Preferential enrichment of diglycerides over triglycerides in a free fatty acid matrix

Abstract

The present disclosure generally relates to the enrichment of diglycerides (DAGs) over triglycerides (TAGs) in a naturally derived free fatty acid matrix. More particularly, the disclosure relates to the in-situ generation of surfactants naturally present in natural oils and fats, thereby effectively partitioning diglycerides and triglycerides, as minor lipid components, out of the oil phase into an aqueous extraction solution in which the extract is appreciably enriched in DAGs relative to TAGs. The oleaginous matrix can be further separated by inducing phase segregation to reform the neutral form of the surfactants, resulting in a separated concentrated fatty formulation enriched with the DAG composition relative to TAG despite statistical distribution in the starting mixture.

Description

PREFERENTIAL ENRICHMENT OF DIGLYCERIDES OVER TRIGLYCERIDES IN A FREE FATTY ACID MATRIXCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of priority to United States Provisional Patent Application No. 63 / 514,279 filed July 18, 2023, entitled “PREFERENTIAL ENRICHMENT OF DIACYLGLYCERIDES OVER TRIACYLGLYCERIDES IN A FREE FATTY ACID MATRIX”, the disclosure of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD OF THE DISCLOSURE

[0002] The present disclosure generally relates to a methods and systems for preferential enrichment of diglycerides (DAGs) over triglycerides (TAGs) in a free fatty acid (FFA) matrix.BACKGROUND

[0003] In most naturally derived oils, diglycerides are not separated from triglycerides despite the potential benefits such as nutritional and health benefits. The ability to separate these components from naturally derived fats and oils, at scale, enables access to unique formulations that can be leveraged toward functional food ingredients production. Overall, the choice of separation process will depend on the specific properties of the diglycerides and triglycerides being separated, as well as the desired level of separation and purity. However, none of the techniques used to separate DAGs from TAGs are scalable. A liquid-liquid separation process in which diglycerides are separated from triglycerides is described herein and provides a scalable, low-cost, and efficient method for doing so.

[0004] Triglycerides and diglycerides are two types of lipids that are found in the body and in many foods. Although they have some similarities in their structure and function, there are also some critical differences between the two. Triglycerides are composed of three fatty acids attached to a glycerol backbone, while diglycerides are composed of two fatty acids attached to a glycerol backbone. This difference in structure affects their physical and chemical properties, including their solubility in water and their ability to form micelles. Triglycerides are the primary form in which fats are stored in the body and are a major source of energy. Triglycerides are broken downby enzymes called lipases into diglycerides and fatty acids, which can then be absorbed into the bloodstream. Diglycerides can be further broken down into monoacylglycerides and fatty acids by additional lipases, which can also be absorbed into the bloodstream.

[0005] Diglycerides have been shown to have a variety of biological activities that are distinct from those of triglycerides and are involved in a variety of cellular signaling pathways. For example, diglycerides can act as second messengers to help regulate a range of physiological processes, including insulin secretion and smooth muscle contraction. Diglycerides can also activate certain protein kinase C enzymes, which play a key role in regulating cell growth, differentiation, and apoptosis.

[0006] The important structure-function differences between triglycerides and diglycerides highlight the need for a separation process of the two classes of molecules from natural products and synthetic mixtures. In most naturally derived oils, diglycerides are not separated from triglycerides despite the potential benefits such as nutritional and health benefits. The separation of diglycerides from triglycerides is necessary to better understand their individual properties and effects on the body. Diglycerides have also been shown to have several health benefits, including improving blood glucose levels and reducing the risk of obesity and heart disease. The ability to separate these components from naturally derived fats and oils, at scale, enables access to unique formulations that can be leveraged toward functional food manufacturing. For example, Diglycerides are easier to digest than triglycerides, which means they can provide a source of energy more quickly. This can be especially beneficial for athletes or people who need a quick energy boost.

[0007] In the food industry, diglycerides are often used as emulsifiers to improve the texture and stability of certain foods and a naturally derived diglyceride enriched product stream could facilitate the tunability of organoleptic properties in food. Furthermore, separating diglycerides from triglycerides can improve the stability and shelf life of certain types of foods, such as baked goods and fried foods.

