ATCC 32766 (32766 (ATCC 32766)). Incorporated herein is the ATCC 32766 Product Sheet.

[0069] In further examples, fermenting the mixture is performed in a bioreactor.

[0070] In certain examples, the Rhodotorula yeast is present in an amount of from 0.5 to 1.5 % by weight. In certain examples, the Rhodotorula yeast is present in an amount of from 0.5% to 0.55%, 0.55% to 0.60%, 0.60% to 0.65%, 0.65% to 0.70%, 0.70% to 0.75%, 0.75% to 0.80%, 0.80% to 0.85%, 0.85% to 0.90%, 0.90% to 0.95%, 0.95% to 1.00%, 1.00% to 1.05%, 1.05% to 1.10%, 1.10% to 1.15%, 1.15% to 1.20%, 1.20% to 1.25%, 1.25% to 1.30%, 1.30% to 1.35%, 1.35% to 1.40%, 1.40% to 1.45%, or 1.45% to 1.50% by weight. In certain examples, the Rhodotorula yeast is present in an amount of from 0.5% to 0.55%, 0.5% to 0.60%, 0.5% to 0.65%, 0.5% to 0.70%, 0.5% to 0.75%, 0.5% to 0.80%, 0.5% to 0.85%, 0.5% to 0.90%, 0.5% to 0.95%, 0.5% to 1.00%, 0.5% to 1.05%, 0.5% to 1.10%, 0.5% to 1.15%, 0.5% to 1.20%, 0.5% to 1.25%, 0.5% to 1.30%, 0.5% to 1.35%, 0.5% to 1.40%, 0.5% to 1.45%, or 0.5% to 1.50% by weight. In certain examples, the Rhodotorula yeast is present in an amount of from 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1.0%, 1.0% to 1.1%, 1.1% to 1.2%, I.2% to 1.3%, 1.3% to 1.4%, or 1.4% to 1.5% by weight. " In certain examples, the Rhodotorula yeast is present in an amount of from 0.5% to 0.75%, 0.75% to 1.0%, 1.0% to 1.25%, or 1.25% to 1.5% by weight.

[0071] In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5 to 15 %. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 5.25%, 5.25% to 5.5%, 5.5% to 5.75%, 5.75% to 6%, 6% to 6.25%, 6.25% to 6.5%, 6.5% to 6.75%. 6.75% to 7%, 7% to 7.25%, 7.25% to 7.5%, 7.5% to 7.75%, 7.75% to 8%, 8% to 8.25%, 8.25% to 8.5%, 8.5% to 8.75%, 8.75% to 9%, 9% to 9.25%, 9.25% to 9.5%, 9.5% to 9.75%, 9.75% to 10%, 10% to 10.25%, 10.25% to 10.5%, 10.5% to 10.75%, 10.75% to 11%, 11% to 11.25%, 11.25% to 11.5%, 11.5% to 11.75%, I I.75% to 12%, 12% to 12.25%, 12.25% to 12.5%, 12.5% to 12.75%, 12.75% to 13%, 13% to 13.25%, 13.25% to 13.5%. 13.5% to 13.75%, 13.75% to 14%, 14% to 14.25%, 14.25% to 14.5%, or 14.5% to 15% by weight. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 5.25%, 5% to 5.5%, 5% to 5.75%, 5% to 6%, 5% to 6.25%, 5% to 6.5%, 5% to 6.75%, 5% to 7%, 5% to 7.25%, 5% to 7.5%, 5% toAttorney Docket No. 11214-012WO1 7.75%. 5% to 8%, 5% to 8.25%. 5% to 8.5%, 5% to 8.75%. 5% to 9%, 5% to 9.25%. 5% to 9.5%, 5% to 9.75%, 5% to 10%, 5% to 10.25%, 5% to 10.5%, 5% to 10.75%, 5% to 11%, 5% to 11.25%, 5% to 11.5%, 5% to 11.75%, 5% to 12%, 5% to 12.25%, 5% to 12.5%, 5% to 12.75%, 5% to 13%, 5% to 13.25%, 5% to 13.5%, 5% to 13.75%, 5% to 14%, 5% to 14.25%, 5% to 14.5%, or 5% to 15% by weight. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, or 14% to 15% by weight. " In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 7%, 7% to 9%, 9% to 11%, 11% to 13%, or 13% to 15% by weight. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 10%, 10% to 15% by weight.

[0072] In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 to 20 pg / g. In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 pg / g to 6 pg / g, 6 pg / g to 7 pg / g, 7 pg / g to 8 pg / g, 8 pg / g to 9 pg / g, 9 pg / g to 10 pg / g, 10 pg / g to 11 pg / g, 11 pg / g to 12 pg / g, 12 pg / g to 13 pg / g, 13 pg / g to 14 pg / g, 14 pg / g to 15 pg / g, 15 pg / g to 16 pg / g, 16 pg / g to 17 pg / g, 17 pg / g to 18 pg / g, 18 pg / g to 19 pg / g, or 19 pg / g to 20 pg / g. In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 pg / g to 8 pg / g, 8 pg / g to 11 pg / g, 11 pg / g to 14 pg / g, 14 pg / g to 17 pg / g, or 17 pg / g to 20 pg / g. In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 pg / g to 10 pg / g, 10 pg / g to 15 pg / g, or 15 pg / g to 20 pg / g. In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 pg / g to 6 pg / g, 5 pg / g to 7 pg / g, 5 pg / g to 8 pg / g, 5 pg / g to 9 pg / g, 5 pg / g to 10 pg / g, 5 pg / g to 11 pg / g, 5 pg / g to 12 pg / g, 5 pg / g to 13 pg / g, 5 pg / g to 14 pg / g, 5 pg / g to 15 pg / g, 5 pg / g to 16 pg / g, 5 pg / g to 17 pg / g, 5 pg / g to 18 pg / g, 5 pg / g to 19 pg / g, or 5 pg / g to 20 pg / g.

[0073] In certain examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 to 1400 pg / g. In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 250 pg / g, 250 pg / g to 300 pg / g, 300 pg / g to 350 pg / g, 350 pg / g to 400 pg / g, 400 pg / g to 450 pg / g, 450 pg / g to 500 pg / g, 500 pg / g to 550 pg / g, 550 pg / g to 600 pg / g, 600 pg / g to 650 pg / g, 650 pg / g to 700 pg / g, 700 pg / g to 750 pg / g, 750 pg / g to 800 pg / g, 800 pg / g to 850 pg / g, 850 pg / g to 900 pg / g, 900 pg / g to 950 pg / g, 950 pg / g to 1000 pg / g, 1000 pg / g to 1050 pg / g, 1050 pg / g to 1100 pg / g, 1100 pg / g to 1150 pg / g, 1150 pg / g to 1200 pg / g, 1200 pg / g to 1250 pg / g, 1250 pg / g to 1300 pg / g, 1300 pg / g to 1350 pg / g, or 1350 pg / g to 1400 pg / g. In further examples, when the yeast is RhodotorulaAttorney Docket No. 11214-012WO1 glutinis, the P-carotene is present in an amount of from 200 pg / g to 250 pg / g, 200 pg / g to 300 pg / g, 200 pg / g to 350 pg / g, 200 pg / g to 400 pg / g, 200 pg / g to 450 pg / g, 200 pg / g to 500 pg / g, 200 pg / g to 550 pg / g, 200 pg / g to 600 pg / g, 200 pg / g to 650 pg / g, 200 pg / g to 700 pg / g, 200 pg / g to 750 pg / g, 200 pg / g to 800 pg / g, 200 pg / g to 850 pg / g, 200 pg / g to 900 pg / g, 200 pg / g to 950 pg / g, 200 pg / g to 1000 pg / g, 200 pg / g to 1050 pg / g, 200 pg / g to 1100 pg / g, 200 pg / g to 1150 pg / g, 200 pg / g to 1200 pg / g, 200 pg / g to 1250 pg / g, 200 pg / g to 1300 pg / g, 200 pg / g to 1350 pg / g, or 200 pg / g to 1400 pg / g.

[0074] In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 400 pg / g, 400 pg / g to 600 pg / g, 600 pg / g to 800 pg / g, 800 pg / g to 1000 pg / g, 1000 pg / g to 1200 pg / g, or 1200 pg / g to 1400 pg / g. In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 500 pg / g, 500 pg / g to 800 pg / g, 800 pg / g to 1100 pg / g, or 1100 pg / g to 1400 pg / g. In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 600 pg / g, 600 pg / g to 1000 pg / g, or 1000 pg / g to 1400 pg / g.

[0075] In specific examples, when the Rhodotorula yeast is combined with the sugar beet molasses, the Rhodotorula yeast is Rhodotorula mucilaginosa.

[0076] In some examples, when the Rhodotorula yeast is combined with the sorghum syrup, the Rhodotorula yeast is Rhodotorula glutinis.

[0077] Method of Making a Personal Care Product

[0078] Also disclosed herein is a method of making a personal care product comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and either of a sugar beet molasses or a sorghum syrup to thereby produce a yeast cell comprising P-carotene; wherein the mineral salt comprises (NH4)2SO4 in an amount of from 0.1 to 0.15 % by weight, KH2PO4 in an amount of from 0.05 to 0.10 % by weight, and MgSC>4 in an amount of from 0.4 to 0.5 % by weight; collecting the yeast cell from the mixture after fermenting; and combining the yeast cell with a personal care product ingredient.

[0079] In some examples, the yeast cell comprises carotenes and xanthophylls including P-carotene, tomline, torularhodin, lutein and other carotenes. In further examples, the yeast cell is not lysed. In certain examples, the P-carotene is not isolated from the mixture. In specific examples, the mixture after fermenting comprises intact yeast cells, yeast cell components, or a combination thereof. In some examples, the mixture after femrenting comprises a major portion of intact yeast cells. The yeast cells act as micro-encapsulated agents in personal care products.

[0080] In some examples, the mineral salt comprises (NH₄)₂SO₄h in an amount of from 0.10% to 0.11%, 0.11% to 0.12%, 0.12% to 0.13%, 0.13% to 0.14%, or 0.14% to 0.15% by weight. InAttorney Docket No. 11214-012WO1 further examples, the mineral salt comprises (NPUhSCh in an amount of from 0.10% to 0.105%, 0.105% to 0.11%, 0.11% to 0.115%, 0.115% to 0.12%, 0.12% to 0.125%, 0.125% to 0.13%, 0.13% to 0.135%, 0.135% to 0.14%, 0.14% to 0.145%, or 0.145% to 0.15% by weight. In some examples, the mineral salt comprises (NPU SCU in an amount of from 0.10% to 0.12%, 0.10% to 0.13%, or 0.10% to 0.14% by weight. In further examples, the mineral salt comprises (NIDhSCU in an amount of from 0.10% to 0.115%, 0.10% to 0.125%, 0.10% to 0.135%, or 0.10% to 0.145%, by weight.

[0081] In further examples, the mineral salt comprises KII2PO4 in an amount of from 0.05% to 0.055%, 0.055% to 0.060%, 0.060% to 0.065%, 0.065% to 0.070%, 0.070% to 0.075%, 0.075% to 0.080%, 0.080% to 0.085%, 0.085% to 0.090%, 0.090% to 0.095%, or 0.095% to 0.10% by weight. In some examples, the mineral salt comprises KH2PO4 in an amount of from 0.05% to 0.06%, 0.06% to 0.07%, 0.07% to 0.08%, 0.08% to 0.09%, or 0.09% to 0.10% by weight. In further examples, the mineral salt comprises KH2PO4 in an amount of from 0.05% to 0.060%, 0.05% to 0.065%, 0.05% to 0.070%, 0.05% to 0.075%, 0.05% to 0.080%, 0.05% to 0.085%, 0.05% to 0.090%, or 0.05% to 0.095%, by weight.

[0082] In certain examples, the mineral salt comprises MgSO₄ in an amount of from 0.40% to 0.41%, 0.41% to 0.42%, 0.42% to 0.43%, 0.43% to 0.44%, 0.44% to 0.45%, 0.45% to 0.46%, 0.46% to 0.47%, 0.47% to 0.48%, 0.48% to 0.49%, or 0.49% to 0.50% by weight. In certain examples, the mineral salt comprises MgSCU in an amount of from 0.40% to 0.42%, 0.42% to 0.44%, 0.44% to 0.46%, 0.46% to 0.48%, or 0.48% to 0.50% by weight. In certain examples, the mineral salt comprises MgSO₄ in an amount of from 0.40% to 0.42%, 0.40% to 0.43%, 0.40% to 0.44%, 0.40% to 0.45%, 0.40% to 0.46%, 0.40% to 0.47%, 0.40% to 0.48%, or 0.40% to 0.49% by weight.

[0083] In some examples, the (NFU SC is present in an amount of from 0.12 to 0.14 %, the KH2PO4 is present in an amount of from 0.06 to 0.08 % by weight, and the MgSC is present in an amount of from 0.41 to 0.43% by weight.

[0084] In some examples, the mineral salt comprises (NH₄)₂SO₄h in an amount of from 0.120% to 0.122%, 0.122% to 0.124%, 0.124% to 0.126%, 0.126% to 0.128%, 0.128% to 0.130%, 0.130% to 0.132%, 0.132% to 0.134%, 0.134% to 0.136%, or 0.136% to 0.140% by weight. In further examples, the mineral salt comprises (NFU^SC in an amount of from 0.120% to 0.122%, 0.120% to 0.124%, 0.120% to 0.126%, 0.120% to 0.128%, 0.120% to 0.130%, 0.120% to 0.132%, 0.120% to 0.134%, 0.120% to 0.136%, 0.120% to 0.138%, or 0.120% to 0.140% by weight. In some examples, the mineral salt comprises (NPUhSCh in an amount of from 0.120% to 0.125%, 0.125% to 0.130%, 0.130% to 0.135%, and 0.135% to 0.140% by weight. In furtherAttorney Docket No. 11214-012WO1 examples, the mineral salt comprises (NtU SC in an amount of from 0.120% to 0.125%, 0.120% to 0.130%, 0.120% to 0.135%, and 0.120% to 0.140% by weight.

[0085] n some examples, the mineral salt comprises KH2PO4 in an amount from 0.060% to 0.062%, 0.062% to 0.064%, 0.064% to 0.066%, 0.066% to 0.068%, 0.068% to 0.070%, 0.070% to 0.072%, 0.072% to 0.074%, 0.074% to 0.076%, 0.076% to 0.078%, or 0.078% to 0.080% by weight. In some examples, the mineral salt comprises KH2PO4 in an amount from 0.060% to 0.062%, 0.060% to 0.064%, 0.060% to 0.066%, 0.060% to 0.068%, 0.060% to 0.070%, 0.060% to 0.072%, 0.060% to 0.074%, 0.060% to 0.076%, 0.060% to 0.078%, or 0.060% to 0.080% by weight. In some examples, the mineral salt comprises KH2PO4 in an amount from 0.060% to 0.065%, 0.060% to 0.070%, 0.060% to 0.075%, 0.060% to 0.080% by weight. In some examples, the mineral salt comprises KH2PO4 in an amount from 0.060% to 0.065%, 0.065% to 0.070%, 0.070% to 0.075%, 0.075% to 0.080% by weight.

[0086] In some examples, the mineral salt comprises MgSCU in an amount from 0.410% to 0.411%, 0.411% to 0.412%, 0.412% to 0.413%. 0.413% to 0.414%, 0.414% to 0.415%, 0.415% to 0.416%, 0.416% to 0.417%, 0.417% to 0.418%, 0.418% to 0.419%, 0.419% to 0.420%, 0.420% to 0.421%, 0.421% to 0.422%, 0.422% to 0.423%, 0.423% to 0.424%, 0.424% to 0.425%, 0.425% to 0.426%, 0.426% to 0.427%, 0.427% to 0.428%, 0.428% to 0.429%, 0.429% to 0.430% by weight. In some examples, the mineral salt comprises MgSCh in an amount from 0.410% to 0.415%, 0.415% to 0.420%, 0.420% to 0.425%, 0.425% to 0.430% by weight. In some examples, the mineral salt comprises MgSOi in an amount from 0.410% to 0.415%, 0.410% to 0.420%, 0.410% to 0.425%, and 0.410% to 0.430% by weight. In some examples, the mineral salt comprises MgSCh in an amount from 0.410% to 0.411%, 0.410% to 0.412%, 0.410% to 0.413%, 0.410% to 0.414%, 0.410% to 0.415%, 0.410% to 0.416%, 0.410% to 0.417%, 0.410% to 0.418%, 0.410% to 0.419%, 0.410% to 0.420%, 0.410% to 0.421%, 0.410% to 0.422%, 0.410% to 0.423%, 0.410% to 0.424%, 0.410% to 0.425%, 0.410% to 0.426%, 0.410% to 0.427%, 0.410% to 0.428%, and 0.410% to 0.430% by weight.

[0087] In some examples, the method further comprises deactivating the Rhodotorula yeast after fermenting the mixture.

[0088] In certain examples, the Rhodotorula yeast is Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula rubra, or any combination thereof.

[0089] In further examples, the Rhodotorula yeast is Rhodotorula mucilaginosa.

[0090] In certain examples, the Rhodotorula mucilaginosa is deposited under ATCC Accession No. 32763.

[0091] In specific examples, the Rhodotorula yeast is Rhodotorula glutinis.Attorney Docket No. 11214-012WO1 In some examples, the Rhodotorula glutinis is deposited under ATCC Accession No. 32766.

[0092] In further examples, fermenting the mixture is performed in a bioreactor.

