Bioactive lipids, systems and methods of obtaining bioactive lipids

Abstract

Disclosed herein are improved walnut tissues and embryos, methods of obtaining fatty acid compounds from walnut tissues, methods of cultivating walnut tissues, and fatty acid compounds.

Description

Attorney Docket No. 11716-008WO1 BIOACTIVE LIPIDS, SYSTEMS AND METHODS OF OBTAINING BIOACTIVE LIPIDS CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application 63 / 639,010, filed April 26, 2024, the contents of which are hereby incorporated in its entirety. FIELD OF THE INVENTION

[0002] Disclosed herein are improved walnut tissues and embryos, methods of obtaining fatty acid compounds from walnut tissues. Also disclosed are fatty acid compounds and bioactive lipids intended as unconventional lipid and / or fatty acid, i.e. bearing atoms and / or chemical functional groups and / or degree of unsaturation and / or structural modifications, on the carbon chain and / or carboxylic moiety, which might confer specific molecular bioactivity in the context of human and plant health. BACKGROUND

[0003] Institutional dietary guidelines claimed both by US and EU health and agricultural agencies promote the daily consumption of walnuts to improve heart health and to reduce cardiovascular diseases (CVD). Walnuts are enriched in healthy compounds like lipids which contribute to reduced risk of cancer, regulation of metabolic dysfunctions which include obesity and diabetes, improvement of cognitive abilities, support of gut health, amelioration of male fertility and age-related cognitive decline. All these conditions are part of a major social concern regarding global health, and as the human population lives longer, in many countries, the aging rate is surpassing natality as fertility is declining.

[0004] Consumer demand for tree nuts, such as almonds, pistachios, and walnuts, continues to increase globally. Based on available data from the USDA Economic Research Service, the per capita consumption of shelled tree nuts has increased from 3.8 pounds in 2010 to almost 6 pounds in 2021. Shelled tree nuts, which consist of only the edible portion of the nut, are popular among consumers for various reasons. They have cultural significance, can be utilized in many diverse culinary applications, are a tasty, nutritional, and convenient snack, and are increasingly recognized for their health benefits. Government health agencies worldwide encourage the consumption of tree nuts, describing them as nutrient-dense foods that provide essential dietary components and bioactive compounds. The health- promoting compounds rich in tree nuts, typically lacking in Western diets, are associated with lowering cardiovascular disease mortality, diabetes, and cancer risk.

[0005] Regarding walnuts specifically, their worldwide consumption experienced the same growth as the broader group of tree nuts in the last decade. The expansion of walnut industries across the globe to 1Attorney Docket No. 11716-008WO1 meet consumer demand resulted in a total industry value of $7.53B in 2022. The global walnut industry is projected to grow at a rate of 5.15 % between 2023 and 2029, reaching an approximate value of $10.71B before the turn of the next decade. Walnuts continue to be enjoyed by consumers due to their unique taste, texture, and aroma, as well as their convenience as a highly nutritious snack food. Furthermore, incisive marketing has utilized health research outcomes and institutional health claims to drive consumer interest based on the associated health benefits of walnut consumption (Lockyer et al. 2022). Consumption of 43 g of walnuts daily is supported by the Food and Drug Administration (FDA) for reducing the risk of coronary heart disease (CHD), and 30 g per day by the EU and UK for improving vascular function. Other benefits associated with daily walnut consumption include reducing the risk of certain high-mortality-rate cancers, regulating metabolic dysfunction conditions such as diabetes and obesity, and supporting cognitive function and gut health. A systematic review of cohort studies and randomized controlled trials published from 2017 to 2021 indicates the consistent association of walnut consumption with positive public health outcomes, specifically, improvement of blood lipid profiles and reduced cardiovascular disease (CVD). While further evidence for these benefits is accumulating in other areas of ongoing health research, more work is needed to draw firm conclusions about the benefits of walnut consumption on health. This is mainly because these health outcomes' molecular mechanisms still need to be completely understood or fully discovered. Compositional studies of the walnut kernels, made up of the meat (cotyledons and embryo) and the pellicle (seed coat), have revealed each to be rich sources of bioactive compounds: essential lipids and potent, antioxidant phenolics, respectively.