[0008] Triglycerides and diglycerides can be separated using various chromatographic techniques, which are based on the differential interactions of these molecules with a stationary phase and a mobile phase. The choice of method for separating triglycerides and diglycerides depends on the specific application and the desired level of separation and purification. One commonly used chromatographic method for separating triglycerides and diglycerides is high-performance liquid chromatography (HPLC). HPLC uses a stationary phase, such as silica gel or a polymer, and a mobile phase, such as a solvent or a mixture of solvents, to separate the different components of a mixture based on their chemical properties. In HPLC, triglycerides and diglycerides can be separated based on differences in their polarity, size, and shape. Another method for separating triglycerides and diglycerides is thin-layer chromatography (TLC). TLC is a simple and inexpensive technique that uses a thin layer of stationary phase, such as silica gel or alumina, coated on a flat surface. A sample is spotted on the stationary phase, and a mobile phase, such as a solvent or a mixture of solvents, is used to move the components of the mixture along the stationary phase. In TLC, triglycerides and diglycerides can be separated based on differences in their solubility and polarity. A liquid-liquid separation technique that can be used to separate diglycerides from triglycerides is chromatography using a liquid stationary phase. For example, reverse-phase chromatography uses a stationary phase that is more polar than the mobile phase, and the different components of the mixture will interact differently with the stationary phase based on their polarity. By selecting the appropriate stationary and mobile phases, it is possible to separate diglycerides from triglycerides.

[0009] Overall, the choice of separation process will depend on the specific properties of the diglycerides and triglycerides being separated, as well as the desired level of separation and purity. However, none of the methods described above are scalable. A liquid-liquid separation process in which diglycerides are separated from triglycerides is described herein and provides a scalable, low-cost and efficient method for doing so.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Various embodiments of the present disclosure will be understood more fully from the detailed description given below and from the accompanying drawing, in which:

[0011] FIG. 1 is a graph showing enrichment of DAG relative to TAG in Examples 1-3.DETAILED DESCRIPTION

[0012] The following disclosure provides many different embodiments or examples. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0013] In general, free fatty acids (FFAs) are important biomolecules that play essential roles in various physiological processes in the body. Naturally derived FFA food formulations can serve as a major source of energy as FFAs can be oxidized in the mitochondria to generate ATP, which is the primary source of energy for cellular processes. The enrichment of diglycerides in an FFA matrix can potentially serve as a synergistic plant derived fatty food formulation in which FFAs stimulate signaling pathways that lead to the release of hormones such as glucagon and leptin to regulate glucose and energy metabolism as digestible lipids in the form of DAGs are simultaneously delivered as a source of energy for the body serving consumers abiding by specialized diets such as the ketogenic diet.

[0014] Furthermore, FFAs are also important for skin health as they maintain the barrier function of the skin by regulating the production of sebum, which is a mixture of FFAs, cholesterol, and other lipids that helps to prevent moisture loss from the skin. A potent formulation containing FFAs, TAGs and DAGs can serve the cosmetic industry through naturally derived ingredients.

[0015] It is important to highlight that the distribution of DAGs and TAGs can affect the physical and chemical properties of the oil or fat, including its melting point, viscosity, and oxidative stability and that the TAG:DAG ratio in both product streams (i.e., the processed oil and the aqueous DAG enriched formulation) can be modulated. The removal of DAGs from fats has important implications for the food industry and other applications where the oil or fat is used as DAGs can impact the crystallization behavior of fats and a process in which their relative distribution ratio can be modulated is beneficial for producing shelf-stable products with superior crystallization properties. Furthermore, as DAGs are used as emulsifiers, the ability to tune their concentration through this process enables pathways to tune the organoleptic properties of the fats as integrated in food formulations.

[0016] Embodiments of the present disclosure provide in-situ generation of surfactants naturally present in natural oils and fats, thereby effectively partitioning diglycerides (DAGs) and triglycerides (TAGs), as minor lipid components, out of the oil phase into an aqueous extraction solution in which the extract is appreciably enriched in DAGs relative to TAGs. The oleaginous matrix can be further separated by inducing phase segregation to reform the neutral form of the surfactants, resulting in a separated concentrated fatty formulation enriched with the DAG composition relative to TAG despite statistical distribution in the starting mixture. The net result is a fast, scalable, efficient production of a unique formulation derived from a natural oil or fat thatcan be utilized as a functional food ingredient or cosmetic formulation. Overall, while both triglycerides and diglycerides are important lipids in the body, their differences in structure and function can lead to distinct biological effects and may have implications for human health and disease. A liquid-liquid separation process in which diglycerides are separated from triglycerides is described herein and provides a scalable, low-cost, and efficient method for doing so.