[0093] In certain examples, the Rhodotorula yeast is present in an amount of from 0.5 to 1.5 % by weight. In certain examples, the Rhodotorula yeast is present in an amount of from 0.5% to 0.55%, 0.55% to 0.60%, 0.60% to 0.65%, 0.65% to 0.70%, 0.70% to 0.75%, 0.75% to 0.80%, 0.80% to 0.85%, 0.85% to 0.90%, 0.90% to 0.95%, 0.95% to 1.00%, 1.00% to 1.05%, 1.05% to 1.10%, 1.10% to 1.15%, 1.15% to 1.20%, 1.20% to 1.25%, 1.25% to 1.30%, 1.30% to 1.35%, 1.35% to 1.40%, 1.40% to 1.45%, or 1.45% to 1.50% by weight. In certain examples, the Rhodotorula yeast is present in an amount of from 0.5% to 0.55%, 0.5% to 0.60%, 0.5% to 0.65%, 0.5% to 0.70%, 0.5% to 0.75%, 0.5% to 0.80%, 0.5% to 0.85%, 0.5% to 0.90%, 0.5% to 0.95%, 0.5% to 1.00%, 0.5% to 1.05%, 0.5% to 1.10%, 0.5% to 1.15%, 0.5% to 1.20%, 0.5% to 1.25%, 0.5% to 1.30%, 0.5% to 1.35%, 0.5% to 1.40%, 0.5% to 1.45%, or 0.5% to 1.50% by weight. In certain examples, the Rhodotorula yeast is present in an amount of from 0.5% to 0.6%, 0.6% to 0.7%, 0.7% to 0.8%, 0.8% to 0.9%, 0.9% to 1.0%, 1.0% to 1.1%, 1.1% to 1.2%, I.2% to 1.3%, 1.3% to 1.4%, or 1.4% to 1.5% by weight. " In certain examples, the Rhodotorula yeast is present in an amount of from 0.5% to 0.75%, 0.75% to 1.0%, 1.0% to 1.25%, or 1.25% to 1.5% by weight.

[0094] In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5 to 15 %. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 5.25%, 5.25% to 5.5%, 5.5% to 5.75%, 5.75% to 6%, 6% to 6.25%, 6.25% to 6.5%, 6.5% to 6.75%, 6.75% to 7%, 7% to 7.25%, 7.25% to 7.5%, 7.5% to 7.75%, 7.75% to 8%, 8% to 8.25%, 8.25% to 8.5%, 8.5% to 8.75%, 8.75% to 9%, 9% to 9.25%, 9.25% to 9.5%, 9.5% to 9.75%, 9.75% to 10%, 10% to 10.25%, 10.25% to 10.5%, 10.5% to 10.75%, 10.75% to 11%, 11% to 11.25%, 11.25% to 11.5%, 11.5% to 11.75%, II.75% to 12%, 12% to 12.25%, 12.25% to 12.5%, 12.5% to 12.75%, 12.75% to 13%, 13% to 13.25%, 13.25% to 13.5%, 13.5% to 13.75%, 13.75% to 14%, 14% to 14.25%, 14.25% to 14.5%, or 14.5% to 15% by weight. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 5.25%, 5% to 5.5%, 5% to 5.75%, 5% to 6%, 5% to 6.25%, 5% to 6.5%, 5% to 6.75%, 5% to 7%, 5% to 7.25%, 5% to 7.5%, 5% to 7.75%, 5% to 8%, 5% to 8.25%, 5% to 8.5%, 5% to 8.75%, 5% to 9%, 5% to 9.25%, 5% to 9.5%, 5% to 9.75%, 5% to 10%. 5% to 10.25%. 5% to 10.5%, 5% to 10.75%, 5% to 11%, 5% to 11.25%, 5% to 11.5%, 5% to 11.75%, 5% to 12%, 5% to 12.25%, 5% to 12.5%, 5% to 12.75%, 5% to 13%, 5% to 13.25%, 5% to 13.5%, 5% to 13.75%, 5% to 14%, 5% to 14.25%, 5% toAttorney Docket No. 11214-012WO1 14.5%, or 5% to 15% by weight. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, or 14% to 15% by weight. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 7%, 7% to 9%, 9% to 11%, 11% to 13%, or 13% to 15% by weight. In specific examples, either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5% to 10%, 10% to 15% by weight.

[0095] In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 to 20 pg / g. In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 pg / g to 6 pg / g, 6 pg / g to 7 pg / g, 7 pg / g to 8 pg / g, 8 pg / g to 9 pg / g, 9 pg / g to 10 pg / g, 10 pg / g to 11 pg / g, 11 pg / g to 12 pg / g, 12 pg / g to 13 pg / g, 13 pg / g to 14 pg / g, 14 pg / g to 15 pg / g, 15 pg / g to 16 pg / g, 16 pg / g to 17 pg / g, 17 pg / g to 18 pg / g, 18 pg / g to 19 pg / g, or 19 pg / g to 20 pg / g. In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 pg / g to 8 pg / g, 8 pg / g to 11 pg / g, 11 pg / g to 14 pg / g, 14 pg / g to 17 pg / g, or 17 pg / g to 20 pg / g. In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 pg / g to 10 pg / g, 10 pg / g to 15 pg / g, or 15 pg / g to 20 pg / g. In further examples, when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 pg / g to 6 pg / g, 5 pg / g to 7 pg / g, 5 pg / g to 8 pg / g, 5 pg / g to 9 pg / g, 5 pg / g to 10 pg / g, 5 pg / g to 11 pg / g, 5 pg / g to 12 pg / g, 5 pg / g to 13 pg / g, 5 pg / g to 14 pg / g, 5 pg / g to 15 pg / g, 5 pg / g to 16 pg / g, 5 pg / g to 17 pg / g, 5 pg / g to 18 pg / g, 5 pg / g to 19 pg / g, or 5 pg / g to 20 pg / g.

[0096] In certain examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 to 1400 pg / g. In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 250 pg / g, 250 pg / g to 300 pg / g, 300 pg / g to 350 pg / g, 350 pg / g to 400 pg / g, 400 pg / g to 450 pg / g, 450 pg / g to 500 pg / g, 500 pg / g to 550 pg / g, 550 pg / g to 600 pg / g, 600 pg / g to 650 pg / g, 650 pg / g to 700 pg / g, 700 pg / g to 750 pg / g, 750 pg / g to 800 pg / g, 800 pg / g to 850 pg / g, 850 pg / g to 900 pg / g, 900 pg / g to 950 pg / g, 950 pg / g to 1000 pg / g, 1000 pg / g to 1050 pg / g, 1050 pg / g to 1100 pg / g, 1100 pg / g to 1150 pg / g, 1150 pg / g to 1200 pg / g, 1200 pg / g to 1250 pg / g, 1250 pg / g to 1300 pg / g, 1300 pg / g to 1350 pg / g, or 1350 pg / g to 1400 pg / g. In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 250 pg / g, 200 pg / g to 300 pg / g, 200 pg / g to 350 pg / g, 200 pg / g to 400 pg / g, 200 pg / g to 450 pg / g, 200 pg / g to 500 pg / g, 200 pg / g to 550 pg / g, 200 pg / g to 600 pg / g, 200 pg / g to 650 pg / g, 200 pg / g to 700 pg / g, 200 pg / g to 750 pg / g, 200 pg / g to 800 pg / g, 200 pg / g to 850 pg / g, 200 pg / g to 900 pg / g, 200 pg / g toAttorney Docket No. 11214-012WO1 950 pg / g, 200 pg / g to 1000 pg / g, 200 pg / g to 1050 pg / g, 200 pg / g to 1100 pg / g. 200 pg / g to 1150 pg / g, 200 pg / g to 1200 pg / g, 200 pg / g to 1250 pg / g, 200 pg / g to 1300 pg / g, 200 pg / g to 1350 pg / g, or 200 pg / g to 1400 pg / g.

[0097] In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 400 pg / g, 400 pg / g to 600 pg / g, 600 pg / g to 800 pg / g, 800 pg / g to 1000 pg / g, 1000 pg / g to 1200 pg / g, or 1200 pg / g to 1400 pg / g. In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 500 pg / g, 500 pg / g to 800 pg / g, 800 pg / g to 1100 pg / g, or 1100 pg / g to 1400 pg / g. In further examples, when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 pg / g to 600 pg / g, 600 pg / g to 1000 pg / g, or 1000 pg / g to 1400 pg / g.

[0098] In specific examples, when the Rhodotorula yeast is combined with the sugar beet molasses, the Rhodotorula yeast is Rhodotorula mucilaginosa.

[0099] In some examples, when the Rhodotorula yeast is combined with the sorghum syrup, the Rhodotorula yeast is Rhodotorula glutinis.

[0100] Composition

[0101] Personal Care Product

[0102] Provided herein is a personal care product comprising the yeast cell comprising P-carotene disclosed herein, wherein the personal care product comprises sunscreen, lip balm, lotion, or shampoo.

[0103] In some examples, personal care products include, but are not limited to, sunscreen, lip balm, lotion, shampoo, deodorant, shaving products, toothpaste, mask, exfoliator, cleanser, fragrances, eye cream, hair care products, body wash, makeup, hair dye, and hair gel, for example.

[0104] In some examples, the personal care product comprises the yeast cell in a concentration of 10% or less (e.g., 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.5% or less, or0.1% or less). In some examples, the personal care product comprises the yeast cell in a concentration of 5% or less. In some examples, the personal care product comprises the yeast cell in a concentration of 2% or less. In some examples, the personal care product comprises the yeast cell in a concentration of 1% or less.

[0105] In some examples, the personal care product comprises the yeast cell in a concentration of from 0.1 to 10%. In some examples, the personal care product comprises the yeast cell in a concentration of from 0.1 to 5%. In some examples, the personal care product comprises the yeast cell in a concentration of from 0.1 to 2%. In some examples, the personal care product comprises the yeast cell in a concentration of from 0.1 to 1%.Attorney Docket No. 11214-012WO1

[0106] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0107] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0108] EXAMPLES

[0109] The following examples are set forth below to illustrate the methods and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention, which are apparent to one skilled in the art.

[0110] Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric. There are numerous variations and combinations of reaction conditions, e.g., component concentrations, temperatures, pressures, and other reaction ranges and conditions that can be used to optimize the product purity and yield obtained from the described process. Only reasonable and routine experimentation will be required to optimize such process conditions.

[0111] Example 1: Optimization of beta-carotene production and nutritional characterization of Rhodoturola Glutinis 32766 in a sorghum syrup under submerged fermentation

[0112] Introduction. Discussed herein is utilizing an agricultural resource, sorghum syrup, to produce natural P-carotene via Rhodotorula glutinis fermentation. Response surface methodology (RSM) was used for bioproccss optimization of -carotcnc production in shake flasks and scaled up to 300ml in media bottles and then 5L in benchtop bioreactors. The RSM-predicted concentrations of media ingredients were 9.18% sorghum syrup, 0.96% yeast extract, 0.07% KH2PO4, 0.13% (NFL^SCU and 0.42% MgSOi with a predicted P-carotene production of 1,003 μg / g of yeast cells on day 10 of fermentation. HPLC confirmed the P-carotene. The 300ml scale-up resulted in 1, 153pg / g of P-carotene and the 5L scale-up in l,753.33pg / g on day 10, which were 15% and 75% enhanced production respectively compared to the predicted yield. Proximate analysis of the fermentation product indicated the presence of other nutrient molecules in carotenoid production in R. glutinis, i.e., vitamins, and minerals. Results showAttorney Docket No. 11214-012WO1 sorghum syrup is a suitable feedstock for R. glutinis fermentation. The several nutrients present in the fermentation product allow for the wide applicability of the product.

[0113] Carotenoids are lipophilic pigments found widely distributed in plants, animals, and microorganisms. As of today, over 1200 naturally occurring molecules of carotenoids have been isolated and characterized (Carotenoid Database, n.d.). Carotenoids are a major class of diverse pigments responsible for the red, yellow, and orange coloration seen in nature and are one of the most studied pigments (Rodriguez-Amaya, 2019). Diverse life forms including plants, bacteria, algae, and fungi produce carotenoids (Ashokkumar et al., 2023). Examples of these carotenoids include P-carotene, torulene, astaxanthin, and torularhodin (Rapoport et al., 2021). Carotenoids work by effectively “quenching” reactive oxygen species and offer protective health benefits in the form of antioxidants (Fiedor & Burda, 2014). Although synthetic carotenoids are widely available and inexpensive, the production of these synthetic carotenoids depends on unsustainable petrochemical processes (Lourengo-Lopes et al., 2021). Natural carotenoids are also preferred over synthetic carotenoids due to increased chemophobia and conscious health practices among consumers worldwide (Dufosse et al., 2005). Additionally, this increased preference for natural carotenoids allows for the use of inexpensive and abundantly available substrates for microbial growth. Estimated to be valued at $2 billion in 2022 with an annual growth rate of 5.7% (2023 to 2028), the carotenoid market is predicted to be $2.7 billion by 2027. As the most studied and consumed carotenoid, -carotene was valued at $261 million in 2010 and U. S. $334 million in 2018 (Global Carotenoids Market Report and Forecast 2023- 2028, n.d.).

[0114] Rhodotorula species and their unique pathways have generated several valuable bioproducts, including carotenoids. Rhodotorula is an advantageous yeast species for fermentation because they are fast-growing and non-fastidious (Grigore et al., 2023).

[0115] Rhodotorula carotenoid producers include but are not limited to, Rhodotorula glutinis, Rhodotorula graminis, Rhodotorula taiwanensis, and Rhodotorula babjevae. Rhodotorula species like Rhodotorula glutinis can produce several valuable compounds such as enzymes, microbial oils like oleic, linoleic, palmitic, and stearic acid, as well as carotenoids (Kot et al., 2016). R. glutinis uses a variety of carbon sources for growth like glucose, trehalose, galactose, etc. The lipid content of their biomass can be as high as 72% and is affected by factors such as the carbon source, the strain used, the C / N ratio, and nitrogen sources. Additionally, their cultivation does not require long periods of growth like their plant counterparts. Rhodotorula also does not require large volumes of water, excess land use, harmful fertilizers, etc. (Kim et al., 1999; Vend et al., 2013).Attorney Docket No. 11214-012WO1 There is a growing interest in maximizing the use of natural resources and agro-industrial products and wastes to produce high-value bioproducts like P-carotene. These agricultural co-and by-products can serve as feedstocks for red yeast cultivation to produce carotenoids. Sweet sorghum or sorghum cane (Sorghum bicolor (L.) Moencli) is an option due to its very large geographic suitability, short growing season (3-5 months), low fertilizer and water inputs compared to other crops and high agronomic stability to temperature fluctuations and drought resistance (Teetor et al., 2011). Sorghum syrup is an agricultural commodity prepared from 100% pure natural juice extracted from the stalks of the sorghum plant. Sorghum syrup also contains vitamins, up to 52.7% of the daily iron, up to 1710mg of potassium, low sodium, and higher amounts of magnesium compared to other syrups (Eggleston et aL, 2022). These qualities make sorghum and its syrup an ideal feedstock for yeast fermentation because of its cost, ease of access, and nutritional content.

[0116] Employed herein was bioprocess optimization to maximize P-carotene yields in the sorghum-based media. Through optimization, carotenoid production can be enhanced using low-cost culture media (Ananda & Vadlani, 2011; Bhosale, 2004). Microbial cultivation and yield optimization procedures to produce high-quality carotenoids are more cost-effective. The availability of several agricultural products or agricultural co- and by-products offers opportunities for value addition. According to the Agricultural Marketing Resource Center (2023). value-added agriculture includes production or manufacturing processes, marketing, or services that increase the value of primary agricultural commodities for example by increasing the appeal of the commodity to the consumer, resulting in the willingness of consumers to pay a premium over similar but undifferentiated products. Value addition results in higher returns than similar, undifferentiated products, allows entry or expansion of new, high-value markets, and creates new product identities. For economic viability and value-addition, explored herein is the suitability of sorghum syrup as the primary carbon source to produce P-carotene by red yeast R. glutinis fermentation. Accordingly, sorghum syrup, when used as the substrate for R. glutinis fermentation, will support the production of p-carotene. The objectives herein were to evaluate the carotenoid yield from R. glutinis fermentation in an optimized sorghum syrup medium and to evaluate the fermentation for its other nutritional characteristics.

[0117] Materials and Methods

[0118] Microorganism and growth conditions. Rhodotorula glutinis (ATCC 32766) used herein was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). It was grown in a 250mL flask containing lOOmL of sterile potato dextrose broth and incubated at 20°C at 200 rpm for 7 days. R. glutinis was also maintained on potato dextrose agar (PDA) platesAttorney Docket No. 11214-012WO1 incubated at room temperature. Finally, culture broth was used as the inoculum for the evaluations that followed.

[0119] Process optimization. A central composite design (CCD) was employed to optimize variable factors, i.e., the concentrations of sorghum syrup, yeast extracts, and mineral salts in fermentation media. The CCD was used to determine the optimum conditions or the cultivation of P-carotene at the shake flask level, by defining the effects and the interaction between the various media components on the yeast growth and the production of the P-carotene. The experiments were conducted in 250mL Erlenmeyer flasks with a working volume of 100ml. The flasks were sterilized at 120°C for 30 minutes and cooled to room temperature. About 1ml of actively growing culture was added to each flask and incubated for 10 days at 200 rpm and 25°C. On day 10, all flasks were centrifuged, the supernatant discarded, and cell pellets were freeze-dried and used for P-carotene quantification.

[0120] Carotenoid extraction and quantification. Carotenoids were manually extracted from lyophilized cells using tetrahydrofuran and acid-washed sand. All samples were then filtered into vials using 0.2µm filters. High-performance liquid chromatography (HPLC) analysis of the extracted carotenoids was performed in a Shimadzu HPLC equipped with a UV-Vis detector. A 90: 10 ratio of acetonitrile and methanol was used as the mobile phase, with a flow rate of 1.5 mL / min. Detection was carried out at 450nm and a calibration curve was prepared using pure beta-carotene standard obtained from Sigma Aldrich and used to determine concentration of beta-carotene in samples. (Fig. SI). A Phenomenex Luna C8 column was used for the separation of carotenoids.

[0121] Validation of optimized medium. The optimal media composition points obtained from the CCD were used to prepare 2 liters of media. Aliquots of 300mL were added to media glass bottles in duplicates and inoculated with 1 % yeast inoculum. All media bottles were incubated on a shaker for 10 days at 25°C and 200 rpm. Sampling from each bottle was undertaken on days 5, day 7, and day 10. P-carotene from samples were analyzed via HPLC, as previously described.