[0006] Among the tree nuts, walnut meat is particularly rich in polyunsaturated fatty acids (PUFAs), in particular, alpha-linolenic acid (ALA; 18:3, n-3) and linoleic acid (LA; 18:2, n-6). The replacement of dietary saturated fats with unsaturated fats, especially omega-3 fatty acids (ω3FAs) like ALA, is connected to tangible health benefits. The reduction of low-density-lipoprotein cholesterol (LDL-C), mediated through upregulation of lipo-protein-lipase (LPL), significantly decreases hypertriglyceridemia and the incidence of CVD. PUFAs are also involved in cellular energetics by regulating cellular glucose and fatty acid metabolism. Interaction of PUFAs with peroxisome proliferator-activated receptors (PPARs) leads to transcriptional activation of genes involved in energy homeostasis, such as fatty acid β- oxidation, that support proper mitochondrial function. Persistent mitochondrial dysfunction and disruption of energy homeostasis are linked to cardiovascular complications, as well as chronic inflammation and neurodegenerative conditions. In addition to their essential role in energy metabolism, incorporating PUFAs into cellular membranes provides protective and stabilizing effects. This is crucial in high-fat-content tissues, such as nerves and the retina, where lack of ω3FAs has severe impacts on organ functionality, leading to neurodegenerative conditions (Alzheimer's disease, dementia, etc.) and retinal deteriorative conditions, respectively. Additional mechanisms of action include 2Attorney Docket No. 11716-008WO1 modulation of inflammation status and resolution of local inflammation. Incorporation of ω3FAs into membranes displaces pro-inflammatory arachidonic acid (AA), as well as competes with AA for enzymatic production of autocoids (i.e., signaling molecules acting as local hormones with brief duration to suppress inflammation), like eicosanoids. This mechanism operates alongside the activation of PPAR- gamma, which mediates inflammatory gene expression and NFkB activation to further resolve inflammation. Although these mechanisms of action have been extensively characterized, they cannot fully explain the broad spectrum of salutary effects associated with walnut consumption, requiring further research.

[0007] Distinct from PUFA-rich walnut meat, the pellicle (seed coat), with its vast array of phenolic compounds, has received significant attention. Phenolic compounds (or phenolics), which encompass a large family of biomolecules containing a phenol structure, are thought to act synergistically with lipids to provide the health benefits associated with walnut consumption. Phenolic compounds are potent antioxidants used to mitigate oxidative stress, which, if left unmanaged, can result in oxidative damage to cellular components, resulting in reduced functionality. Oxidative damage is at the heart of many high-mortality conditions (particularly in the West); this includes heart disease, cancers, and chronic inflammation. The walnut pellicle is especially abundant with phenolics, even when compared with other tree nuts, being especially enriched in compounds from the hydrolyzable tannin and flavonoid pathways. Some examples are gallic acid, ellagic acid, quercetin, and catechin, among many others. While this has been the primary focus of prior walnut pellicle research, the lipid fraction of the pellicle has yet to be thoroughly explored.

[0008] As with all plant tissues, the walnut pellicle comprises cells and thus contains ample lipids. These include the “standard” lipids, such as phospholipids (PLs), galactolipids, and triacylglycerols (TAGs), which are found in cellular membranes and lipid droplets for energy storage, together with fatty acids like palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1Δ9), linoleic acid (18:2Δ9,12), and α-linolenic acid (18:3Δ9,12,15). Generally speaking, seed coats are known to accumulate many potent and unusual bioactive compounds to protect the seed within. Indeed, bioactive compounds have been discovered in the seed coat of other plant species therefore this might extends to lipids. Lipid structures are diversified in plants by modulating carbon chain lengths, the number of unsaturations (double bonds) and their chain position, and the addition of carbon-chain modifications (i.e., hydroxylation, epoxidation, methylation, etc.), with countless combinations of these structural features. This population, therefore, represents a hidden fraction of “unusual lipids”, having peculiar structural features and lower abundance but with unique and often unexplored bioactivity for both plant and human health. Two of the best-understood classes of these “unusual lipids” are oxylipins and endocannabinoids because, in animals, they serve as the backbone of numerous signaling pathways and 3Attorney Docket No. 11716-008WO1 as homeostatic regulators for physiological processes. These include the resolution of inflammation and pain, activation of immune functions, and, via the gut-brain axis, improvement of cognitive health, mood, and behavioral disorders. Because of this critical functionality and to distinguish them from standard, more structural lipids, these unusual lipids are termed “bioactive”. In animals, these bioactive lipids are synthesized endogenously from common lipids, like PUFAs, in small amounts. Unfortunately, these bioactive lipids are highly unstable, reducing the potential positive effects on health achieved at higher concentrations. Because these bioactive lipids can be absorbed from the diet and serve such vital functions, they can be regarded as “essential bioactive dietary lipids”. Due to the abundance of precursor oils found in walnuts and the protective purpose of the walnut pellicle, this tissue is a likely reservoir of these potent bioactive lipids.