[0017] A significant benefit of the micellar solubilization processed described herein is that unlike liquid-liquid extraction processes in which a volatile polar organic solvent such as ethanol is used to selectively extract diglycerides, the extracted diglycerides in the micellar solution can be recovered via pH modulation as opposed to solvent evaporation, which is a highly energy intensive process. Thus, the two-stage process described herein provides a scalable, low- cost production method of unique naturally derived formulations.

[0018] The solvent extraction described herein involves adding a solvent to a mixture of diglycerides and triglycerides to selectively dissolve one of the components. For example, a polar solvent such as water can be used to selectively extract diglycerides, while leaving the triglycerides behind as the bulk oil / fat stream. The extracted diglycerides can then be recovered by inducing a phase separation of the oleaginous diglyceride from the aqueous extract.

[0019] The statistical distribution of diglycerides (DAGs) relative to triglycerides (TAGs) in natural oils and fats can vary depending on the source of the oil or fat and can also be influenced by processing factors such as temperature, pressure, and enzymatic or chemical treatments. For example, high-temperature processing and enzymatic hydrolysis can increase the DAG content in some oils and fats. Generally, the DAG content is relatively low compared to the TAG content, but it can vary from less than 1% to more than 10% of the total glyceride content. For example, soybean oil typically contains around 1-3% DAGs and 90-95% TAGs, while palm oil can have up to 10-15% DAGs and 85-90% TAGs. The DAG content of canola oil is typically around 1-2%, while olive oil can have up to 2-3% DAGs.

[0020] The process described herein utilizes the in-situ generation of a surfactant mixture, derived from the bulk natural oil / fat starting mixture, as the extractive matrix in which diglycerides are selectively partitioned from the bulk triglyceride phase into a separate immiscible liquid phase. Despite the appreciable difference in distribution percentage (i.e., TAGs present in large excess relative to DAGs), the separated extract is selectively enriched in diglycerides over triglycerides.

[0021] The first step of the process comprises of the generation of surfactants derived from the crude oil mixture. Crude vegetable oils are a mixture of triacylglycerols (triglycerides), diacylglycerols (diglycerides), monoacylglycerols, free fatty acids, phospholipids, pigments, sterols, and tocopherols. Thus, crude oils and fats contain a variety of natural surfactants, including phospholipids, glycolipids, and proteins and free fatty acids which can be further activated as surfactants through saponification with bases.

[0022] The second step incorporates micellar solubilization of the diglyceride into a liquid solution that is immiscible with the oil to enable the actual separation of the diglyceride from the bulk oil mixture into a polar formulation which may be directly used as a product. However, a third step may be applied to separate the bulk polar fluid from the oily diglyceride / surfactant matrix to generate a more concentrated formulation of diglycerides.

[0023] In some embodiments, surfactants in the form of fatty acid salts were generated by reacting crude oil which, naturally contained FFAs, diglycerides and triglycerides, with a stoichiometric amount of a strong base, such as sodium hydroxide (NaOH) at 60°C. The amount of base required depended on the concentration of free fatty acids in the crude oil and may be delivered as dilute aqueous solution. The generated surfactants may be used to promote preferential micellar solvation of the present diglycerides in the aqueous phase which phased out of the bulk oil-water mixture due to the density differential.

[0024] In some embodiments, the amount of base in the reactant solution is from 0.2 to 2.0, from 0.5 to 1.2, about 0.5, about 1.0, or about 1.2 molar equivalents, based on the amount of FFA in the oil. In some embodiments, the reactant solution has a pH of greater than 7, greater than 8, greater than 9, greater than 10, greater than 11, or greater than 12.

[0025] The process described herein preferentially extracts DAGs over TAGs from the crude oil such that a weight ratio of TAGs to DAGs (TAG:DAG ratio) in the crude oil is higher than a ratio of TAGs to DAGs in the extracted oil (or the aqueous extract). In some embodiments, the TAG:DAG ratio in the crude oil is at least 5, at least 10, at least 12, at least 15, at least 20, at least 25, at least 30, at least 35, or greater than 35. In some embodiments, the TAG:DAG ratio in the extracted oil or the aqueous extract is at most 10, at most 6, at most 5, at most 4, at most 3, less than 5, less than 4, or less than 3. In some embodiments a ratio between the TAG:DAG ratio of the crude oil and the TAG:DAG ratio of the extracted oil or aqueous extract is 2 to 20, 5 to 15, 5 to 10, at least 2, at least 5, or at least 10.