[0122] Fermentation in Benchtop Bioreactors. A batch- fed run was performed using benchtop bioreactors (7L Eppendorf-New Brunswick Bioflo 115, Eppendorf AG, Germany). Triplicates of a working volume of 5L of media were prepared, consisting of 459g sorghum syrup, 6.5g (NH4)2SO4, 3.5g KH2PO4, 21g MgSO4, and 48g yeast extract. The bioreactors were set after sterilization and a 1 % yeast inoculum was added. The batch-fed conditions maintained for fermentation were as follows: temperature (25°C), agitation (200 rpm), and dissolved oxygen level (1 vvm). All three bioreactors were integrated with supervisory control and data acquisition (SCADA) software system and data was gathered from all bioreactors every 10 minutes duringAttorney Docket No. 11214-012WO1 the 10-day fermentation. The fermentation profile of the bioreactor variables such as pH, DO, temperature, agitation speed were recorded.

[0123] Nutritional Characterization of the Fermented Product. At the end of fermentation on day 10, the broth of the bioreactor samples was harvested and centrifuged and biomass (cells) collected were stored at -80°C. All triplicate bioreactor samples were freeze dried. All samples were analyzed for vitamins (P-carotene, riboflavin, niacin, pantothenic acid, and pyridoxine), total amino acid profile, total fatty acid profile, crude fat, crude protein, crude fiber, and minerals. The methods used for proximate analyses are as follows: P-carotene was evaluated using the AACC-86-06 mod method, B vitamins were assessed by using the LS / MS / MS Journal of Chromatography 2003 method, D vitamins were assessed using the AOAC 2016.05 method, E vitamins were evaluated used the Journal CRNFS 2016 Volume 4, 92 method. Crude proteins were analyzed using the AOAC 990.03 method, crude fats were analyzed using the AOAC 2003.05 method, and total amino acids were evaluated by the AOAC 994.12 and AOAC 988.15 methods. Total lipids and fatty acids were assessed using the AOAC 996.06 method and glucosamine was analyzed using the Anal Chem ACTA June 2011:695 method. The mineral contents were analyzed using the AOAC 985.01 method.

[0124] Results and Discussion

[0125] Optimizing culture medium for fi-carotene production. Bioprocess optimization involving 31 sorghum syrup-based experimental culture media revealed a combination of nutrients that supported the successful production of P-carotene from R. glutinis. The P-carotene yields for all 31 combinations of media from RSM are listed in Table 1.

[0126] The highest P-carotene concentration was 1,378 pg / g of P-carotene for combination #30 from Table 2.

[0127] Table 1. Beta-carotene concentrations obtained from sorghum syrup fermented R. glutinis 32766 via RSM.

[0128] Flask # P-carotene cone (pg / g)

[0129] 1 628.43

[0130] 2 184.09

[0131] 3 1045.77

[0132] 4 658.48

[0133] 5 503.51

[0134] 6 471.26

[0135] 7 868.34

[0136] 8 1041.49

[0137]

[0138] 9 570.95Attorney Docket No. 11214-012WO1 10 1022.71

[0139] 11 793.47

[0140] 12 1053.39

[0141] 13 1101.09

[0142] 14 803.96

[0143] 15 1183.43

[0144] 16 563.64

[0145] 17 1031.84

[0146] 18 536.58

[0147] 19 172.04

[0148] 20 1106.12

[0149] 21 519.50

[0150] 22 169.43

[0151] 23 1145.05

[0152] 24 355.17

[0153] 25 276.14

[0154] 26 726.96

[0155] 27 158.40

[0156] 28 592.75

[0157] 29 239.55

[0158] 30 1378.12

[0159]

[0160] 31 392.74

[0161] Table 2. Design Matrix from the central composite design (CCD) used during process optimization of the media components for the production of beta-carotene from sorghum-fermented R. glutinis.

[0162] Sorghum Syrup (NH4)2SO4KH2PO4MgSO4Yeast Extract Flask #

[0163] (g) (mL) (mL) (mL) (g) 1 4.83 0.15 0.01 0.26 1.00

[0164] 2 6.72 0.05 0.10 0.43 0.28

[0165] 3 10.00 0.50 0.01 0.10 1.00

[0166] 4 1.00 0.30 0.10 0.38 0.56

[0167] 5 2.58 0.50 0.06 0.41 0.10

[0168] 6 10.00 0.27 0.06 0.26 0.10

[0169] 7 1.00 0.05 0.04 0.27 0.46

[0170] 8 3.39 0.22 0.04 0.50 0.73

[0171] 9 5.19 0.23 0.05 0.10 0.62

[0172] 10 1.00 0.50 0.01 0.50 0.87

[0173] 11 1.00 0.30 0.10 0.38 0.56

[0174] 12 1.00 0.50 0.09 0.10 1.00

[0175] 13 2.08 0.50 0.04 0.24 0.63

[0176] 14 7.93 0.50 0.01 0.36 0.37

[0177]

[0178] 15 10.00 0.13 0.03 0.50 0.74Attorney Docket No. 11214-012WO1 16 1.00 0.05 0.10 0.10 0.10

[0179] 17 1.00 0.05 0.09 0.50 1.00

[0180] 18 7.17 0.43 0.08 0.42 1.00

[0181] 19 1.00 0.42 0.01 0.10 0.10

[0182] 20 7.17 0.43 0.08 0.42 1.00

[0183] 21 5.19 0.23 0.05 0.10 0.62

[0184] 22 10.00 0.05 0.01 0.10 0.10

[0185] 23 10.00 0.05 0.10 0.15 1.00

[0186] 24 10.00 0.50 0.03 0.10 0.10

[0187] 25 1.65 0.40 0.08 0.12 0.10

[0188] 26 7.93 0.50 0.10 0.10 0.33

[0189] 27 10.00 0.27 0.06 0.26 0.10

[0190] 28 4.83 0.15 0.01 0.26 1.00

[0191] 29 10.00 0.50 0.10 0.50 0.10

[0192] 30 8.49 0.05 0.10 0.48 1.00

[0193]

[0194] 31 3.25 0.21 0.01 0.50 0.10

[0195] To identify a medium composition, three-dimensional plots with two factors were generated. The P-carotene yield was affected by medium composition, with a tenfold difference between the best and the weakest medium (1,378 pg / g and 158 pg / g respectively). Figs. 3A and 3B show the effect of varying sorghum syrup, yeast extract, and mineral salt ratios and concentrations. Both sorghum syrup and yeast extract concentration positively influenced the P-carotene production, indicating that increasing concentrations of both ingredients in the media would result in the increased production of P-carotene. The optimum points for the production of P-carotene can be seen in Fig 2a with the predicted minimum average P-carotene to be produced at the optimal points to be 1,003.38µg / g. The interaction of the media components also corresponds to the findings, suggesting that an increase in the concentration of sorghum syrup positively influences cell biomass, while an increase in the yeast extract positively influences the concentration of the target bioproduct, P-carotene. Despite low concentrations of minerals present in the fermentation media, their specific concentrations impacted yield. The exact functions of inorganic salts in carotenogenesis have not been fully understood, however, their addition to media contributes to improved carotenoid yields (Han et al., 2002).

[0196] The minerals were found to have a positive influence on the carotenoid concentration (Fig. 3B). Chen et al. (2006) reported that the production of carotenoids in Rhodotorula sphaeroides was enhanced in media containing MgSCh, Na2HPC>4, FeSC, and Na2CO.

[0197] Choudhari and Singhal (2008) reported that KH2PO4 and MgSCh were useful for P-carotene production in Blakeslea trispora. At low concentrations of nitrogen sources (KNO3 incorporated with (NH4)2SO4), Phaffia rhodozyma was able to produce astaxanthin (Ni et al., 2007; Parajo etAttorney Docket No. 11214-012WO1 al., 1998). (NH4)2SO4 was used herein as the mineral source of nitrogen coupled with organic sources in yeast extract and sorghum syrup. The overall optimization studies show that the high concentration of yeast extract increases the production of carotenoids while a high concentration of sorghum syrup increases the cell biomass or cell dry weight of R. glutinis. The results present a ratio for optimal production of P-carotene. The validated results show that fermentation time in an optimized medium impacts yield (Fig. 3). The -carotene yields on days 5, 7, and 10 were 476, 1,173, and 1,153 pg / g, respectively. The day 10 yield on validation of 1,153 pg / g P-carotene was slightly higher than the predicted day 10 yield of 1,003.38µg. However, the best yield was observed on day 7, exceeding the predicted amount as described previously.

[0198] Batch-mode fermentation. To evaluate productivity under medium-scale, batch-mode conditions, used herein was a 7L benchtop fermenter containing 5L of the optimized medium. The overall trend in the three replicates for the 10 days was similar. As fermentation progressed, P-carotene concentration increased. The dissolved oxygen of all the fermenters decreased to less than 15 % within 24 hours of the fermentation period, indicating that R. glutinis was actively respiring the oxygen in the fermenters. The batch-mode fermentation of R. glutinis in triplicates resulted in an average of 1,008, 1,227, and 1,753mg / kg of P-carotene on day 5, day 7, and day 10 of fermentation respectively (Fig 4).

[0199] Red yeast nutrient content. R. glutinis- vnvtA& X sorghum syrup enriched in carotenoids was evaluated for its nutritional composition. Apart from providing P-carotene in the form of vitamin A, there were also other vitamins, fatty acids, crude fats, crude proteins, amino acids, and minerals in the R. glutinis fermented product (Table 3).

[0200] Table 3. Proximate nutritional analyses profile of R. glutinis (ATCC 32766) fermented in a sorghum syrup-based media.

[0201] Nutrient Average Standard Standard Vitamins deviation Error P-carotene (as pro-vitamin A)(mg / kg) 11800.00 2705.55 1562.05 Riboflavin (Vitamin B2) (mg / kg) 6.57 0.51 0.29 Niacin (Vitamin B3) (mg / kg) 20.03 2.11 1.22 Pantothenic acid (mg / kg) 117.67 10.79 6.23 Pyridoxine (B6) as pyridoxine HO (mg / kg) 0.19 0.08 0.05 Biotin (mg / kg) 2.09 0.03 0.02 Folic acid (mg / kg) 0.63 0.11 0.06 Cobalamin (B 12) as cyanocobalamin (mg / kg) 0.04 0.02 0.01 Vitamin D2 (ergocalciferol) (mg / kg) 0.38 0.14 0.08

[0202] Proteins

[0203]

[0204] Crude protein % 17.20 2.62 1.51Attorney Docket No. 11214-012WO1 Crude fats % 0.53 0.40 0.23 Amino acids

[0205] Cysteine % 0.13 0.03 0.02 Methionine % 0.20 0.04 0.02 Tryptophan % 0.15 0.04 0.02 Lipids and Fatty Acids

[0206] Palmitic (C16:0) g / 100g 0.07 0.05 0.03 Stearic (C18:0) g / 100g 0.02 0.02 0.01 Oleic (C18:1, Cis)g / 100g 0.34 0.24 0.14 Linoleic (C18:2, Trans) g / 100g 0.05 0.05 0.03 alpha-Linoleic (C18:3, alpha) g / lOOg 0.01 0.02 0.01 Lignoceric (C24:0) g / 100g 0.01 0.01 0.01 Saturated fat total g / 100g 0.11 0.09 0.05 Polyunsaturated fat (total) g / 100g 0.06 0.06 0.04 Monounsaturated fats (total) g / 100g 0.35 0.25 0.14 Omega 3 fatty acids (total) g / 100g 0.01 0.02 0.01 Omega 6 fatty acids (total) g / 100g 0.05 0.05 0.03 Omega 9 fatty acids (total) g / 100g 0.34 0.24 0.14 Glucosamine (HCL total) mg / kg 3073.00 1559.24 900.23 Minerals

[0207] Sulphur (total) % 1.67 0.24 0.14 Phosphorus (total) % 0.55 0.06 0.04 Potassium (total) % 1.39 0.22 0.13 Magnesium (total) % 1.02 0.13 0.07 Calcium (total) % 0.16 0.02 0.01 Sodium (total) % 0.28 0.04 0.02

[0208] Iron (total) ppm 54.00 31.59 18.24 Manganese (total) ppm 8.83 1.47 0.85 Copper (total) ppm 12.20 3.99 2.30 Zinc (total) ppm 22.83 5.01 2.89 Sulphur (total) % 1.67 0.24 0.14 Phosphorus (total) % 0.55 0.06 0.04

[0209]

[0210] Potassium (total) % 1.39 0.22 0.13

[0211] Vitamin A - Sorghum syrup fermentation of R. glutinis resulted in an average of 11,800 mg / kg of P-carotene. Animal species handle -carotene differently as it pertains to its absorption, accumulation in blood and tissues, and metabolism to Vitamin A. Dairy cows are reported to have early lactation when supplemented up to 2,000mg of P-carotene daily from day 21 before calving date (Kawashima et al., 2010). The effects of age and dietary P-carotene in domestic animals like dogs have been investigated. P-carotene supplemented in the diet of older dogs significantly restored the immune responses compared to older dogs whose diets were not supplemented and their younger counterparts (Massimino et aL, 2003).Attorney Docket No. 11214-012WO1 Niacin - Sorghum syrup fermentation of R. glutinis resulted in an average of 20 mg / kg of niacin. Niacin (Vitamin B3) contributes molecules to the metabolic process of animals and human beings. This is due to its incorporation into the coenzymes NAD and NADP. Notably to dairy cows, niacin in the rumen due to the action of gastrointestinal microorganism is a source. Niacin is involved in various energy-yielding pathways and for synthesis of amino acid and fatty acids; for which it is useful for milk production (Erickson, 2020). As a result, supplementation of niacin in lactating cattle has beneficial effects on growth of cattle. Cattle supplemented with 12g of niacin per head per day can increase milk production by approximately 11b. Supplementing the dairy animals with the dose of niacin will not only protect them from various metabolic diseases but will also help them defend from severe heat stress; leading to augmentation of their health, production potential and economy (Panda et al., 2017).

[0212] Pantothenic Acid - Sorghum syrup fermentation of R. glutinis resulted in an average of 118 mg / kg of pantothenic acid. Pantothenic acid, or vitamin B5, is a water-soluble vitamin that contributes to produce coenzyme A. This vitamin is found in many plants and animal food sources, therefore, in humans, the deficiency is rare. On the other hand, the consequences of pantothenic acid deficiency in animals have presented as low blood glucose, rapid breathing and heart rates, and convulsions in (National Research Council (U. S.), 2016). Poultry developed skin irritation, feather abnormalities, and spinal nerve damage associated with the degeneration of the myelin sheath (Tang et al., 2019).

[0213] While the human diet only requires 5 mg of pantothenic acid per, domesticated animals like cats and dogs require between 10 to 20 mg / kg of pantothenic acid (National Research Council (U. S.), 2016). The pantothenic acid production herein from R. glutinis supports the possibilities of the development of feed blends in the pet food industry as well as human nutraceutical development.

[0214] Pyridoxine - Sorghum syrup fermentation of R. glutinis resulted in an average of 0.1 mg / kg of pyridoxine. Pyridoxine, or vitamin B6 is used for energy production and the metabolizing of carbohydrates and fatty acids. Pyridoxine is also a useful element for boosting the immune system (Harvard School of Public Health, 2019). Humans require 1.5-1.7 mg of pyridoxine in the diet, while the required amount for animals varies. Animals, whether farmed or domesticated, get their pyridoxine from many grains, however pyridoxine is not always bioavailable. As a result, many grazing animals like cattle can synthesize their own pyridoxine while young grazers acquire it via their mother’s milk (Parra et al., 2018). In the swine industry, supplementing the pigs’ diet with pyridoxine has benefits. Pyridoxine has increased the daily weight gains of swine and may also help sows to breed quickly after weaning offspringAttorney Docket No. 11214-012WO1 (Woodworth et aL, 2000). As for pet animals, pyridoxine supplementation may slow or stop tumor development in cats and has been used to treat seizures in dogs. The recommended pyridoxine allowance is 0.375 mg (National Research Council (U. S.), 2016). The value-added bioproduct provides only half of this recommended dosage.

[0215] Biotin - Sorghum syrup fermentation of R. glutinis resulted in an average of 2 mg / kg of biotin. Biotin, or vitamin B7 is used for animals. This vitamin is useful to a plethora of metabolic pathways including keratin and lipid synthesis that are used for cattle horn production (Zempleni et al., 2009). Glucose synthesis, the main source for milk production in cattle, also uses biotin. Notably, while ruminants have microbes in the rumen that produce biotin, trials have shown that cattle, especially dairy cattle respond to biotin supplementation (Riveron-Negrete & Fernandez-Mejia, 2017). These responses suggest that the biotin present in the rumen is not fully bioavailable to the animal (Weiss, 2019).

[0216] Folic Acid - Sorghum syrup fermentation of R. glutinis resulted in an average of 0.63 mg / kg of pantothenic acid. Folic acid is used as a vitamin for normal metabolic functions in domestic and companion animals alike. Folic acid is needed in processes like DNA synthesis and red blood cell production (Scientific Opinion on the Safety and Efficacy of Folic Acid as a Feed Additive for All Animal Species I EFSA, 2012). Deficiencies in this vitamin usually occur because of small intestine disease or damage set on by chronic use of certain drugs like trimethoprim (Folic Acid I VCA Animal Hospitals, n.d.). Folic acid supplementation, especially for animals, is very effective.