[0009] Oleoylethanolamide, (OEA, molecular formula C20H39NO2) is a monounsaturated fatty acid, specifically oleic acid (C18:1) with the carboxylic moiety conjugated to an ethanolamine molecule. OEA is classified as an N-acylethanolamine (NAE) and is known to mediate many important physiological functions in both mammals and plants. In the case of humans, OEA is a potent ligand binding to three main receptors: 1) The peroxisome proliferator-activated receptor alpha (PPAR-α, EC50 of ~120 nM); 2) G protein-coupled receptor-119 (GPR119, EC50 ~3 μM), and 3) the transient receptor potential cation channel vanilloid-1 (TRPV1, EC50 of ∼2 μM). OEA-induces GPR119 activation, significantly increasing GLP- 1 secretion from intestinal L-cells thus enhancing insulin levels which in turn inhibit glucose-dependent glucagon secretion and as a consequence increase satiety. Most of the studies conducted with OEA supplementation over the last two decades have focused on regulation of metabolic dysfunction related to lipid and sugar metabolism and its effectiveness as a treatment for obesity and eating disorders. OEA supplementation has been shown to reduce food intake and body weight gain in obese rodents and humans

[0010] Significant and noteworthy metabolic and pharmacological effects have been associated with OEA supplementation that include but that are not limited to neuroprotection, anti-inflammatory action, the amelioration of mood disorders, infertility and the improvement of cognitive function. These studies suggest that OEA is working as lipid messenger of the gutbrain axis, as OEA produced or assimilated in the gut can travel through the vagal nerve stimulating the vagal sensory nerves via TRPV1 activation in the brain. Interestingly, TRPV1 has not only analgesic and thermosensation effects but also can contribute to the regulation of energy hemostasis through control of feeding and energy expenditure.

[0011] OEA is endogenously produced in human body primarily in the small intestine, OEA is highly unstable as it can be degraded into oleic acid and ethanolamine by two different groups of intracellular enzymes including fatty acid amide hydrolase (FAAH) and N-acylethanolamine-hydrolyzing acid amidase 4Attorney Docket No. 11716-008WO1 (NAAA) in the intestinal epithelium. OEA catabolism is tightly regulated by fasting or feeding state; however genetic and exogenous factors like stress, diseases, lifestyle can significantly affect OEA levels in the human body. OEA supplementation in human diet can be considered as essential to assure body homeostasis and good mental health. OEA can be absorbed from food directly, therefore can be regarded as “essential bioactive dietary lipid.

[0012] Considering the above perspective, the exploration of edible dietary sources highly enriched in OEA is critical for its incorporation in a daily human diet. This is very challenging as NAE-enriched food sources are limited and in general poorly consumed in a Western diet. OEA doses reported in literature showing therapeutic effects (in the case of obesity treatments, improvement of glycemic status, energy metabolism and anti-inflammatory treatments) ranged between 125 to 400 mg daily, which is an incredibly high dose compared to the reasonable amount of NAEs that humans may consume in a daily meal. Indeed, while Mediterranean and Vegetarian diets account for about 300 ug / day of NAEs, typical Western diet account for a 2- to 4-fold lower daily rate (8 ug / day). Therefore, OEA supplementation in daily diet is recommended, especially for people in a food insecure areas, i.e. household-level economic and social condition of limited or uncertain access to adequate and nutritious food; indeed, food insecurity has been recently considered as an important risk factor of obesity and chronic inflammation and for susceptibility to diseases.