[0026] This process can be done as a continuous flow chemical process in which the crude oil and the basic aqueous solution are simultaneously injected into a high-throughput microchannel reactor where diffusion rates and mass transfer between oleaginous and aqueous solutions are accelerated, and the saponification reaction occurs instantaneously. In a microfluidic device where the reaction is promoted to occur in channels ranging from 100-20 micrometers in width, the enhanced interfacial surface area accelerates the formation of micellar aggregates in which the DAGs are solubilized and partitioned into the aqueous phase. The reaction products are collected in a vessel wherein the reaction products include a first raffinate including the oil having DAGs and FFAs removed therefrom and a first aqueous stream including the now separated FFA salts and DAGs.

[0027] EXAMPLE 1

[0028] Crude cocoa butter, comprising of 91.35 wt.% TAGs, 3.23% DAGs and 3.65 wt.%FFA (1 molar equiv.), was heated to 80°C and continuously pumped into a microchannel reactor through one injection inlet. In a second injection inlet, an aqueous sodium hydroxide solution was also simultaneously injected, and the flowrate modulated to deliver 1.0 molar equivalent (relative to the starting FFA concentration) of NaOH as an aqueous solution. The oil and aqueous solution were allowed to pass through the microchannel reactor and collected from a separator vessel adjoined at the bottom to analyze the reaction efficiency as determined by the percentage of FFA removed from the cocoa butter and the relative distribution of DAGs and TAGs co-extracted in the aqueous stream. The FFA weight percentage in the bulk separated oil phase was reduced to 0.8 wt.%. Due to the removal of appreciable DAGs into the aqueous phase, the remaining DAG wt.% in the resultant cocoa butter phase was reduced to 2.98 wt.% and the TAG was increased to 95 wt.% indicating an increase in the TAG:DAG ratio in the cocoa butter from 28:1 in the crude to 32:1 in the processed cocoa butter.

[0029] The aqueous phase was subjected to additional processing in order to further separate the oleaginous components from the bulk aqueous phase and co-extracted water-soluble components. The separation of the lipids from the aqueous matrix was achieved via drop-wise addition of acids such as phosphoric acid, sulfuric acid, hydrochloric acid, or combinations thereof. The addition of acid reprotonated the FFA salts to regenerate the non-polar acid form as an olagenous layer in which DAGs and co-extracted MAGs are embedded. The resulting MAG:DAG ratio in this matrix ranged from 2:1 to 5:1.

[0030] EXAMPLE 2

[0031] The concentration of the lipid fractions from the aqueous stream generated in Example 1 was conducted by first promoting the reprotonation of the FFA through drop-wise addition of concentrated phosphoric acid at 60°C which resulted in immediate phasing of an oleaginous layer which upon cooling solidified and filtered and analyzed via HPLC to indicate that the MAG:DAG ratio was reduced from 28:1 in the crude cocoa butter to 3:1 in the recovered lipid formulation resulting in a naturally derived matrix significantly enriched in diglyceride content without necessitating chemical hydrolysis or decomposition of triglycerides.

[0032] EXAMPLE 3

[0033] Additional samples were subjected to the procedures described in Examples 1 and 2 above. The analysis of the treated samples is summarized in Table 1 below, which also lists the TAG to DAG ratio of the starting material.

[0034] Table 1. Compositional analysis of separated DAG enriched free fatty acid formulations.

[0035] As shown above, the separation and enrichment process was able to reduce the T / D ratio by an order of magnitude. FIG. 1 also depicts the reduction in T / D ratio.

[0036] Although various embodiments have been shown and described, the disclosure is not limited to such embodiments and will be understood to include all modifications and variations as would be apparent to one of ordinary skill in the art. Therefore, it should be understood that the disclosure is not intended to be limited to the particular forms disclosed; rather, the intention is tocover all modifications, equivalents, and alternatives falling within the spirit and scope of the disclosure as defined by the appended claims.

Claims

CLAIMSWhat is claimed is:

1. A method comprising: contacting a first oil comprising triglycerides and diglycerides with an aqueous solution to transfer at least a portion of the triglycerides and the diglycerides to the aqueous solution, thereby forming an aqueous extract; and isolating the at least a portion of the triglycerides and the diglycerides from the aqueous extract to yield a second oil; wherein a weight ratio of triglycerides to diglycerides in the first oil is higher than a weight ratio of triglycerides to di glycerides in the second oil.