[0217] Amino Acids: Cystine, Methionine, and Tryptophan - The biosynthesis of cysteine occurs in animals and plants via the trans-sulphuration pathway from methionine, in the presence of adequate nitrogen and sulfur (Hesse et al., 2004). Consequently, since cysteine is synthesized from methionine via the trans-sulphuration pathway, its requirement is usually considered together with methionine. This was seen in R. glutinis with the production of 0.13% ± 0.02 and 0.20% ± 0.02 respectively, for every 100g. The U. S. National Research Council (1994) reports the combined requirements of cysteine and methionine for poultry to be 0.72% for grower poultry (National Research Council (U. S.), 1994). Cysteine and methionine are important sulfur-containing amino acids that are useful for animal health. In the case of the poultry industry, both amino acids contribute to the growth and development of broilers. The two are major protein constituents of feathers and hair, with methionine occurring in greater percentage in muscle while cysteine is higher in feather keratin (Pacheco et al., 2018). Currently, synthetic methionine and cysteine supplements are used in the poultry industry, whose production depends on a petroleum derivative - propylene. However, natural supplements for use in the poultryAttorney Docket No. 11214-012WO1 production industry can be developed as microorganisms like R. glutinis can produce the substance via fermentation (Fanatico & Ellis, 2016). Tryptophan is another essential amino acid for all animals and humans that was found present in R. glutinis. Tryptophan is essential in the production of serotonin and melatonin (Kaklińska-Czaplińska et al., 2019). Because tryptophan serves as a precursor for serotonin and melatonin in the diet, a tryptophan deficiency could affect the behavioral responses, e.g., pecking behavior and stress in poultry, increased aggression in pet animals, and poor lactation in cattle (Moehn et al., 2012).

[0218] Tryptophan and its supplementation in the diet of different animal groups has been determined to improve the amino acid balance and promote the poultry’s growth performance through enhancing appetite, feed efficiency, and protein synthesis (Jo et aL, 2021). In addition, supplemented tryptophan also improves the immune response or the immunomodulatory activity of poultry to various diseases. Furthermore, tryptophan also has a strong relationship with lysine, and the appropriate ratio of both amino acids is known to improve growth performance (Linh et al., 2021).

[0219] Glucosamine - Glucosamine is an important nutrient for proper bone and joint support in humans and animals (Neil et al., 2005). Glucosamine deficiency often presents osteoarthritis. Both humans and pet animals, especially dogs, require about 1,500mg of glucosamine (Dahmer & Schiller, 2008). Interestingly, Weimer et al. (2014) reported the extension of lifespan of aging mice and nematodes when provided with glucosamine. In this sorghum syrup fermentation of R. glutinis, the carotenoid production was accompanied by 3,073mg / kg of glucosamine which is twice the minimum required dosage.

[0220] Minerals - There is a growing interest in the use of minerals in the diets of animals due to studies uncovering the great benefits of their use. Minerals are used in many metabolic processes in animals and humans alike. In human nutrition, minerals are responsible for structural functions involving the skeleton and soft tissues and for regulatory functions including neuromuscular transmission, blood clotting, oxygen transport, and enzymatic activity (Health, 1989). In animal nutrition, minerals are obtained via the diet, however, the bioavailability of these minerals is varied, therefore supplementation of minerals is often recommended for ruminants, poultry, and swine (Cuellar Saenz, 2021). Sorghum syrup fermented by R. glutinis was evaluated for its mineral composition and the findings are outlined in Table 3. Organically chelated minerals are more stable than inorganic salt forms when used in nutrition. Organically chelated minerals are better bioavailable than their inorganic counter parts. Due to this reason organic trace minerals (OTM) are developed though microbial fermentation as inexpensive route (Byrne et al 2021; Chen et al 2022)Attorney Docket No. 11214-012WO1 Phosphorus and Calcium - Phosphorus and calcium work together as the main constituents of bone minerals. Additionally, calcium is important to muscle function while phosphorus is used for many metabolic processes throughout the body of animals and humans (Manopriya et al., 2022). In dairy cows, the requirement for calcium is about 0.30% while the phosphorus requirement is 0.20% (Harty, 2022). Notably, the phosphorus and calcium found in R. glutinis fermented sorghum syrup were found to be 0.55 ± 0.04% and 0.16 ± 0.01% respectively.

[0221] Potassium - Potassium works in tandem with sodium in the diet to regulate osmotic pressure and transport of nutrients in and out of cells. Potassium can leach out of animal forages therefore it is recommended to supplement with 1% potassium to remedy or prevent deficiency (Preston & John, 1985). Sorghum syrup fermented by R. glutinis resulted in 1.39 ± 0.13% potassium which is higher than the required potassium percentage for dairy cattle (0.70%).

[0222] Magnesium - Magnesium in R. glutinis fermented sorghum syrup was found to be 1.02 ± 0.07%. This is ten times more than the required magnesium in gestating cattle which requires 0.1% magnesium. For lactating cows, 0.2% is required. Magnesium is essential to the enzyme and nervous system functions as well as carbohydrate metabolism (Harty, 2022).

[0223] Sodium - Sodium works with chlorine ions in extracellular fluid to maintain acid-base balance, osmotic regulation, muscle contraction, and absorption of glucose in animals and humans (Harty, 2022). Upon the evaluation of the mineral content of sorghum syrup fermented by R. glutinis was found to contain 0.28 ± 0.02% sodium. While the recommended intake for humans is much higher (2,300mg) (EFSA Panel on Nutrition, Novel Foods and Food Allergens (ND A) et al., 2019), the sodium in R. glutinis fermented sorghum syrup was above the sodium requirement for dairy cattle at 0.07%.

[0224] Trace Mineral Supplementation - Trace minerals present in R. glutinis fermented sorghum syrup are iron, copper, zinc, and manganese. Iron is needed for hemoglobin production to prevent weight loss, depressed immunity, and anemia (Pajarillo et al., 2021). Sorghum syrup fermented by R. glutinis contained 54.00 ± 18.24 mg / kg of iron compared to the 8-18mg required in humans and the 50mg required for optimal health in cattle (Rasby et al., 2011;

[0225] Relative Bio availability of Trace Minerals in Production Animal Nutrition: A Review - PMC, n.d.). Copper is essential to growth and development and when deficient, there is reduced fertility and reduced hair pigmentation in cattle. The human diet only requires 900pg of copper (Harvard School of Public Health, 2022) and cattle only 10 mg (Rasby et al., 2011).

[0226] Interestingly, sorghum syrup fermented by R. glutinis contained 12.20 ± 2.30mg / kg. Zinc is a component of many enzymes and is important for immunity, male reproductive health, and skinAttorney Docket No. 11214-012WO1 and hoof health in cattle (Zigo et al., 2022). Interestingly, sorghum syrup fermented by R. glutinis was found to produce 22.83 ± 2.89mg / kg however, cattle require 30mg while humans require 8-11 mg in the diet.

[0227] These trace minerals are organically chelated to the proteins present in R. glutinis.

[0228] Chelation increases the bioavailability of minerals to both animals and humans ultimately supporting improved metabolic functions (Byrne & Murphy, 2022).

[0229] Conclusion. Nutritious sorghum syrup can serve as an excellent substrate for red yeast fermentation to produce carotenoids. Media optimization using RSM provided the following concentrations of media components (9.18% sorghum syrup, 0.13% (NH4)2SO4, 0.07% KH2PO4, 0.42% MgSO4 and 0.96% yeast extract). The scale-up resulted in yields much higher (up to 1,753μg / g) than the predicted yield of P-carotene of 1,003 μg / g. In addition to the healthpromoting P-carotene, which was the primary bioproduct of interest, also determined herein was that the fermentation product is nutritionally dense with other nutrients such as glucosamine, vitamins and organic trace minerals.

[0230] References

[0231] Ananda, N., & Vadlani, P. V. (2011). Substrates Influence Stimulatory Effect of Mevalonic Acid on Carotenoid Production in Red Yeasts. Cereal Chemistry Journal, 88(3), 310-314. https: / / doi.org / 10.1094 / CCHEM-10-10-0149

[0232] Ashokkumar, V., Flora, G., Sevanan, M., Sripriya, R., Chen, W. H., Park, J.-H., Rajesh banu, J., & Kumar, G. (2023). Technological advances in the production of carotenoids and their applications- A critical review. Bioresource Technology, 367, 128215.

[0233] https: / / doi.org / 10.1016 / j.biortech.2022.128215

[0234] Bhosale, P. (2004). Environmental and cultural stimulants in the production of carotenoids from microorganisms. Applied Microbiology and Biotechnology, 63, 351-361. https: / / doi.org / 10.1007 / s00253-003-1441-1

[0235] Byrne, L., & Muiphy, R. A. (2022). Relative Bioavailability of Trace Minerals in Production Animal Nutrition: A Review. Animals: An Open Access Journal from MDPI,

[0236]

[0237] 1981. https: / / doi.org / 10.3390 / ani12151981

[0238] Carotenoid Database, (n.d.). Retrieved April 2, 2024, from http: / / carotenoiddb.jp / Cuellar Saenz, J. A. (2021). Importance of minerals in animal nutrition. Veterinaria Digital. http: / / https%253A%252F%252Fwww.veterinariadigital.com%252Fen%252Farticulos%252Fim portance-of-minerals-in-animal-nutrition%252FAttorney Docket No. 11214-012WO1 Dufosse, L., Galaup, P., Yaron, A., Arad, S., Blanc, P., Murthy, K., & Gokare, R. (2005). Microorganisms and microalgae as sources of pigments for food use: A scientific oddity or an industrial reality? Trends in Food Science & Technology, 16, 389-406. https: / / doi.org / 10.1016 / j.tifs.2005.02.006

[0239] EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA), Turck, D., Castenmiller, J., de Henauw, S., Hirsch-Ernst, K.-I., Kearney, J., Knutsen, H. K., Maciuk, A., Mangelsdorf, I., McArdle, H. J., Pelaez, C., Pentieva, K., Siani, A., Thies, F., Tsabouri, S., Vinceti, M., Aggett, P., Fairweather-Tait, S., Martin, A.,... Naska, A. (2019). Dietary reference values for sodium. EFSA Journal, 17(9), e05778. https: / / doi.org / 10.2903 / j.efsa.2019.5778 Eggleston, G., Triplett, A., Bett-Garber, K., Boue, S., & Bechtel, P. (2022).

[0240] Macronutrient and mineral contents in sweet sorghum syrups compared to other commercial syrup sweeteners. Journal of Agriculture and Food Research, 7, 100276.

[0241] https: / / doi.org / 10.1016 / j.jafr.2022.100276

[0242] Erickson, P. (2020, July 6). Feeding Nicotinic Acid (Niacin) to Dairy Cows. Extension. https: / / extension.unh.edu / resource / feeding-nicotinic-acid-niacin-dairy-cows

[0243] Fanatico, A., & Ellis, K. (2016). Organic Poultry Production: Providing Adequate Methionine - ATTRA - Sustainable Agriculture, https: / / attra.ncat.org / publication / organic-poultry-production-providing-adequate-methionine /

[0244] Fiedor, J., & Burda, K. (2014). Potential role of carotenoids as antioxidants in human health and disease. Nutrients, 6(2), 466-488. https: / / doi.org / 10.3390 / nu6020466

[0245] Folic Acid I VC A Animal Hospitals, (n.d.). Retrieved March 23, 2023, from https: / / vcahospitals.com / know-your-pet / folic-acid

[0246] Global Carotenoids Market Report and Forecast 2023-2028. (n.d.). Retrieved November 29, 2022, from https: / / www.expertmarketresearch.com / reports / carotenoids-market Grigore, D.-M., Ungureanu-Iuga, M., Pogurschi, E. N., & Babeanu, N. E. (2023).

[0247] Transforming Rhodotorula sp. Biomass to Active Biologic Compounds for Poultry Nutrition. Agriculture, 13(6), Article 6. https: / / doi.org / 10.3390 / agriculturel3061159

[0248] Han, J.-R., Xu, J., & Zhou, X.-M. (2002). Influence of inoculum type, inorganic salt and nitrogen to carbon ratio on sclerotium formation and carotenoid production in surface culture of Penicillium sp. PT95. Journal of Basic Microbiology, 42(A), 254-259. https: / / doi.org / 10.1002 / 1521-4028(20()208)42:4<254:: AID-JOBM254>3.0. CO;2-Y

[0249] Harty, A. (2022). Cow Mineral Nutrition: Macro Minerals and Their Importance. https: / / extension.sdstate.edu / cow-mineral-nutrition-macro-minerals-and-their-importanceAttorney Docket No. 11214-012WO1 Harvard School of Public Health. (2019, June 4). Vitamin B6. The Nutrition Source. https: / / www.hsph.harvard.edu / nutritionsource / vitamin-b6 /

[0250] Harvard School of Public Health. (2022, July 25). Copper. The Nutrition Source. https: / / www.hsph.harvard.edu / nutritionsource / copper /

[0251] Health, N. R. C. (US) C. on D. and. (1989). Minerals. In Diet and Health: Implications for Reducing Chronic Disease Risk. National Academies Press (US).

[0252] https: / / www.ncbi.nlm.nih.gov / books / NBK218735 /

[0253] Hesse, H., Nikiforova, V., Gakiere, B., & Hoefgen, R. (2004). Molecular analysis and control of cysteine biosynthesis: Integration of nitrogen and sulphur metabolism. Journal of Experimental Botany, 55(401), 1283-1292. https: / / doi.org / 10.1093 / jxb / erh136

[0254] Jo, J.-H., Lee, J.-S., Ghassemi Nejad, J., Kim, W.-S., Moon, J.-O., & Lee, H.-G. (2021). Effects of Dietary Supplementation of Acetate and L-Tryptophan Conjugated Bypass Amino Acid on Productivity of Pre- and Post-Partum Dairy Cows and Their Offspring. Animals, 11(6), Article 6. https: / / doi.org / 10.3390 / ani11061726

[0255] Kaźmierczak-Barańska, J., Gałarek, P., Chirumbolo, S., Chartrand, M. S., & Bjørklund, G. (2019). How important is tryptophan in human health? Critical Reviews in Food Science and Nutrition, 59(1), 72-88. https: / / doi.org / 10.1080 / 10408398.2017.1357534

[0256] Kawashima, C., Nagashima, S., Sawada, K., Schweigert, F., Miyamoto, A., & Kida, K. (2010). Effect of β-Carotene Supply During Close-up Dry Period on the Onset of First Postpartum Luteal Activity in Dairy Cows. Reproduction in Domestic Animals, 45(6), e282-e287. https: / / doi.org / 10.1111 / j.1439-0531.2009.01558.x

[0257] Kim, C.-H., Kim, S.-W., & Hong, S.-I. (1999). An integrated fermentation-separation process for the production of red pigment by Serratia sp. KH-95. Process Biochemistry, 35(5), 485-490. https: / / doi.org / 10.1016 / S0032-9592(99)00091-6

[0258] Kirti K., A. S. (2014). Colorful world of microbes: Carotenoids and their applications. Advances in Biology, 1-14.

[0259] Kot, A. M., Blazejak, S., Kurcz, A., Gientka, I., & Kieliszek, M. (2016). Rhodotorula glutinis — Potential source of lipids, carotenoids, and enzymes for use in industries. Applied Microbiology and Biotechnology, 100(14), 6103-6117. https: / / doi.org / 10.1007 / s00253-016-7611-8

[0260] Linh, N. T„ Guntoro, B., & Hoang Qui, N. (2021). Immunomodulatory, behavioral, and nutritional response of tryptophan application on poultry. Veterinary World, 14(8), 2244-2250. https: / / doi.org / 10.14202 / vetworld.2021.2244-2250Attorney Docket No. 11214-012WO1 Louren^o-Lopes, C., Carreira-Casais, A., Fraga-Corral, M., Garcia-Oliveira, P., Soria, A., Jarboui, A., Barral, M., Otero, P., Simal-Gandara, J., & Prieto, M. A. (2021). Carotenoids as Natural Colorful Additives for the Food Industry. In Natural Food Additives. IntechOpen. https: / / doi.org / 10.5772 / intechopen.101208

[0261] Manopriya, S., Aberathna, A. a. a. U., Satharasinghe, D. A., Jayasooriya, L. J. P. a. P., Mantilaka, M. M. M. G. P. G., Fernando, C. a. N., Jayaweera, B. P. A., Weerathilake, W. a. D. V., Prathapasinghe, G. A., Liyanage, J. A., & Premarathne, J. M. K. J. K. (2022). Importance of Phosphorus in Farm Animals. Iranian Journal of Applied Animal Science, 12(2), 203-210.

[0262] Massimino, S., Kearns, R. J., Loos, K. M., Burr, J., Park, J. S., Chew, B., Adams, S., & Hayek, M. G. (2003). Effects of age and dietary beta-carotene on immunological variables in dogs. Journal of Veterinary Internal Medicine, 77(6), 835-842. https: / / doi.org / 10.1111 / j.1939-1676.2003.tb02523.x

[0263] Moehn, S., Pencharz, P. B., & Ball, R. O. (2012). Lessons learned regarding symptoms of tryptophan deficiency and excess from animal requirement studies. The Journal of Nutrition, 142(12), 2231S-2235S. https: / / doi.org / 10.3945 / jn.112.159061

[0264] Murthy, K. (2005). Production of Beta- Carotene from Cultured Dunaliella Sp. And Evaluation Of Biological Activities.

[0265] National Research Council (U. S.) (Ed.). (1994). Nutrient requirements of poultry (9th rev. ed). National Academy Press.

[0266] National Research Council (U. S.). (2016). National Research Council: Nutrient requirements for Cats and Dogs.