[0013] There remains a need for improved systems and methods for obtaining bioactive lipids from natural sources. There remains a need for improved systems and methods for obtaining polyunsaturated fatty acids and derivatives (i.e., oxylipin derivatives) from natural sources. There remains a need for improved systems and methods for obtaining oleoylethanolamide from natural sources. There remains a need for improved polyunsaturated fatty acid compounds, including polyunsaturated fatty acid amide compounds and derivatives (i.e., oxylipin derivatives of fatty acid amides). There remains a need for improved supplement compositions providing nutritionally important compounds. There remains a need for improved supplement compositions providing polyunsaturated fatty acids. There remains a need for improved supplement compositions providing polyunsaturated fatty acid amide compounds, including oleoylethanolamide. BRIEF DESCRIPTION OF THE FIGURES

[0014] Figure 1 depicts a workflow diagram representing the steps if the experimental design for untargeted metabolomic analysis of walnut seed coat.

[0015] Figure 2 depicts a diagram representing the walnut processing waste stream generating “Blower Fluff (BF)” and “Sorting room Meal (SRM)” samples respectively.

[0016] Figure 3 depicts metabolites in a walnut tissue. 5Attorney Docket No. 11716-008WO1

[0017] Figure 4 depicts a lipid profile in a walnut tissue.

[0018] Figure 5 depicts a lipid profile in a walnut tissue.

[0019] Figure 6 depicts an oxylipin profile in a walnut tissue.

[0020] Figure 7 depicts a DC-FA profile in a walnut tissue.

[0021] Figure 8 depicts a fatty acid amide profile in a walnut tissue.

[0022] Figure 9 depicts a hydroxy fatty acid profile in a walnut tissue.

[0023] Figure 10 depicts molecular processes intrinsically related to stress resilience and the physiological processes associated with programmed desiccation of the walnut pellicle.

[0024] Figure 11 depicts a graphical display of linoleate-derived oxylipins and acylethanolamides in walnut embryos.

[0025] Figure 12 depicts oleoylethanolamide levels in walnut embryos across developmental stages.

[0026] Figure 13 depicts linoleoylethanolamide levels in walnut embryos across developmental stages.

[0027] Figure 14 depicts synthetic routes to obtain compounds of the disclosure.

[0028] Figure 15 depicts images of walnut embryos in different development stages. Figure 15A depicts early-stage walnut embryos; Figure 15B depicts mid-stage walnut embryos; Figure 16C depicts late-stage walnut embryos. DETAILED DESCRIPTION

[0029] Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0030] 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. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includesfrom the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will 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.

[0031] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. 6Attorney Docket No. 11716-008WO1

[0032] Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. “Exemplary” means “an example of” and is not intended to convey an indication of a preferred or ideal embodiment. “Such as” is not used in a restrictive sense, but for explanatory purposes.

[0033] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0034] Disclosed herein are methods for obtaining lipid compounds from a walnut tissue composition including the step of separating the lipid from the walnut tissue. In certain implementations, the lipid compound includes a fatty acid compound, for example an unsaturated fatty acid compound like a polyunsaturated fatty acid compound.

[0035] In some implementations, the fatty acid compound is a hydroxyalkyl amide of a fatty acid compound, for example a hydroxyalkyl amide of an unsaturated fatty acid compound, such as a hydroxyalkyl amide of a polysaturated fatty acid compound. In some implementations the hydroxyalkyl amides have the formula *-NH(CH2)nOH, where *- represents the bond to the carbonyl of the fatty acid compound, and n is an integer from 2-6, preferably 2-4, more preferably 2-3, and even more preferably 2. In certain implementations the hydroxyalkyl amide has the formula *-NHCH2CH2OH, which may alternatively be designated a 2-ethanolamide.

[0036] In some implementations, the fatty acid compound is a carboxyalkyl amide of a fatty acid compound, for example a carboxyalkyl amide of an unsaturated fatty acid compound, such as a hydroxyalkyl amide of a polysaturated fatty acid compound. In some implementations the carboxyalkyl amides have the formula *-NH(CH2)mCOOH, where *- represents the bond to the carbonyl of the fatty acid compound, and m is an integer from 1-6, preferably 1-3, more preferably 1-2, and even more preferably 1. In certain implementations the hydroxyalkyl amide has the formula *-NHCH2COOH, which may alternatively be designated a glycine amide.