2. The method of claim 1, wherein the aqueous solution has a pH of greater than 10.

3. The method of claim 1, wherein the first oil further comprises free fatty acids and the aqueous solution comprises from 0.2 to 2.0 molar equivalents of a base relative to the amount of free fatty acids in the first oil.

4. The method of claim 3, wherein the base is sodium hydroxide.

5. The method of claim 1, wherein isolating comprises contacting the aqueous extract with an acid.

6. The method of claim 5, wherein contacting the aqueous extract with an acid comprises introducing the aqueous extract and the acid into a microchannel reactor comprising a hollow conduit and a plurality of fibers disposed within the hollow conduit.

7. The method of claim 5, wherein contacting the aqueous extract with the acid comprises dropwise addition of the acid to the aqueous extract.

8. The method of claim 5, wherein the acid comprises phosphoric acid, sulfuric acid, hydrochloric acid, or combinations thereof9. The method of claim 1, wherein the first oil is cocoa butter.

10. The method of claim 1, wherein the contacting the first oil with the aqueous solution comprises using a microchannel reactor.

11. The method of claim 10, wherein the microchannel reactor comprises a hollow conduit having a plurality of fibers disposed therein.

12. The method of claim 2, wherein the contacting comprises forming micelles that enhance solubility of the diglycerides in the aqueous solution.

13. The method of claim 1, wherein the first oil comprises free fatty acids and the contacting transfers at least a portion of the free fatty acids from the first oil into the aqueous solution.

14. The method of claim 13, wherein the second oil comprises diglycerides embedded in a free fatty acid matrix.

15. The method of claim 1, wherein contacting the first oil and the aqueous solution comprises introducing the first oil and the aqueous solution into a first end of a microchannel reactor comprising a hollow conduit having a plurality of fibers disposed therein, receiving reaction products proximate a second end of the microchannel reactor, the reaction products comprising the aqueous extract and the first oil less the at least a portion of the triglycerides and the diglycerides, and separating the reaction products; wherein the first oil is cocoa butter, the first oil comprises free fatty acids, the aqueous solution has a pH of greater than 10, and the aqueous extract comprises at least a portion of the free fatty acids; andwherein isolating the at least a portion of the triglycerides and the diglycerides from the aqueous extract comprises mixing the aqueous extract with an acid to form the second oil phase, which comprises diglycerides embedded in a free fatty acid matrix.

16. The method of claim 1, wherein the weight ratio of triglycerides to diglycerides in the first oil is at least two times the weight ratio of triglycerides to diglycerides in the second oil.

17. The method of claim 16, wherein the weight ratio of triglycerides to diglycerides in the first oil is at least 12 and the weight ratio of triglycerides to diglycerides in the second oil is less than 6.

18. A system comprising: a microchannel reactor comprising a hollow conduit having a plurality of fibers disposed therein; an oil supply in fluid communication with a first end of the microchannel reactor and comprising a first oil comprising triglycerides, diglycerides, and free fatty acids, wherein a first ratio is defined between a weight of the triglycerides in the first oil to a weight of the di glycerides in the first oil; a caustic supply in fluid communication with the first end of the microchannel reactor and comprising an aqueous solution having a pH of greater than 7, the aqueous solution capable of extracting at least a portion of the triglycerides, diglycerides, and free fatty acids such that a weight ratio of the extracted triglycerides to the extracted diglycerides is less than the first ratio; a collection vessel in fluid communication with a second end of the microchannel reactor opposite the first end and configured to receive reaction products comprising an oil phase comprising the first oil less a portion of the triglycerides, diglycerides, and free fatty acids and an aqueous phase comprising the aqueous solution and the portion of the triglycerides, diglycerides, and free fatty acids; and an acid supply comprising a second aqueous solution having a pH of less than 7.

19. The system of claim 18, wherein the collection vessel is further in fluid communication with the first end of the microchannel reactor or a first end of a second microchannel reactor comprising a second hollow conduit having a second plurality of fibers disposed therein and the collection vessel is configured to supply the aqueous phase to the first end of the microchannel reactor or the first end of the second microchannel reactor; and wherein the acid supply is in fluid communication with the first end of the microchannel reactor or the first end of the second microchannel reactor.

20. The system of claim 18, wherein the first oil is cocoa butter and the aqueous solution comprises sodium hydroxide.

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