[0267] https: / / scholar.google.coni / scholar_lookup?title=Nutrient+Requirements-i-of-i-Cats-i-and-i-Dogs&p ublication_year=2006&

[0268] Pacheco, L. G., Sakomura, N. K., Suzuki, R. M., Dorigam, J. C. P., Viana, G. S., Van Milgen, J., & Denadai, J. C. (2018). Methionine to cystine ratio in the total sulfur amino acid requirements and sulfur amino acid metabolism using labelled amino acid approach for broilers. BMC Veterinary Research, 14(1), 364. https: / / doi.org / 10.1186 / s12917-018-1677-8 Pajarillo, E. A. B., Lee, E., & Kang, D.-K. (2021). Trace metals and animal health: Interplay of the gut microbiota with iron, manganese, zinc, and copper. Animal Nutrition, 7(3), 750-761. https: / / doi.org / 10.1016 / j.aninu.2021.03.005

[0269] Panda, S., Panda, N., Panigrahy, K. K., Gupta, S. K., Mishra, S. P., & Laishram, M. (2017). Role of niacin supplementation in dairy cattle: A review. Asian Journal of Dairy and Food Research, 36(02). https: / / doi.org / 10.18805 / ajdfr.v36i02.7949Attorney Docket No. 11214-012WO1 Parajo, J. C., Santos, V., & Vazquez, M. (1998). Optimization of carotenoid production by Phaffia rhodozyma cells grown on xylose. Process Biochemistry, 33(2), 181-187. https: / / doi.org / 10.1016 / S0032-9592(97)00045-9

[0270] Parra, M., Stahl, S., & Hellmann, H. (2018). Vitamin B6 and Its Role in Cell Metabolism and Physiology. Cells, 7(7), 84. https: / / doi.org / 10.3390 / cells7070084

[0271] Preston, R. 1., & John, L. R. (1985). Potassium in Animal Nutrition. In Potassium in Agriculture (pp. 595-617). John Wiley & Sons, Ltd. https: / / doi.org / 10.2134 / 1985.potassium.c26 Rapoport, A., Guzhova, I., Bemetti, L., Buzzini, P., Kieliszek, M., & Kot, A. M. (2021). Carotenoids and Some Other Pigments from Fungi and Yeasts. Metabolites, 11(2). https: / / doi.org / doi.org / 10.3390 / metaboll020092

[0272] Rasby, R. J., Berger, A. L., Bauer, D. E., & Brink, D. R. (2011). Minerals and Vitamins for Beef Cows.

[0273] Relative Bioavailability of Trace Minerals in Production Animal Nutrition: A Review — PMC. (n.d.). Retrieved April 4, 2023, from https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC9367456 /

[0274] Riveron-Negrete, L., & Fernandez-Mejia, C. (2017). Pharmacological Effects of Biotin in Animals. Mini Reviews in Medicinal Chemistry, 17(6), 529-540. https: / / doi.org / 10.2174 / 1389557516666160923132611

[0275] Rodriguez-Amaya, D. B. (2019). Update on natural food pigments — A mini-review on carotenoids, anthocyanins, and betalains. Food Research International, 124, 200-205. https: / / doi.org / 10.1016 / j.foodres.2018.05.028

[0276] Scientific Opinion on the safety and efficacy of folic acid as a feed additive for all animal species I EFSA. (2012, May 16). https: / / www.efsa.europa.eu / en / efsajournal / pub / 2674 Shahbandeh, M. (2024, May 28). Sugar beet production value in the U. S. 2022. Statista. Tang, W., Wang, Y., Zhang, J., Cai, Y., & He, Z. (2019). Biosynthetic Pathway of Carotenoids in Rhodotorula and Strategies for Enhanced Their Production. 29(4), 507-517. https: / / doi.org / 10.4014 / jmb.1801.01022

[0277] Teetor, V. H., Duclos, D. V., Wittenberg, E. T., Young, K. M., Chawhuaymak, J., Riley, M. R., & Ray, D. T. (2011). Effects of planting date on sugar and ethanol yield of sweet sorghum grown in Arizona. Industrial Crops and Products, 34(2), 1293-1300. https: / / doi. Org / 10.1016 / j.indcrop.2010.09.010

[0278] Venil, C. K., Zakaria, Z. A., & Ahmad, W. A. (2013). Bacterial pigments and their applications. Process Biochemistry, 48(7), 1065-1079.

[0279] https: / / doi.org / 10.1016 / j.procbio.2013.06.006Attorney Docket No. 11214-012WO1 Weiss, B. (2019). Effect of Supplemental Biotin on Performance of Lactating Dairy Cows -DAIReXNET. https: / / dairy-cattle.extension.org / effect-of-supplemental-biotin-on-performance-of-lactating-dairy-cows /

[0280] What is Value-added Agriculture? (2023). Agricultural Marketing Resource Center. https: / / www.agmrc.org / business-development / valueadded-agriculture / what-is-value-added-agriculture

[0281] Woodworth, J., Goodband, R., Nelssen, J., Tokach, M., & Musser, R. (2000). Added dietary pyridoxine, but not thiamin, improves weanling pig growth performance. Journal of Animal Science, 78, 88-93. https: / / doi.org / 10.2527 / 2000.78188x

[0282] Zempleni, J., Wijeratne, S. S. K., & Hassan, Y. I. (2009). Biotin. BioFactors (Oxford, England), 35(1), 36-46. https: / / doi.org / 10.1002 / biof.8

[0283] Zigo, M., Kerns, K., Sen, S., Essien, C., Oko, R., Xu, D., & Sutovsky, P. (2022). Zinc is a master-regulator of sperm function associated with binding, motility, and metabolic modulation during porcine sperm capacitation. Communications Biology, 5(1), Article 1. https: / / doi.org / 10.1038 / s42003-022-03485-8.

[0284] Example 2: Enhancing P-carotene Yields from Rhodotorula mucilaginosa via Bioprocess Optimization with Sugar Beet Molasses

[0285] Carotenoids are valuable pigments useful in food, pharmaceutics, and animal health. Carotenoids are responsible for the red, yellow, and orange coloration in diverse life forms such as plants, animals, algae, and fungi. Microbially produced carotenoids have great advantages such as weather-independent production, rapid microbial growth, and color variations. Sugar beet molasses is widely produced in the US Midwest region, valued at over US $2 billion.

[0286] Carotenogenic microbes include Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula rubra, and many others. The sugar industry makes up to 30% sugar beet molasses for every 1kg produced.

[0287] Materials and methods used to investigate P-carotene production in R. mucilaginosa in sugar beet molasses substrate are shown in FIG. 6. Herein the suitability of sugar beet molasses as the primary carbon source in R. mucilaginosa fermentation for P-carotene production in an optimized media is developed using the Response Surface Methodology (RSM). FIG. 7A shows an RSM-optimized experiment with predicted concentrations of 8.746 pg / g and 114pg / g of P-carotene and total carotenoids, respectively. FIGS. 7B-7C show three-dimensional contour plots illustrating the interaction of the media components on the concentrations of P-carotene and total carotenoids in R. mucilaginosa. FIG. 8 shows a bar graph showing the actual concentration of P-carotene (16.722 pg / g) and total carotenoids (137.249 pg / g). The data on the graph outlines theAttorney Docket No. 11214-012WO1 average yields from three validated samples of the optimized media. Further, herein the optimization process is validated and the productivity and scalability of the process via fermentation in bioreactors determined. FIG. 9 shows a schematic of the fermentation and quantification process. There is wide applicability to several industries with quick turnaround time, productive, and scalable. The microbial P-carotene product is natural, safe for public use, and requires no petrochemical intermediates. Multiple carotenogenic microbes are available but there is also the possibility for product yield improvement via strain development.

[0288] Example 3: Enhancing p-carotene yields from Rhodotorula yeast via bioprocess optimization with sorghum syrup

[0289] Carotenoids are responsible for the red, yellow, and orange coloration seen in diverse life forms such as plants, animals, algae, and fungi. Carotenoids are one of the most studied pigments and have excellent benefits to industries like food, pharmaceutics, and animal health. Carotenogenic microbes include Rhodotorula glutinis, Rhodotorula rubra, and Rhodotorula mucilaginosa. Microbially produced carotenoids have great advantages such as weatherindependent production, rapid microbial growth, color variations, similar health benefits, and availability of cost-effective fermentation substrates. The carotenoids market is valued at $US 2 billion and is expected to be almost $US 3 billion in 2027.

[0290] The suitability of sorghum syrup as the primary carbon source in R. glutinis fermentation for P-carotene production was determined in an optimized media developed from the use of the Response Surface Methodology. The optimization process over 3 time points - day 5, day 7, and 10, was validated. The productivity and scalability of the process via benchtop fermentation was analyzed.

[0291] After process optimization, the highest yield of P-carotene was found to be 1378 pg / g compared to a predicted value of 1003 pg / g. The optimized medium of that sample consisted of 9% sorghum syrup, 1 % yeast extract, and 0.07 - 0.4 % mineral salts.

[0292] Process optimization revealed that the yeast extract positively influences the yields of P-carotene. Sorghum syrup influences cell biomass, and the mineral salts have little to no impact on the process(es). Validation experiments yielded 476, 117, and 1153 pg / g of P-carotene at the time points (days 5, 7, and 10). Benchtop fermentation yielded 1008, 1227, and 1753 pg / g.

[0293] Example 4: Sustainable p-carotene production from sorghum syrup using Rhodotorula glutinis: bioprocess optimization and scale-up

[0294] Abstract: P-Carotene, a key provitamin A carotenoid, is widely used as an antioxidant and natural pigment. Due to animals’ inability to synthesize carotenoids, dietary sources are essential. This study utilized low-cost sorghum syrup for P-carotene production via RhodotorulaAttorney Docket No. 11214-012WO1 glutinis fermentation. Bioprocess optimization using response surface methodology was conducted in shake flasks, then scaled to 300 mL and 7 L fermentations. The optimized medium (9.18% sorghum syrup, 0.96% yeast extract, 0.07% KH2PO4, 0.13% (NH4)2SO4, 0.42% MgSO4 yielded a predicted 1003 pg / g P-carotene after 10 days. Scale-up achieved 1153 pg / g (300 mL) and 1753.33 pg / g (7 L). Nutritional analysis showed the presence of chelated minerals, vitamins, proteins, and glucosamine, enhancing biomass value. These results highlight sorghum syrup as an effective, sustainable substrate for P-carotene production with applications in food, feed, and nutraceutical sectors. Using sorghum syrup as a low-cost substrate, P-carotene production by Rhodotorula glutinis was optimized via response surface methodology and validated at 7 L scale (up to 1,753 pg / g), while profiling the nutrient-dense biomass (protein, minerals, glucosamine) for food / feed applications (Figure 25).

[0295] Introduction. Carotenoids are a diverse group of lipophilic pigments responsible for the red, orange, and yellow hues observed across plants, algae, fungi, and bacteria (Ashokkumar et al., 2023; Rodriguez-Amaya, 2019; Sandmann, 2022). To date, more than 1200 naturally occurring carotenoids have been identified and characterized (Yabuzaki, 2017), including -carotene, torulene, astaxanthin, and torularhodin (Rapoport et al., 2021). Beyond their role as pigments, carotenoids possess strong antioxidant properties, functioning by quenching reactive oxygen species, thereby providing protective health benefits (Di Mascio et al., 1991; Fiedor & Burda, 2014).

[0296] Although synthetic carotenoids are widely available, their production relies on petrochemical processes that raise environmental and health concerns, including potential carcinogenicity (Louren o-Lopes et al., 2021). Growing consumer demand for natural ingredients, driven by chemophobia and health-conscious practices, has increased interest in sustainably produced natural carotenoids (Dufosseet al., 2005).

[0297] The global carotenoids market was valued at approximately USD 2 billion in 2022, with an expected compound annual growth rate of 5.7% from 2023 to 2028, projected to reach USD 2.7 billion by 2027 (Global Carotenoids Market Report and Forecast 2023-2028, n.d.). P-Carotene, the most studied and commercially important carotenoid, alone accounted for USD 261 million in 2010 and USD 334 million in 2018, highlighting its wide applicability and growing demand.

[0298] To reduce production costs and improve sustainability, recent efforts have focused on utilizing agro-industrial byproducts as substrates for microbial carotenoid production (Aksu & Eren, 2005; Sharma & Ghoshal, 2020; Thumkasem et al., 2024). Sweet sorghum (Sorghum bicolor (L.) Moench) is an attractive feedstock option due to its wide geographic adaptability,Attorney Docket No. 11214-012WO1 short growing cycle (3-5 months), low water and fertilizer requirements, and resilience to drought and temperature fluctuations (Teetor et al., 2011). Sorghum syrup, derived from stalk juice, is rich in sugars, vitamins, iron, potassium, and magnesium, making it a cost-effective, nutrient-rich substrate for microbial cultivation (Eggleston et al., 2022).

[0299] Among microbial carotenoid producers, Rhodotorula species, particularly R. glutinis. have gained attention for their ability to synthesize carotenoids, enzymes, and microbial oils such as oleic, linoleic, palmitic, and stearic acids (Grigore et al., 2023; Kot et al., 2016). These yeasts exhibit rapid growth, utilize diverse carbon sources, and require minimal cultivation inputs compared to plant- or algae-based systems (Kim et al., 1999; Venil et al., 2013). Process optimization, including response surface methodology, has been successfully employed to enhance microbial carotenoid production (Ananda & Vadlani, 2011; Bhosale, 2004). Coupling such approaches with low-cost, agricultural substrates like sorghum syrup offers an economically viable strategy for natural P-carotene production.

[0300] The present study investigated the feasibility of using sorghum syrup as the primary carbon source for P-carotene production by R. glutinis. It was hypothesized that sorghum syrup could support both high carotenoid yields and provide additional nutritional value in the final product. The objectives were to (i) optimize P-carotene production in R. glutinis using sorghum syrup-based media, (ii) scale-up the bioprocessing to benchtop bioreactors, and (iii) evaluate the nutritional / biochemical composition of the fermented product.

[0301] Materials and Methods

[0302] Chemicals and Reagents. All chemicals and reagents used in this study were of analytical grade and purchased from Fisher Scientific. HPLC-grade acetonitrile and methanol were used for chromatographic analysis. The P-carotene standard was obtained from Millipore-Sigma.

[0303] Microorganism and Growth Conditions. R. glutinis (ATCC 32766) used in this study was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The strain was cultivated in 250 mL Erlenmeyer flasks containing 100 mL of sterile potato dextrose broth (PDB) and incubated at 25 °C with agitation at 200 rpm for 7 days. The culture was also maintained on potato dextrose agar (PDA) plates at room temperature for routine use. The broth culture was subsequently used as the inoculum for all experimental evaluations.

[0304] Process Optimization. A central composite design (CCD) was employed to optimize the key medium components for P-carotene production. Five independent variables were evaluated: sorghum syrup (1-10%, 100% pure, Muddy Pond Sorghum Mill, Monterey, TN, USA), ammonium sulfate [(NH4)2SO4, 0.01-0.1%], potassium phosphate (KH2PO4, 0.01-0.1%),Attorney Docket No. 11214-012WO1 magnesium sulfate (MgSO4, 0.01-0.1%), and yeast extract (0.1-1%, Research Products International, Mt. Prospect, IL, USA).

[0305] Design-Expert® software version 13 (Stat- Ease Inc., Minneapolis, MN, USA) was used to generate the experimental matrix, analyze the influence of the independent variables on P-carotene production and biomass yield, and develop response surface and contour plots. A design matrix with minimum (-1) and maximum (+1) is provided in Table 4. A detailed 31 experimental matrix is given in Table 5.

[0306] Table 4. Media composition for RSM with minimum (-1) and maximum (+1) for all selected ingredients.

[0307] A B C D E

[0308] Factor Name

[0309] Sorghum Syrup (NH4)2SO4KH2PO4MgSO4Yeast Extract Units % % % % %

[0310] Type Numeric Numeric Numeric Numeric Numeric Subtype Continuous Continuous Continuous Continuous Continuous Minimum 1.0000 0.0500 0.0100 0.1000 0.1000 Maximum 10.00 0.500 0.1000 0.5000 1.0000

[0311] Coded low -1 1.00 -1 ~ 0.05 -1 ~ 0.01 -1 0.10 -1 0.10 Coded high +1 < > 10.00 +1 ~ 0.05 +1 0.10 +1 < > 0.50 +1 < > 1.00 Mean 5.37 0.2900 0.569 0.2904 0.5411

[0312]

[0313] Std. Dev. 3.64 0.1783 0.356 0.1606 0.3739

[0314] Table 5. Design Matrix from the central composite design (CCD) used during process optimization of the media components for the production of P-carotene from sorghum-fermented R. glutinis.

[0315] Run Sorghum Syrup (NH4)2SO4KH2PO4MgSO4Yeast Extract # (g) (mL) (mL) (mL) (g)

[0316] 1 4.83 0.15 0.01 0.26 1.00

[0317] 2 6.72 0.05 0.10 0.43 0.28

[0318] 3 10.00 0.50 0.01 0.10 1.00

[0319] 4 1.00 0.30 0.10 0.38 0.56

[0320] 5 2.58 0.50 0.06 0.41 0.10

[0321] 6 10.00 0.27 0.06 0.26 0.10

[0322] 7 1.00 0.05 0.04 0.27 0.46

[0323] 8 3.39 0.22 0.04 0.50 0.73

[0324] 9 5.19 0.23 0.05 0.10 0.62

[0325] 10 1.00 0.50 0.01 0.50 0.87

[0326] 11 1.00 0.30 0.10 0.38 0.56

[0327] 12 1.00 0.50 0.09 0.10 1.00

[0328] 13 2.08 0.50 0.04 0.24 0.63

[0329] 14 7.93 0.50 0.01 0.36 0.37

[0330] 15 10.00 0.13 0.03 0.50 0.74

[0331]

[0332] 16 1.00 0.05 0.10 0.10 0.10Attorney Docket No. 11214-012WO1 17 1.00 0.05 0.09 0.50 1.00

[0333] 18 7.17 0.43 0.08 0.42 1.00

[0334] 19 1.00 0.42 0.01 0.10 0.10

[0335] 20 7.17 0.43 0.08 0.42 1.00

[0336] 21 5.19 0.23 0.05 0.10 0.62

[0337] 22 10.00 0.05 0.01 0.10 0.10

[0338] 23 10.00 0.05 0.10 0.15 1.00

[0339] 24 10.00 0.50 0.03 0.10 0.10

[0340] 25 1.65 0.40 0.08 0.12 0.10

[0341] 26 7.93 0.50 0.10 0.10 0.33

[0342] 27 10.00 0.27 0.06 0.26 0.10

[0343] 28 4.83 0.15 0.01 0.26 1.00

[0344] 29 10.00 0.50 0.10 0.50 0.10

[0345] 30 8.49 0.05 0.10 0.48 1.00

[0346]

[0347] 31 3.25 0.21 0.01 0.50 0.10

[0348] The CCD was conducted to determine the optimal conditions for 0-carotene production at the shake flask level, assessing both the individual and interactive effects of the media components on yeast growth and pigment synthesis. Experiments were performed in 250 mL Erlenmeyer flasks containing 100 mL of working volume. The flasks were sterilized at 120 °C for 30 minutes and cooled to room temperature before inoculation with 1 mL of an actively growing R. glutinis culture. Flasks were incubated at 25 °C with shaking at 200 rpm for 10 days.