[0037] In certain implementations, fatty acid compound is a saturated fatty acid, mono-unsaturated fatty acid, di-unsaturated fatty, tri-unsaturated fatty acid, tetra-unsaturated fatty acid, or a combination 7Attorney Docket No. 11716-008WO1 thereof. As used herein, a mono-unsaturated fatty acid includes a single element of unsaturation in the lipid chain, e.g., a carbon-carbon double bond, or simply, a double bond. As used herein, a di- unsaturated fatty acid includes two elements of unsaturation in the lipid chain, e.g., two double bonds. A similar definition applies to higher orders of unsaturation. In some implementations, each element of unsaturation is a double bond. In some implementations, an element of unsaturation can be a ring element, for example an epoxide. In some implementations, the unsaturation can be an oxo, i.e., a ketone or aldehyde.

[0038] In some implementations, the lipid can be an omega-3 fatty acid, an omega-6 fatty acid, or a combination thereof. In certain implementations, the lipid can be crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, tetra-unsaturated fatty acids, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosanolpentaenoic acid, docosahexaenoic acid, herring acid, petroselinic acid, paullinic acid, gondoic acid, erucic acid, brassidic acid, nervonic acid, arachidonic acid, or a combination thereof.

[0039] In some implementations, the lipid can be a hydroxyalkyl amide or carboxyalkyl amide of an omega-3 fatty acid, a hydroxyalkyl amide or carboxyalkyl amide of an omega-3 fatty acid, or a combination thereof. In certain implementations, the lipid can be a hydroxyalkyl amide or carboxyalkyl amide of crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, tetra- unsaturated fatty acids, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosanolpentaenoic acid, docosahexaenoic acid, herring acid, petroselinic acid, paullinic acid, gondoic acid, erucic acid, brassidic acid, nervonic acid, arachidonic acid, or a combination thereof.

[0040] In some implementations, the lipid can be a 2-ethanolamide of an omega-6 fatty acid, a 2- ethanolamide of an omega-6 fatty acid, or a combination thereof. In certain implementations, the lipid can be a 2-ethanolamide of crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, tetra-unsaturated fatty acids, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosanolpentaenoic 8Attorney Docket No. 11716-008WO1 acid, docosahexaenoic acid, herring acid, petroselinic acid, paullinic acid, gondoic acid, erucic acid, brassidic acid, nervonic acid, arachidonic acid, or a combination thereof.

[0041] In certain implementations, the lipid includes oleoylethanolamide.

[0042] In certain implementations, the walnut tissue includes the walnut pellicle. In certain implementations the walnut tissue is a walnut embryo, for example a somatic walnut embryo. Mixtures of the tissue types may also be used.

[0043] In some implementations, the walnut tissue can be dehydrated prior to separating the lipid compound from the tissue. In some implementations, the walnut tissue can be dehydrated such that the resulting tissue includes water in an amount of 25 wt.% or less, 20 wt.% or less, 15 wt.% or less, 10 wt.% or less, 5 wt.% or less, 1 wt.% or less, 0.5 wt.% or less, or 0.1 wt.% or less.

[0044] In some implementations the walnut tissue can be crushed or ground to give a walnut tissue powder. The walnut tissue may be dehydrated prior to conversion to a powder, or subsequent to conversion to a powder. The walnut tissue may be partially dehydrated, converted to a powder, and then subjected to further dehydration.

[0045] In some implementations, the drying process includes one or more of the following steps: a) Placing in a desiccator at room temperature b) Placing in a desiccator at higher temperatures (greater than 25 °C and below 50 - 70 °C) c) Grinding in liquid nitrogen and lyophilization to obtain dry powder. d) Drying in culture by increasing the osmotic pressure of the culture medium with by addition of non-metabolizable osmoticum e.g., PEG (polyethylene glycol) or sugar alcohols (sorbitol, mannitol, xylitol etc.).