[0349] At the end of fermentation cultures were centrifuged, the supernatant discarded, and the cell pellets freeze-dried for -carotene quantification. Freeze drying was carried out at -80°C at pressure 2.6 x 104psi using Labconco benchtop freeze-dryer. The relationship between independent variables and responses was modeled using a second-order (quadratic) polynomial equation (Equation 1).

[0350] y = 2xi2- 3X22+ 4X32+ X42- 5x52+ 3xiX2 — 2x2x3 + X3X4

[0351] (1) + 2x4x5 + 5xi - X2 + 3x3 + 4x4 - 2x5 + 7

[0352] where y is the dependent variable and xi = sorghum syrup, X2 = (NFL^SC, X3 = KH2PO4, X4 = MgSCU and xs = Yeast extract, x2i represent the squared influence of each variable, XiXj capture how pairs of variables interact, Xi represent direct, proportional effects, 7 represents the constant term and is the baseline value of y when all Xi = 0

[0353] Carotenoid Extraction and Quantification. Carotenoids were extracted from approximately 0.2 g of freeze-dried R. glutinis cells using 10 mL of tetrahydrofuran. The extraction was performed by grinding the cells with acid-washed sand in a mortar and pestle to facilitate cell disruption and pigment release. The extracts were filtered through 0.2 pm syringe filters into HPLC vials for analysis (Ananda and Vadlani, 2011).Attorney Docket No. 11214-012WO1 Shimadzu High-performance liquid chromatography (HPLC) equipped with LC-40D HPLC Pump, DGU-405 5-channel Degasser, SPD-M40 Photodiode Array Detector, SIL-40C auto-sampler and CTO-40C Column Oven, CBM-40 System Controller and LabSolutions software was used for analysis. A method described in Ananda and Vadlani (2011) was used with slight modification. Carotenoid separation was achieved using a Phenomenex Luna C8 column (150 mm x 4.6 mm), with a mobile phase comprising acetonitrile and methanol (90:10, v / v) at a flow rate of 1.5 mL / min. Detection was carried out at 450 nm, and P-carotene concentrations were quantified using a calibration curve prepared with a pure P-carotene standard (Millipore-Sigma, St. Louis, MO, USA). A sample chromatogram extracted at 455 nm is given in Figure 19.

[0354] Validation of Optimized Medium in Media Bottles. The optimal medium composition determined through the central composite design (CCD) was used to prepare 2 liters of fermentation medium. Aliquots of 300 mL were dispensed into sterile one liter glass media bottles in duplicates and inoculated with 1% (v / v) of actively growing R. glutinis culture. All bottles were incubated on an orbital shaker at 25 °C and 200 rpm for 10 days. Samples were collected from each bottle on days 5, 7, and 10, and P-carotene content was quantified using IIPLC as described previously.

[0355] Validation in Benchtop Bioreactors. A batch-mode fermentation was conducted using three 7 L bench-top bioreactors (BioFlo® 115, Eppendorf AG, Germany), each operated in triplicate. The working volume for each bioreactor was 5 L, prepared with the optimized medium composition comprising 459 g sorghum syrup, 6.5 g (NH4)2SO4, 3.5 g KH2PO4, 21 g MgSCU, and 48 g yeast extract. Following sterilization, the bioreactors were inoculated with 1% (v / v) of a 48-hour-old R. glutinis culture (Figure 20).

[0356] Fermentation was carried out under controlled bat ch- mode conditions: temperature at 25 °C, agitation at 200 rpm, and aeration maintained at 1 vvm. All bioreactors were integrated with the BioCommand® supervisory control and data acquisition (SCAD A) system, which collected real-time data from each unit at 10- minute intervals over the 10-day fermentation period.

[0357] Samples were drawn on day 5, day 7 and harvest day of 10. Samples were centrifuged and yeast cell pellets were freeze dried. Freeze dried samples were used for P-carotene estimation. The day 10 freeze dried samples were also used for nutritional characterization. The fermentation profiles, including pH, dissolved oxygen (DO), temperature, and agitation speed, were continuously monitored and recorded.

[0358] Nutritional Characterization of the Fermented Product. At the end of the 10-day fermentation, the culture broth was harvested by centrifugation, and the resulting biomass wasAttorney Docket No. 11214-012WO1 stored at -80 °C. All biomass samples from the triplicate bioreactors were freeze-dried and analyzed for nutritional composition, including vitamins (riboflavin, niacin, pantothenic acid, and pyridoxine), total amino acids, total fatty acids, crude fat, crude protein, crude fiber, glucosamine, and minerals. B vitamins were quantified using the LC-MS / MS method described by the (Vinas et al 2003), while vitamin D and vitamin E were analyzed using AO AC Methods 2016.05 and the CRNFS (2016, Vol. 4, p. 92) method, respectively. Crude protein and crude fat were determined by AOAC Methods 990.03 and 2003.05, and total amino acids were measured using AOAC Methods 994.12 and 988.15. Total lipids and fatty acids were analyzed following AOAC Method 996.06, glucosamine was assessed using the (Pastorini et al. 2011), and mineral content was determined using AOAC Method 985.01.

[0359] Statistical Analysis. The statistical analysis was conducted using Statistical Analysis Software (SAS, version 9.4, Cary, NC). PROC ANOVA was used to compare the treatments and Tukey pairwise differences was used. For correlation, PROC CORR was used. Significance was set at P = 0.05.

[0360] Results and Discussion

[0361] Optimizing Culture Medium for P-carotene Production. Bioprocess optimization using a central composite design (CCD) with 31 experimental combinations of sorghum syrup-based culture media revealed the optimal conditions for P-carotene production by R. glutinis. The P-carotene yields and percent dry cell weight for all experimental runs were summarized in Table 7. -Carotene production ranged from 158 pg / g to 1 378 pg / g of dry cell weight, while dry cell weight ranged from 0.3% to 2.22%. The contour plots in Figure 16a-Figure 16c illustrated the interaction between sorghum syrup and yeast extract concentrations and their influence on overall desirability keeping mineral concentrations at 0.07% KH2PO4, 0.13% (NFU SCh, and 0.42% MgSCh, (Figure 16a), dry cell weight (Figure 16b), and P-carotene production (Figure 16c). The results of the Analysis of Variance (ANOVA) for the response surface model are presented in Table 6. The model was statistically significant (p < 0.0002), and the lack-of-fit was non-significant (F = 1.44, p = 0.366), indicating that the model adequately described the experimental data. The optimization results showed that achieving both high cell biomass and P-carotene production required a balance between carbon and nitrogen sources. Although a desirability of 0.68 was achieved for dry cell weight (1.78% dry weight), the maximum predicted P-carotene yield of 1003.38 pg / g of dried yeast cells was not fully attained under those conditions, indicating a trade-off between biomass accumulation and pigment production (Figure 17a).Attorney Docket No. 11214-012WO1 The data further demonstrated that an increase in sorghum syrup concentration positively influenced cell biomass production, while an increase in yeast extract concentration enhanced flcarotene yields (Figure 17b). These trends were in contrast with earlier findings, where nitrogen limitation stimulated carotenoid biosynthesis, while adequate nitrogen levels promoted cell growth (Anzola-Rojas et al., 2015: Mata-Gomez et al., 2014; Singh et al., 2020). Sharma and Ghoshal (2020) also observed improved biomass and carotenoid production in Rhodotorula mucilaginosa when agricultural waste hydrolysates with higher sugar concentrations were utilized. It was well established that the carbon-to-nitrogen (C / N) ratio regulated carotenoid biosynthesis in oleaginous yeasts such as Rhodotorula spp. Ratios near 20: 1 favored carotenoid accumulation by channeling acetyl-CoA toward the mevalonate pathway, while higher C / N ratios ( > 50: 1) promoted fatty acid synthesis as a cellular response to excess carbon avail ability (Paul, Bohacz, et al., 2023; Tkacova et al., 2022). In this study, the optimized medium contained approximately 9.18% sorghum syrup (carbon source) and 0.96% yeast extract (nitrogen source), corresponding to an estimated C / N ratio of ~20:l to 25:1, which supported carotenoid production. Detailed concentrations of the optimized media components and their corresponding desirability values were provided in Table 8. The correlation between the predicted and experimental values for P-carotene yield and dry cell weight was illustrated in Figure 21. The regression model demonstrated a strong agreement between predicted and experimental dry cell weight values, while -carotene yields exhibited higher variability, likely due to the sensitivity of carotenoid biosynthesis to subtle changes in media composition. The final regression models representing the combined effects of the independent variables on P-carotcnc yield and dry cell weight were provided in Equations 2 and 3, respectively.

[0362] Carotenoid = +182.25203 - 3.32468SorghumSyrup +

[0363]

[0364] 102.7I735(AW4)2SO4 + 674.8389673O4+ 257.89752MgSO4+ 701.143367^

[0365] Cell Dry wt. = +0.201416 + 0. 435641 SorghumSyrup - Y32362(NH4)2SO4- 0.5158T9KH2PO4- 0.934599MgSO4+

[0366] 0.068443 YeastExtract - 0.136133SorghumSyrup*(NH4)2SO4- Q.269\35SorghumSyrup*KH2PO - (}X}11514SorghumSyrup*MgSO4- ()X)94919SorghumSyrup*YeastExtract - 1 A22 3(NH4) SO4*KEI2PO4 +

[0367] (3) 0.4645Q2(NH4)2SO4*MgSO4 + Q.506l29(NH4)2SO4^YeastExtract + V9.22349KH2PO4*MgSO4- l. S3830KH2PO4*YeastExtract - 0.30821 1 > / *YeastExtract - 0.019160SorghumSyrup2+ 2X)0214((NH4)2SO4)2- 23.5^4(KH2PO4)2+ 0) 442916(MgSO4)2+

[0368] 0

[0369]

[0370] .3 \6454YeastExtract2Attorney Docket No. 11214-012WO1 Table 6. ANOVA for response surface method (RSM) model for variables in the study Source Sum of squares df Mean square F-value P-value

[0371] Model 2.26E+06 5 4.53E+05 7.54 0.0002 Significant A-sorghum syrup 4357.14 1 4357.14 0.0726 0.7898

[0372] B-(NH4)2SO4 9816.29 1 9816.29 0.1635 0.6894

[0373] C-KH2PO4 17050.31 1 17050.31 0.284 0.5988

[0374] D-MgSO448711.35 1 48711.35 0.8115 0.3763

[0375] E- yeast extract 1.96E+06 1 1.96E+06 32.64 <0.0001

[0376] Residual 1.50E+06 25 60029.94

[0377] Lack of Fit 1.28E+06 20 63927.48 1.44 0.3668 Not significant Pure Error 2.22E+05 5 44439.78

[0378]

[0379] Cor total 3.77E+06 30

[0380] Table 7. P-carotene concentrations obtained from sorghum syrup fermented R. glutinis 32766 via RSM.

[0381] Run # P-carotene cone (pg / g) Cell dry wt. (%)

[0382] 1 628.43 1.2

[0383] 2 184.09 1.84

[0384] 3 1045.77 1.18

[0385] 4 658.48 0.42

[0386] 5 503.51 0.68

[0387] 6 471.26 1.56

[0388] 7 868.34 0.4

[0389] 8 1041.49 0.92

[0390] 9 570.95 1.12

[0391] 10 1022.71 0.36

[0392] 11 793.47 0.44

[0393] 12 1053.39 0.5

[0394] 13 1101.09 0.76

[0395] 14 803.96 1.1

[0396] 15 1183.43 1.16

[0397] 16 563.64 0.28

[0398] 17 1031.84 0.62

[0399] 18 536.58 1.2

[0400] 19 172.04 0.3

[0401] 20 1106.12 1.1

[0402] 21 519.50 1.2

[0403] 22 169.43 2.22

[0404] 23 1145.05 1.34

[0405] 24 355.17 1.7

[0406] 25 276.14 0.44

[0407] 26 726.96 0.96

[0408] 27 158.40 1.4

[0409] 28 592.75 1.28

[0410] 29 239.55 1.36

[0411] 30 1378.12 1.16

[0412]

[0413] 31 392.74 0.66Attorney Docket No. 11214-012WO1 Table 8. RSM optimal point prediction for beta-carotene production

[0414] Numbe Sorghu (NH4)2S KH2P MgS Yeast Caroteno Desirabili r in 04 O404 Extra id ty

[0415] Syrup ct

[0416] 1 9.188 0.140 0.071 0.429 0.968 1003.379 1.000 Selecte d 2 3.385 0.216 0.037 0.500 0.730 858.989 1.000

[0417] 3 7.930 0.500 0.100 0.102 0.330 532.062 1.000

[0418] 4 10.000 0.050 0.010 0.100 0.100 256.794 1.000

[0419] 5 1.000 0.303 0.100 0.384 0.564 771.643 1.000

[0420] 6 1.000 0.421 0.010 0.100 0.100 324.850 1.000

[0421] 7 7.930 0.500 0.010 0.356 0.365 562.074 1.000

[0422] 8 1.000 0.500 0.089 0.100 1.000 1017.111 1.000

[0423] 9 2.575 0.500 0.063 0.410 0.100 443.484 1.000

[0424] 10 2.080 0.500 0.039 0.236 0.626 753.057 1.000

[0425] 11 10.000 0.271 0.064 0.256 0.100 355.812 1.000

[0426] 12 1.000 0.050 0.036 0.270 0.465 603.738 1.000

[0427] 13 4.825 0.151 0.010 0.264 1.000 957.723 1.000

[0428] 14 1.647 0.397 0.082 0.116 0.100 372.695 1.000

[0429] 15 1.000 0.500 0.010 0.500 0.865 972.776 1.000

[0430] 16 1.000 0.050 0.100 0.100 0.100 347.451 1.000

[0431] 17 3.250 0.207 0.010 0.500 0.100 398.572 1.000

[0432] 18 5.185 0.230 0.054 0.100 0.622 686.745 1.000

[0433] 19 1.000 0.050 0.085 0.500 1.000 1071.618 1.000

[0434] 20 10.000 0.500 0.015 0.100 1.000 937.082 1.000

[0435] 21 7.165 0.432 0.080 0.422 1.000 1066.981 1.000

[0436] 22 10.000 0.500 0.100 0.500 0.100 466.911 1.000

[0437] 23 10.000 0.050 0.100 0.148 1.000 960.937 1.000

[0438] 24 10.000 0.498 0.025 0.100 0.100 313.110 1.000

[0439] 25 10.000 0.127 0.033 0.500 0.744 834.484 1.000

[0440] 26 6.715 0.050 0.096 0.432 0.280 537.242 1.000

[0441] 27 4.925 0.271 0.093 0.134 0.777 835.807 1.000

[0442] 28 2.445 0.443 0.069 0.304 0.778 890.071 1.000

[0443] 29 3.220 0.223 0.044 0.248 0.731 800.524 1.000

[0444] 30 2.217 0.149 0.045 0.267 0.169 407.729 1.000

[0445] 31 1.103 0.056 0.062 0.394 0.627 767.563 1.000

[0446] 32 4.110 0.271 0.098 0.310 0.300 553.118 1.000

[0447] 33 9.784 0.055 0.098 0.473 0.774 886.094 1.000

[0448] 34 9.551 0.436 0.079 0.445 0.373 625.330 1.000

[0449] 35 3.472 0.469 0.095 0.369 0.183 506.379 1.000

[0450] 36 1.132 0.159 0.056 0.140 0.288 470.998 1.000

[0451] 37 5.518 0.248 0.041 0.456 0.683 813.042 1.000

[0452] 38 8.504 0.190 0.026 0.349 0.601 702.381 1.000

[0453] 39 6.842 0.082 0.016 0.435 0.572 692.029 1.000

[0454] 40 6.485 0.104 0.010 0.160 0.772 761.185 1.000

[0455] 41 7.304 0.113 0.041 0.474 0.545 701.256 1.000

[0456] 42 5.908 0.315 0.029 0.459 0.147 436.090 1.000

[0457] 43 8.478 0.100 0.024 0.126 0.660 676.380 1.000

[0458]

[0459] 44 5.805 0.200 0.095 0.481 0.521 736.639 1.000Attorney Docket No. 11214-012WO1 7.123 0.133 0.039 0.452 0.447 628.563 1.000

[0460] 4.761 0.150 0.041 0.268 0.600 699.963 1.000

[0461] 7.811 0.139 0.057 0.130 0.479 578.596 1.000

[0462] 8.551 0.071 0.033 0.445 0.797 856.816 1.000

[0463] 2.872 0.180 0.027 0.145 0.199 386.712 1.000

[0464] 1.274 0.133 0.078 0.120 0.601 696.707 1.000

[0465] 2.623 0.177 0.069 0.284 0.981 999.690 1.000

[0466] 5.516 0.310 0.086 0.249 0.870 928.667 1.000

[0467] 9.622 0.079 0.019 0.446 0.213 435.766 1.000

[0468] 2.464 0.262 0.081 0.213 0.475 644.097 1.000

[0469] 9.404 0.354 0.060 0.310 0.156 416.746 1.000

[0470] 6.016 0.389 0.029 0.258 0.595 705.406 1.000

[0471] 1.826 0.490 0.071 0.453 0.703 884.412 1.000

[0472] 6.768 0.417 0.028 0.428 0.665 798.102 1.000

[0473] 2.347 0.052 0.077 0.195 0.558 672.923 1.000

[0474] 2.411 0.460 0.032 0.447 0.378 623.451 1.000

[0475] 7.317 0.470 0.080 0.108 0.227 446.764 1.000

[0476] 7.260 0.426 0.051 0.241 0.182 425.965 1.000

[0477] 2.761 0.250 0.040 0.471 0.329 577.526 1.000

[0478] 2.696 0.058 0.032 0.204 0.235 418.032 1.000

[0479] 3.795 0.249 0.086 0.130 0.373 548.409 1.000

[0480] 7.624 0.226 0.021 0.468 0.674 787.347 1.000

[0481] 2.613 0.306 0.081 0.325 0.838 931.519 1.000

[0482] 7.272 0.096 0.063 0.480 0.565 730.016 1.000

[0483] 7.646 0.461 0.054 0.174 0.499 635.019 1.000

[0484] 9.259 0.381 0.083 0.200 0.187 429.139 1.000

[0485] 8.907 0.492 0.010 0.243 0.471 603.288 1.000

[0486] 3.453 0.155 0.029 0.211 0.530 632.224 1.000

[0487] 3.726 0.120 0.095 0.110 0.513 633.901 1.000

[0488] 1.677 0.257 0.052 0.473 0.949 1025.466 1.000

[0489] 2.923 0.392 0.044 0.372 0.899 968.653 1.000

[0490] 9.064 0.352 0.021 0.295 0.701 769.996 1.000

[0491] 3.545 0.459 0.091 0.185 0.580 733.101 1.000

[0492] 6.105 0.290 0.073 0.484 0.390 638.932 1.000

[0493] 7.434 0.283 0.079 0.334 0.510 683.375 1.000

[0494] 9.915 0.274 0.058 0.282 0.455 608.065 1.000

[0495] 2.264 0.223 0.027 0.104 0.558 633.984 1.000

[0496] 4.502 0.200 0.045 0.201 0.954 938.807 1.000

[0497] 3.953 0.133 0.084 0.154 0.423 576.239 1.000

[0498] 6.532 0.469 0.049 0.312 0.650 778.300 1.000

[0499] 4.196 0.428 0.062 0.287 0.767 865.646 1.000

[0500] 4.462 0.391 0.014 0.375 0.914 954.503 1.000

[0501] 8.724 0.255 0.043 0.328 0.262 476.977 1.000

[0502] 2.824 0.499 0.019 0.293 0.464 637.548 1.000

[0503] 9.116 0.259 0.068 0.184 0.319 495.559 1.000

[0504] 3.741 0.347 0.090 0.340 0.875 967.228 1.000

[0505] 8.133 0.216 0.037 0.239 0.681 741.058 1.000

[0506] 3.721 0.425 0.026 0.315 0.906 947.392 1.000

[0507]

[0508] 9.408 0.427 0.085 0.342 0.415 631.391 1.000Attorney Docket No. 11214-012WO1 94 1.361 0.486 0.017 0.459 0.681 834.859 1.000

[0509] 95 3.900 0.278 0.059 0.107 0.242 434.826 1.000

[0510] 96 2.712 0.148 0.026 0.307 0.196 422.285 1.000

[0511] 97 5.348 0.253 0.085 0.245 0.160 423.374 1.000

[0512] 98 8.051 0.243 0.093 0.406 0.173 469.731 1.000

[0513] 99 7.305 0.426 0.073 0.369 0.211 494.574 1.000

[0514]

[0515] 100 9.003 0.430 0.056 0.170 0.446 591.079 1.000

[0516] Despite the low concentrations of minerals present in the fermentation medium, their specific levels appeared to influence P-carotene production. While the precise role of inorganic salts in carotenogenesis remains not fully understood, previous studies have demonstrated that the addition of minerals to the culture medium can enhance carotenoid yields (Han et al., 2002). It is plausible that minerals play a critical role in regulating enzyme activities involved in carotenogenic pathways.

[0517] In this study, mineral salts were observed to have a positive influence on P-carotene production but a negative effect on cell biomass (Figure 17b). Similar trends have been reported in the literature. For example, Chen et al. (2006) demonstrated enhanced carotenoid production in Rhodotorula sphaeroides cultured in media supplemented with MgSCF, Na2HPC>4, FeSCL, and Na2CO<. reported that KH2PO4 and MgSC were essential for P-carotene synthesis in Blakeslea trispora. Likewise and Parajo et al. (1998) showed that Phaffia rhodozyma produced astaxanthin under nitrogen-limited conditions when KNO3 and (NH4)2SO4 were used as nitrogen sources which stimulated P-carotene production.

[0518] In alignment with these findings, the present study incorporated (NH4)2SO4 as the primary inorganic nitrogen source, supplemented by organic nitrogen sources from yeast extract and sorghum syrup. The optimization experiments revealed that higher yeast extract concentrations significantly enhanced P-carotene production, while higher concentrations of sorghum syrup primarily increased cell biomass (dry cell weight) of R. glutinis. It is possible that sorghum syrup, being rich in naturally occurring minerals, reduced the requirement for additional inorganic mineral supplementation during fermentation, particularly for carotenoid biosynthesis.

[0519] Batch-mode Validation in Media Bottles and Benchtop Bioreactors. Validation experiments using media bottles further confirmed the influence of fermentation duration on P-carotene yields (Figure 18). P-Carotene concentrations on days 5, 7, and 10 were 476, 1173, and 1153 pg / g of dry cell weight, respectively. Interestingly, the day 10 yield of 1153 pg / g slightly exceeded the predicted yield of 1003.38 pg / g, confirming the accuracy of the response surface model. However, the highest yield was recorded on day 7, surpassing both the predicted andAttorney Docket No. 11214-012WO1 final day 10 yields. There was no statistically significant difference among days with respect to P- carotene production is concern, but highest was noticed on day 10.

[0520] These media bottle -carotene production profile followed a trend consistent with previous studies by Sharma and Ghoshal (2020) and Ochoa- Vinals et al. (2024), where carotenoid production peaked at intermediate fermentation times, followed by a slight decline, possibly due to product degradation or metabolic shifts during extended cultivation.

[0521] The overall fermentation process trends for the three bioreactor replicates over the 10-day period were consistent, as illustrated in the SCADA profiles (Figure 22 - Figure 24). As fermentation progressed, P-carotene concentrations steadily increased (Figure 18). Notably, the dissolved oxygen (DO) levels in all fermenters decreased to below 15% within the first 24 hours, indicating active growth of R. glutinis during the early stages of fermentation (Figure 22 -Figure 24 ). The pH of the fermentation medium initially measured 5.5 across all bioreactors and decreased to approximately 4.0 within 24 hours, suggesting rapid utilization of carbohydrates present in the medium. This initial pH drop corresponded with the observed decline in DO levels, further supporting the indication of vigorous microbial activity. The pH remained stable in the range of 4.5 to 5.0 for the remainder of the 10-day fermentation period. Batch-mode fermentation of R. glutinis, conducted in triplicate bioreactors, resulted in average P-carotene concentrations of 1008 pg / g, 1227 pg / g, and 1753 pg / g of dry cell weight on days 5, 7, and 10, respectively (Figure 18). P-carotene production overall is significantly higher in fermenter than bottle (P = 0.0051). Considering the cost of fermentation, it is important to reduce the duration and based on the benchtop bioreactor as well as media bottle validation statistically ideal to stop the fermentation on day 7.

[0522] Nutritional Characterization of Freeze-Dried R. Glutinis Fermented Biomass. In addition to P-carotene enrichment, R. glutinis fermented sorghum syrup was evaluated for proximate composition and mineral content. The fermented product contained P-carotene as a source of provitamin A, along with crude proteins, crude fats, amino acids, fatty acids, and essential minerals (Table 9), adding to the overall value proposition of utilizing R. glutinis fermentation in a sorghum syrup-based medium.

[0523] The protein content of the freeze-dried yeast biomass was approximately 17%, indicating its potential as a protein source for food and feed applications. Interestingly, lipid levels were relatively low, measured at 0.53%, despite R. glutinis being widely reported as a high lipid-accumulating yeast (Maza et al., 2020). Additionally, no significant levels of fatty acids or free amino acids were detected in the fermented samples, which may be attributed to the specificAttorney Docket No. 11214-012WO1 fermentation conditions optimized for carotenoid production rather than lipid or protein accumulation.

[0524] Table 9. Nutritional analyses profile of R. glutinis (ATCC 32766) fermented in a sorghum syrup-based media.

[0525] Nutritional Profile ( Units are next to the nutrients) Range of nutrient (with Std Frr) Fatty acids

[0526] Palmitic (Cl 6:0) g / lOOg 0.07+0.03

[0527] Stearic (C18:0) g / 100g 0.02±0.01

[0528] Oleic (Cl 8:1, Cis) g / lOOg 0.34+0.14

[0529] Linoleic (C18:2, Trans) g / lOOg O. O5±O. O3

[0530] alpha-Linoleic (C18:3, alpha) g / lOOg 0.01+0.1

[0531] Lignoceric (C24:0) g / lOOg 0.01+0.01

[0532] Saturated fat total g / 100g 0.11+0.05

[0533] Polyunsaturated fat (total) g / 100g 0.06+0.04

[0534] Monounsaturated fats (total) g / 100g 0.35±0.14

[0535] Omega 3 fatty acids (total) g / 100g 0.01+0.01

[0536] Omega 6 fatty acids (total) g / 100g O. O5±O. O3

[0537] Omega 9 fatty acids (total) g / 100g 0.34+0.14

[0538] Glucosamine (HCL total) mg / kg 3073±900

[0539] Other biochemicals

[0540] Crude protein % 17.2±1.5

[0541] Crude fats % 0.53+0.23

[0542] Cysteine % 0.13±0.02

[0543] Methionine % 0.2+0.02

[0544] Tryptophan % 0.15+0.02

[0545] Minerals

[0546] Sulphur (total) % 1.67+0.14

[0547] Phosphorus (total) % 0.55±0.04

[0548] Potassium (total) % 1.39+0.13

[0549] Magnesium (total) % 1.02±0.07

[0550] Calcium (total) % 0.16+0.01

[0551] Sodium (total) % 0.28±0.02

[0552] Iron (total) ppm 54+18.24

[0553] Manganese (total) ppm 8.83±0.85

[0554] Copper (total) ppm 12.2+2.3

[0555] Zinc (total) ppm 22.83±2.89

[0556] Sulphur (total) % 1.67+014

[0557] Phosphorus (total) % 0.55+0.04

[0558]

[0559] Potassium (total) % 1.39+0.13

[0560] Glucosamine. The fermentation process also resulted in the production of glucosamine, measured at 3073 mg / kg (0.3%) in the R. glutinis fermented biomass (Table 9). Glucosamine is a critical structural component of fungal cell walls and is widely recognized as a beneficial nutrient for supporting joint health and bone integrity in both humans and animals (Neil et al.,Attorney Docket No. 11214-012WO1 2005). Comparatively, previous studies reported glucosamine levels ranging from 1.91% to 2.4% during Monascus purpureus fermentation of corn ethanol coproduct wetcake (Nanjundaswamy and Okeke 2014). Although the glucosamine levels observed here are lower, they demonstrate the potential of R. glutinis fermentation to co-produce this value-added compound alongside P-carotene.

[0561] Mineral Composition. Sorghum syrup is inherently rich in minerals such as magnesium, potassium, calcium, phosphorus, zinc, and other trace elements (Eggleston et al., 2022). Minerals play critical roles in both human and animal health, contributing to skeletal structure, enzymatic functions, neuromuscular regulation, oxygen transport, and metabolic processes (Health, 1989; Sampath et al., 2023).

[0562] In animal nutrition, mineral supplementation is often necessary due to variations in mineral bioavailability from natural feed sources. Microbial fermentation has been shown to improve mineral bioavailability by converting inorganic minerals into organically chelated forms, which are more stable and better absorbed in the gastrointestinal tract (Chen et al., 2022).

[0563] Mineral analysis of R. glutinis fermented sorghum syrup confirmed the presence of both macro- and trace minerals (Table 9). Notably, potassium (1.39 ± 0.13%), magnesium (1.02 ± 0.07%), and sodium (0.28 ± 0.02%) were present at appreciable levels. The biomass also contained essential trace minerals including iron (54.00 ± 18.24 mg / kg), copper (12.20 ± 2.30 mg / kg), and zinc (22.83 ± 2.89 mg / kg). These findings suggest that R. glutinis fermentation not only enhances β-carotene production but may also serve as a platform for generating nutrient-dense, mineral-enriched biomass with potential applications in human and animal nutrition.

[0564] Conclusions. This study demonstrated that sorghum syrup, an agricultural co-product derived from sweet sorghum processing, is a viable and cost-effective substrate for R. glutinis fermentation to produce P-carotene. Process optimization using response surface methodology (RSM) identified the optimal medium composition (9.18% sorghum syrup, 0.13% (NH₄)₂SO₄, 0.07% KH₂PO₄, 0.42% MgSO₄, and 0.96% yeast extract). Scale-up studies in bioreactors achieved P-carotene yields as high as 1753 pg / g. In addition to P-carotene, the R. glutinis biomass was enriched with protein, glucosamine, and organically chelated minerals, further enhancing its nutritional value. These results indicate that optimized R. glutinis fermentation of sorghum syrup can serve as a sustainable approach for producing multiple high-value bioproducts, including carotenoids, glucosamine, and mineral-enriched biomass for potential use in food, feed, and nutraceutical industries.

[0565] References

[0566] Aksu, Z., & Eren, A. T. (2005). Carotenoids production by the yeast RhodotorulaAttorney Docket No. 11214-012WO1 mucilaginosa: Use of agricultural wastes as a carbon source. Process Biochemistry, 40 (9), 2985-2991.

[0567] Ananda, N., & Vadlani, P. V. (2011). Substrates Influence Stimulatory Effect of Mevalonic Acid on Carotenoid Production in Red Yeasts. Cereal Chemistry, 88 (3), 310-314.

[0568] Anzola-Rojas, M. P., Goncalves da Fonseca, S., Canedo da Silva, C., Maia de Oliveira, V., & Zaiat, M. (2015). The use of the carbon / nitrogen ratio and specific organic loading rate as tools for improving biohydrogen production in fixed-bed reactors. Biotechnology Reports, 5, 46-54.

[0569] Ashokkumar, V., Flora, G., Sevanan, M., Sripriya, R., Chen, W. H., Park, J. H., Rajeshbanu, J., & Kumar, G. (2023). Technological advances in the production of carotenoids and their applications- A critical review. Bioresource Technology, 367, 128215.

[0570] Bhosale, P. (2004). Environmental and cultural stimulants in the production of carotenoids from microorganisms. Applied Microbiology and Biotechnology, 63, (4), 351-361.

[0571] Chen, H„ Heng, X., Li, K„ Wang., A., Ni, Z„ Gao, E„ Yong, Y„ & Wu, X.. (2022). Complexation of multiple mineral elements by fermentation and its application in laying hens. Frontiers in Nutrition. 9, 1-15.

[0572] Di Mascio, P., Murphy, M., & Sies, II. (1991). Antioxidant defense systems: The role of carotenoids, tocopherols, and thiols. The American Journal of Clinical Nutrition, 53 (1), 194S-200S.

[0573] Dufosse, L., Galaup, P., Yaron, A., Arad, S., Blanc, P., Murthy, K., & Gokare, R. (2005). Microorganisms and microalgae as sources of pigments for food use: A scientific oddity or an industrial reality? Trends in Food Science & Technology, 16, (9), 389-406.

[0574] Eggleston, G., Triplett, A., Bett-Garber, K., Boue, S., & Bechtel, P. (2022).

[0575] Macronutrient and mineral contents in sweet sorghum syrups compared to other commercial syrup sweeteners. Journal of Agriculture and Food Research, 7, 100276.

[0576] Fiedor, J., & Burda, K. (2014). Potential role of carotenoids as antioxidants in human health and disease. Nutrients, 6 (2), 466-488.

[0577] Global Carotenoids Market Report and Forecast 2023-2028. (2023). Retrieved November 29, 2022

[0578] Grigorc, D. M., Ungureanu-Iuga, M., Pogurschi, E. N., & B”abcanu, N. E. (2023).

[0579] Transforming Rhodotorula sp. Biomass to Active Biologic Compounds for Poultry Nutrition. Agriculture, 13 (6), 1159.

[0580] Han, I. R., Xu, I., & Zhou, X. M. (2002). Influence of inoculum type, inorganic salt and nitrogen to carbon ratio on sclerotium formation and carotenoid production in surface culture ofAttorney Docket No. 11214-012WO1 Penicillium sp. PT95. Journal of Basic Microbiology, 42 (4), 254-259.

[0581] Kim, C. H., Kim, S. W., & Hong, S. I. (1999). An integrated fermentation- separation process for the production of red pigment by Serratia sp. KH-95. Process Biochemistry, 35 (5), 485-490.

[0582] Kot, A. M., Bia' zejak, S., Kurcz, A., Gientka, I., & Kieliszek, M. (2016). Rhodotorula glutinis — Potential source of lipids, carotenoids, and enzymes for use in industries. Applied Microbiology and Biotechnology, 100 (14), 6103-6117.

[0583] Lourengo- Lopes, C., Carreira-Casais, A., Fraga-Corral, M., Garcia-Oliveira, P., Soria, A., Jarboui, A., Barral, M„ Otero, P., Simal-Gandara, J., & Prieto, M. A. (2021). Carotenoids as Natural Colorful Additives for the Food Industry. In Natural Food Additives. IntechOpen. https: / / doi.org / 10.5772 / intechopen.101208

[0584] Mata-Gomez, L. C., Montanez, J. C., Mendez-Zavala, A., & Aguilar, C. N. (2014).

[0585] Biotechnological production of carotenoids by yeasts: An overview. Microbial Cell Factories, 13 (1), 12. https: / / doi.org / 10.1186 / 1475-2859-13-12

[0586] Maza, D. D., Vinartab, S. C., Su, Y., Guillamonc, J. M., & Aybar, M. J. (2020). Growth and lipid production of Rhodotorula glutinis R4, in comparison to other oleaginous yeasts. Journal of Biotechnology, 310, 21-31.

[0587] Nanjundaswamy, A., & Okeke, B. (2014). Nutraceutical compositions produced from co-products of corn or milo ethanol fermentation and methods of making and using thereof. US Patent U 89168277B2.