[0046] In some implementations, the walnut tissue, for example dehydrated walnut tissue, optionally in powder form, can be contacted with a solvent such that the lipid compound is extracted into the solvent away from the walnut tissue. Exemplary solvents include water, polar protic solvents, polar aprotic solvents, non-polar solvents, and combinations thereof. In some implementations the solvent can include supercritical carbon dioxide. In some implementations the solvent can be water or water mixed with an alcohol such as methanol, ethanol, or isopropanol. In certain implementations the solvent is a mixture of water and ethanol, for example in a ratio of 3:7 water:ethanol (v / v).

[0047] In some implementations, the walnut tissue (optionally dehydrated and / or powdered) may be pressed to extract lipid compounds. A solvent may be used to facilitate the press operation, including solvents like water or oil, e.g., olive oil, peanut oil, canola oil, vegetable oil, etc. In some implementations, the walnut tissue may be subjected to steam distillation to separate lipid compounds from the walnut tissue. In certain implementations, the walnut tissue is a walnut embryo. In some implementations the walnut tissue is an early-stage walnut embryo, for example a cotyledonary 9Attorney Docket No. 11716-008WO1 translucent somatic embryo which is characterized by globular, heart and torpedo shape. In some implementations the walnut tissue is mid-stage walnut embryo, for example a large translucent cotyledonary somatic embryo. In some implementations the walnut tissue is a late-stage walnut embryo, for example opaque cotyledonary embryos. Images of walnut embryos in different developmental stages are depicted in Figure 15.

[0048] In some implementations, the extraction process includes one or more of the following steps: Oil extraction with pressing; Supercritical fluid extraction; Microwave extraction; Milk (any beverage) production by homogenization of walnut embryos; Extraction of oil bodies and protein bodies

[0049] Also disclosed herein are walnuts and walnut tissue enriched in lipid components, relative to naturally occurring walnuts and walnut tissues. The level of enrichment may be determined by quantitating the wt.% of the lipid component (for example using mass spectrometry, NMR, HPLC, or a combination thereof). In some implementations, the composition is enriched in bioactive lipids including oleoylethanolamide, any oleoylethanolamide derivative and fatty acid amides. In some implementations, the enriched composition is enriched in the compound oleoylethanolamide. In certain implementations, the enriched compositions include oleoylethanolamide in an amount greater than 10 wt.%, greater than 25 wt.%, greater than 50 wt.%, greater than 75 wt.%, greater than 100 wt.%, greater than 200 wt.%, greater than 300 wt.%, greater than 400 wt.%, or greater than 500 wt.% of oleoylethanolamide present in naturally occurring walnut compositions.

[0050] The enriched walnut tissues may be obtained by one or more of the following steps: • Chemical-physical processes to boost the production of bioactive lipids (i.e., exposure to environmental stress) include exposure to increased temperature, exposure to decreased temperature, overexposure to light, under exposure to light, submersion in water, water deprivation, oxygen deprivation, oxygen oversaturation, carbon dioxide deprivation, carbon dioxide oversaturation, and / or supplementing the media with growth regulators (dopamine, jasmonic acid, auxin, cytokinin etc.); and / or • Fermentation with active bacteria like Lactiplantibacillus plantarum, Bifidobacteria, etc.

[0051] In certain implementations, the lipid enriched compositions can be a walnut embryo, preferably a somatic walnut embyro. In certain implementations, the lipid enriched compositions can be a walnut shoot grown from somatic walnut embryo. In certain implementations, the somatic walnut embryo is enriched in bioactive lipids relative to wild type walnut embryos, i.e., those cultivated under conventional conditions. Also disclosed herein are walnut shoots obtained from walnut embryos, 10Attorney Docket No. 11716-008WO1 walnut shoot obtained from walnut embryos enriched in bioactive lipids, and walnut shoot enriched in bioactive lipids.

[0052] In some implementations, the environmental stress can include cultivation in a medium having a CO2content greater than 1,000 ppm, greater than 1,500 ppm, greater than 2,000 ppm, greater than 2,500 ppm, greater than 3,000 ppm, greater than 4,000 ppm, or greater than 5,000 ppm.

[0053] In some implementations, the environmental stress can include cultivation in an atmosphere having a CO2content greater than 1,000 ppm, greater than 1,500 ppm, greater than 2,000 ppm, greater than 2,500 ppm, greater than 3,000 ppm, greater than 4,000 ppm, or greater than 5,000 ppm.