[0588] Neil, K. M., Caron, J. P., & Orth, M. W. (2005). The role of glucosamine and chondroitin sulfate in treatment for and prevention of osteoarthritis in animals. Journal of the American Veterinary Medical Association, 226 (7), 1079-1088.

[0589] Ochoa-Vinals, N., Alonso-Estrada, D., Pacios-Michelena, S., Garcia-Cruz, A., Ramos-Gonzalez, R., Faife-Perez, E., Michelena-Alvarez, L. G., Martinez-Hernandez, J. L., & Ilina, A. (2024). Current Advances in Carotenoid Production by Rhodotorula sp. Fermentation, 10 (4), 190.

[0590] Parajd, J. C., Santos, V., & Vazquez, M.. (1998). Optimization of carotenoid production by Phaffia rhodozyma cells grown on xylose. Process Biochemistry, 33 (2), 181-187.

[0591] Pastorini, E., Vccchiotti, S., Colliva, C., Pcrsiani, S., Rotini, R., Roatti, G., Zaccarelli, L., Claudio Rovati, L., & Roda, A.. (2011). Identification and quantification of glucosamine in rabbit cartilage and correlation with plasma levels by high performance liquid chromatographyelectrospray ionization-tandem mass spectrometry. Analytica Chimica Acta, 695, (1-2), 77-83.

[0592] Paul, D., Bohacz, J., & Bhatia, S. K. (2023). Editorial: Biowaste valorization utilizingAttorney Docket No. 11214-012WO1 microbial systems. Frontiers in Microbiology, 14, 1-3.

[0593] Paul, D., Kumari, P. K., & Siddiqui, N. (2023). Yeast Carotenoids: Cost-Effective Fermentation Strategies for Health Care Applications. Fermentation, 9 (2), 147.

[0594] Rapoport, A., Guzhova, I., Bemetti, L., Buzzini, P., Kieliszek, M., & Kot, A. M. (2021). Carotenoids and Some Other Pigments from Fungi and Yeasts. Metabolites, 11 (2), 1-17. https: / / doi.org / doi.org / 10.3390 / metabol11020092

[0595] Rodriguez-Amaya, D. B. (2019). Update on natural food pigments — A mini-review on carotenoids, anthocyanins, and betalains. Food Research International, 124, 200-205.

[0596] Sampath, V., Sureshkumar, S., Seok, W. J., & Kim, I. H. (2023). Role and functions of micro and macro-minerals in swine nutrition: A short review. Journal of Animal Science and Technology, 65 (3), 479- 489.

[0597] Sandmann, G. (2022). Carotenoids and Their Biosynthesis in Fungi. Molecules (Basel, Switzerland), 27 (4), 1431.

[0598] Sharma, R., & Ghoshal, G. (2020). Optimization of carotenoids production by Rhodotorula mucilaginosa (MTCC-1403) using agroindustrial waste in bioreactor: A statistical approach. Biotechnology Reports, 25, e00407. https: / / doi. Org / 10.1016 / j.btre.2019.e00407 Singh, G., Sinha, S., Kukkala, K., Gaur, N., Bandyopadhyay, K., & Paul, D. (2020). High density cultivation of oleaginous yeast isolates in “mandi” waste for enhanced lipid production using sugarcane molasses as feed. Fuel, 276, 118073. https: / / doi. Org / 10.1016 / j.fuel.2020.118073

[0599] Teetor, V. H., Duclos, D. V., Wittenberg, E. T., Young, K. M., Chawhuaymak, J., Riley, M. R., & Ray, D. T. (2011). Effects of planting date on sugar and ethanol yield of sweet sorghum grown in Arizona. Industrial Crops and Products, 34 (2), 1293-1300. https: / / doi.org / 10.101 6 / j.indcrop.2010.09.010

[0600] Thumkasem, N., Onmee, T., Kongsinkaew, C., Chittapun, S., Pornpukdeewattana, S., Ketudat-Caims, M., Thongprajukaew, K., Antimanon, S., & Charoenrat, T. (2024). Enhanced high P-carotene yeast cell production by Rhodotorula paludigena CM33 and in vitro digestibility in aquatic animals. Scientific Reports, 14 (1), 9188. https: / / doi.org / 10.1038 / s41598-024-59809-7 Tkacova, J., Zara, G., laniri, G., Castoria, R.,vCertik, M., & Mannazzu, I. (2022).

[0601] Impairment of carotenoid biosynthesis through CAR1 gene mutation results in CoQ10, sterols, and phytoene accumulation in Rhodotorula mucilaginosa. Applied Microbiology and Biotechnology, 106 (1), 317-327.

[0602] Venil, C. K., Zakaria, Z. A., & Ahmad, W. A. (2013). Bacterial pigments and their applications. Process Biochemistry’, 48 (7), 1065-1079.Attorney Docket No. 11214-012WO1 https: / / doi.org / 10.1016 / j.procbio.2013.06.006

[0603] Vinas, P., Lbpez-Erroz, C., Balsalobre, N., & Hernandez-Cordoba, M. (2003). Reversed-phase liquid chromatography on an amide stationary phase for the determination of the B group vitamins in baby foods. Journal of Chromatography A, 25, 1007.

[0604] Yabuzaki, J. (2017). Carotenoids Database: Structures, chemical fingerprints and distribution among organisms. Database, 2017, 1- 11. https: / / doi.org / 10.1093 / database / bax004 Example 5: Skin cream formulated with carotenogenic yeast

[0605] Disclosed herein is an example skin cream formulated with carotenogenic yeast.

[0606] The formulations employed 2% and 5% yeast incorporated into the cream / lotion. The yeast biomass was previously sterilized and stored in vacuum packed bags.

[0607] The formulation of the 2% and 5% yeast loading creams are shown in Table 10 and Table 11 respectively.

[0608] The preparation is shown in Figure 26. The glucan and mannan biopolymers in the yeast cell wall provided good consistency for the cream the cream produced good texture. The nice light pink color was appealing color for the product (Figure 26).

[0609] Table 10. Formulation of cream for 2% yeast loading.

[0610] %

[0611] Water 72.2

[0612] Glycerin 4

[0613] Xanthan Gum 0.3

[0614] Olivem1000 4

[0615] Steric Acid 2

[0616] Glyceryl Sterate 2

[0617] Caprylic / Capric Triglyceride 8

[0618] VitE 0.5

[0619] Geogard 1

[0620] Macadamia oil 4

[0621] Carotenogenic yeast 2

[0622]

[0623] Total 100

[0624] Table 11. Formulation of cream for 5% yeast loading.

[0625] %

[0626] Water 72.2

[0627] Glycerin 4

[0628] Xanthan Gum 0.3

[0629] Olivem1000 3

[0630]

[0631] Steric Acid 2Attorney Docket No. 11214-012WO1 Glyceryl Sterate 2

[0632] Caprylic / Capric Triglyceride 6

[0633] VitE 0.5

[0634] Geogard 1

[0635] Macadamia oil 4

[0636] Carotenogenci yeast 5

[0637]

[0638] Total 100

[0639] EXEMPLARY ASPECTS

[0640] In view of the described compositions, devices, systems, and methods, herein below are described certain more particularly described aspects of the inventions. The particularly recited aspects should not, however, be interpreted to have any limiting effect on any different claims containing different or more general teachings described herein or that the “particular” aspects are somehow limited in some way other than the inherent meanings of the language and formulas literally used therein.

[0641] Example 1: A method of making β-carotene, comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and either of a sugar beet molasses or a sorghum syrup to thereby produce the β-carotene; wherein the mineral salt comprises (NH₄)₂SO₄ in an amount of from 0.1 to 0.15 % by weight, KH₂PO₄ in an amount of from 0.05 to 0.10 % by weight, and MgSO₄ in an amount of from 0.4 to 0.5 % by weight.

[0642] Example 2: The method of any example herein, particularly example 1, wherein the Rhodotorula yeast is Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula rubra, or any combination thereof.

[0643] Example 3: The method of any example herein, particularly example 2, wherein the Rhodotorula yeast is Rhodotorula mucilaginosa.

[0644] Example 4: The method of any example herein, particularly example 3, wherein the Rhodotorula mucilaginosa is deposited under ATCC 32763.

[0645] Example 5: The method of any example herein, particularly example 2, wherein the Rhodotorula yeast is Rhodotorula glutinis.

[0646] Example 6: The method of any example herein, particularly example 5, wherein the Rhodotorula glutinis is deposited under ATCC. 32766.

[0647] Example 7: The method of any example herein, particularly examples 1-6, wherein fermenting the mixture is performed in a bioreactor.

[0648] Example 8: The method of any example herein, particularly examples 1-7, wherein the Rhodotorula yeast is present in an amount of from 0.5 to 1.5 % by weight.Attorney Docket No. 11214-012WO1 Example 9: The method of any example herein, particularly examples 1-8, wherein either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5 to 15 %.

[0649] Example 10: The method of any example herein, particularly examples 1-9, wherein the (NH₄)₂SO₄ is present in an amount of from 0.12 to 0.14 %, the KH₂PO₄ is present in an amount of from 0.06 to 0.08 % by weight, and the MgSO₄ is present in an amount of from 0.41 to 0.43% by weight.

[0650] Example 11: The method of any example herein, particularly examples 1-10, wherein when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 to 20 pg / g.

[0651] Example 12: The method of any example herein, particularly examples 1-10, wherein when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 to 1400 pg / g.

[0652] Example 13: The method of any example herein, particularly examples 1-12, wherein when the Rhodotorula yeast is combined with the sugar beet molasses, the Rhodotorula yeast is Rhodotorula mucilaginosa.

[0653] Example 14: The method of any example herein, particularly examples 1-12, wherein when the Rhodotorula yeast is combined with the sorghum syrup, the Rhodotorula yeast is Rhodotorula glutinis.

[0654] Example 15: A method of making a personal care product comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and either of a sugar beet molasses or a sorghum syrup to thereby produce a yeast cell comprising β-carotene; wherein the mineral salt comprises (NH₄)₂SO₄ in an amount of from 0.1 to 0.15 % by weight, KH₂PO₄ in an amount of from 0.05 to 0.10 % by weight, and MgSO₄ in an amount of from 0.4 to 0.5 % by weight; collecting the yeast cell from the mixture after fermenting; and combining the yeast cell with a personal care product ingredient.

[0655] Example 16: The method of any example herein, particularly example 15, wherein the method further comprises deactivating the Rhodotorula yeast after fermenting the mixture.

[0656] Example 17: The method of any example herein, particularly examples 15-16, wherein the Rhodotorula yeast is Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula rubra, or any combination thereof.

[0657] Example 18: The method of any example herein, particularly example 17, wherein the Rhodotorula yeast is Rhodotorula mucilaginosa.

[0658] Example 19: The method of any example herein, particularly example 18, wherein the Rhodotorula mucilaginosa is deposited under ATCC Accession No. 32763.Attorney Docket No. 11214-012WO1 Example 20: The method of any example herein, particularly example 17, wherein the Rhodotorula yeast is Rhodotorula glutinis.

[0659] Example 21: The method of any example herein, particularly example 20, wherein the Rhodotorula glutinis is deposited under ATCC Accession No. 32766.

[0660] Example 22: The method of any example herein, particularly examples 15-21, wherein fermenting the mixture is performed in a bioreactor.

[0661] Example 23: The method of any example herein, particularly examples 15-22, wherein the Rhodotorula yeast is present in an amount of from 0.5 to 1.5 % by weight.

[0662] Example 24: The method of any example herein, particularly examples 15-23, wherein either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5 to 15 %.

[0663] Example 25: The method of any example herein, particularly examples 15-24, wherein the (NH₄)₂SO₄ is present in an amount of from 0.12 to 0.14 %, the KH₂PO₄ is present in an amount of from 0.06 to 0.08 % by weight, and the MgSO₄ is present in an amount of from 0.41 to 0.43% by weight.

[0664] Example 26: The method of any example herein, particularly examples 15-25, wherein when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 to 20 pg / g.

[0665] Example 27: The method of any example herein, particularly examples 15-25, wherein when the yeast is Rhodotorula glutinis, the P-carotene is present in an amount of from 200 to 1400 pg / g.

[0666] Example 28: The method of any example herein, particularly examples 15-27, wherein when the Rhodotorula yeast is combined with the sugar beet molasses, the Rhodotorula yeast is Rhodotorula mucilaginosa.

[0667] Example 29: The method of any example herein, particularly examples 15-27, wherein when the Rhodotorula yeast is combined with the sorghum syrup, the Rhodotorula yeast is Rhodotorula glutinis.

[0668] Example 30: A personal care product comprising the yeast cell comprising P-carotene of any example herein, particularly examples 15-29, wherein the personal care product comprises sunscreen, lip balm, lotion, or shampoo.

[0669] Other advantages which are obvious, and which are inherent to the invention, will be evident to one skilled in the art. It will be understood that certain features and sub-combinations are of utility and may be employed without reference to other features and sub-combinations.Attorney Docket No. 11214-012WO1 This is contemplated by and is within the scope of the claims. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matter herein set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

Claims

Attorney Docket No. 11214-012WO1CLAIMSWhat is claimed is:

1. A method of making β-carotene, comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and either of a sugar beet molasses or a sorghum syrup to thereby produce the β-carotene; wherein the mineral salt comprises (NH₄)₂SO₄ in an amount of from 0.1 to 0.15 % by weight, KH₂PO₄ in an amount of from 0.05 to 0.10 % by weight, and MgSO₄ in an amount of from 0.4 to 0.5 % by weight.

2. The method of claim 1, wherein the Rhodotorula yeast is Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula rubra, or any combination thereof.

3. The method of claim 2, wherein the Rhodotorula yeast is Rhodotorula mucilaginosa.

4. The method of claim 3, wherein the Rhodotorula mucilaginosa is deposited under ATCC 32763.

5. The method of claim 2, wherein the Rhodotorula yeast is Rhodotorula glutinis.

6. The method of claim 5, wherein the Rhodotorula glutinis is deposited under ATCC. 32766.

7. The method of any one of claims 1-6, wherein fermenting the mixture is performed in a bioreactor.

8. The method of any one of claims 1-7, wherein the Rhodotorula yeast is present in an amount of from 0.5 to 1.5 % by weight.

9. The method of any one of claims 1-8, wherein either of the sugar beet molasses or the sorghum syrup are present in an amount of from 5 to 15 %.

10. The method of claims 1-9, wherein the (NH₄)₂SO₄ is present in an amount of from 0.12 to 0.14 %, the KH₂PO₄ is present in an amount of from 0.06 to 0.08 % by weight, and the MgSO₄ is present in an amount of from 0.41 to 0.43% by weight.

11. The method of any one of claims 1-10, wherein when the yeast is Rhodotorula mucilaginosa, the P-carotene is present in an amount of from 5 to 20 pg / g.

12. The method of any one of claims 1-10, wherein when the yeast is Rhodotorula glutinis,Attorney Docket No. 11214-012WO1 the 0-carotene is present in an amount of from 200 to 1400 pg / g.

13. The method of any one of claims 1-12, wherein when the Rhodotorula yeast is combined with the sugar beet molasses, the Rhodotorula yeast is Rhodotorula mucilaginosa.

14. The method of any one of claims 1-12, wherein when the Rhodotorula yeast is combined with the sorghum syrup, the Rhodotorula yeast is Rhodotorula glutinis.

15. A method of making a personal care product comprising fermenting a mixture of a Rhodotorula yeast, a mineral salt, and either of a sugar beet molasses or a sorghum syrup to thereby produce a yeast cell comprising 0-carotene; wherein the mineral salt comprises (NH₄)₂SO₄ in an amount of from 0.1 to 0.15 % by weight, KH2PO4 in an amount of from 0.05 to 0.10 % by weight, and MgSO₄ in an amount of from 0.4 to 0.5 % by weight; collecting the yeast cell from the mixture after fermenting; and combining the yeast cell with a personal care product ingredient.

16. The method of claim 15, wherein the method further comprises deactivating the Rhodotorula yeast after fermenting the mixture.

17. The method of any one of claims 15-16, wherein the Rhodotorula yeast is Rhodotorula mucilaginosa, Rhodotorula glutinis, Rhodotorula rubra, or any combination thereof.

18. The method of claim 17, wherein the Rhodotorula yeast is Rhodotorula mucilaginosa.

19. The method of claim 18, wherein the Rhodotorula mucilaginosa is deposited under ATCC Accession No. 32763.

20. The method of claim 17, wherein the Rhodotorula yeast is Rhodotorula glutinis.

21. The method of claim 20, wherein the Rhodotorula glutinis is deposited under ATCC Accession No. 32766.

22. The method of any one of claims 15-21, wherein fermenting the mixture is performed in a bioreactor.

23. The method of any one of claims 15-22, wherein the Rhodotorula yeast is present in an amount of from 0.5 to 1.5 % by weight.

24. The method of any one of claims 15-23, wherein either of the sugar beet molasses or theAttorney Docket No. 11214-012WO1 sorghum syrup are present in an amount of from 5 to 15 %.

25. The method of claims 15-24, wherein the (NH₄)₂SO₄ is present in an amount of from 0.12 to 0.14 %, the KH2PO4 is present in an amount of from 0.06 to 0.08 % by weight, and the MgSO₄ is present in an amount of from 0.41 to 0.43% by weight.

26. The method of any one of claims 15-25, wherein when the yeast is Rhodotorula mucilaginosa, the 0-carotene is present in an amount of from 5 to 20 pg / g.

27. The method of any one of claims 15-25, wherein when the yeast is Rhodotorula glutinis, the 0-carotene is present in an amount of from 200 to 1400 pg / g.

28. The method of any one of claims 15-27, wherein when the Rhodotorula yeast is combined with the sugar beet molasses, the Rhodotorula yeast is Rhodotorula mucilaginosa.

29. The method of any one of claims 15-27, wherein when the Rhodotorula yeast is combined with the sorghum syrup, the Rhodotorula yeast is Rhodotorula glutinis.

30. A personal care product comprising the yeast cell comprising 0-carotene of any one of claims 1 -29, wherein the personal care product comprises sunscreen, lip balm, lotion, or shampoo.

Images