[0054] In some implementations, the environmental stress can include cultivation in the presence of ethylene.

[0055] In some implementations, the environmental stress can include cultivation in a medium including ethylene at a concentration from 200-10,000 ppm, from 200-5,000 ppm, from 200-2,500 ppm, from 500-2,500 ppm, from 500-1,500 ppm, from 500-1,000 ppm, from 1,000-1,500 ppm, or from 750- 1,250 ppm.

[0056] In some implementations, the environmental stress can include cultivation in an atmosphere including ethylene at a concentration from 200-10,000 ppm, from 200-5,000 ppm, from 200-2,500 ppm, from 500-2,500 ppm, from 500-1,500 ppm, from 500-1,000 ppm, from 1,000-1,500 ppm, or from 750- 1,250 ppm.

[0057] In some implementations, the environmental stress can include cultivation in an environment including jasmonic acid.

[0058] In some implementations, the environmental stress can include cultivation in a medium including jasmonic acid at a concentration from 1-100 ppm, from 1-50 ppm, from 10-50 ppm, from 10- 25 ppm, or from 25-50 ppm.

[0059] In some implementations, the environmental stress can include cultivation in an atmosphere including jasmonic acid at a concentration from 1-100 ppm, from 1-50 ppm, from 10-50 ppm, from 10- 25 ppm, or from 25-50 ppm.

[0060] In some implementations, the environmental stress can include cultivation in a growing medium having a moisture content of less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, or less than 1%.

[0061] In some implementations, the environmental stress can include cultivation in a desiccator.

[0062] In some implementations, the environmental stress can include cultivation in a growing medium including polyethylene glycol.

[0063] In some implementations, the environmental stress can include cultivation in a growing medium including polyethylene glycol, wherein the polyethylene glycol has a MW from 2,000-50,000, from 11Attorney Docket No. 11716-008WO1 2,000-25,000, from 2,000-15,000, from 2,000-10,000, from 2,000-8,000, from 2,000-6,000, or from 2,000-4,000.

[0064] In some implementations, the environmental stress can include cultivation in a growing medium including polyethylene glycol at a concentration from 20-500 mmol / kg, from 20-400 mmol / kg, from 20- 300 mmol / kg, from 20-200 mmol / kg, from 20-100 mmol / kg, from 20-80 mmol / kg, or from 40-80 mmol / kg.

[0065] In some implementations, the environmental stress can include exposure to a temperature from 30-50 °C. from 35-45 °C., from 35-40 °C., from 40-45 °C., or from 37.5-42.5 °C. for a period of 0.1-200 hours, 1-6 hours, 1-36 hours, 1-72 hours, 1-100 hours, 1-150 hours, 1-200 hours, 6-36 hours, 6-72 hours, 6-100 hours, 36-72 hours, 36-100 hours, 72-100 hours, 72-150 hours, or 100-200 hours.

[0066] In some implementations, the environmental stress can include exposure to a temperature from 0-15 °C. from 2-15 °C., from 5-15 °C., from 5-10 °C., from 10-15 °C. or from 7.5-12.5 °C. for a period from 1-14 days, from 3-14 days, from 5-15 days, from 5-10 days, or from 10-15 days.

[0067] In some implementations, the environmental stress can include exposure to light for a period from 1-10 hours / day, from 3-10 hours / day, from 5-10 hours a day from 3-5 hours days, or from 4-7 hours / day.

[0068] In some implementations, combinations of the above-identified environmental stresses can be used. For example, the embryos can be submerged in water containing dissolved polyethylene glycol and jasmonic acid. In some implementations the embryos can be submerged in water and subjected to elevated and / or decreased temperature. In some implementations the embryos can be cultivated in a desiccator in an environment including ethylene, decreased oxygen, and / or increased carbon dioxide.

[0069] In certain implementations, the lipid compositions disclosed herein can be produced by: cultivating somatic embryos in a liquid media supplied with nutrient formulations supporting growth of the embryos and / or development of specific traits and separating the bioactive lipids from the cultured tissue of walnut somatic embryos. In some implementations the growing medium is Driver and Kuniyuki Walnut medium

[0070] Also disclosed herein are methods of cultivating walnut tissues having enriched lipid profiles. In some implementations the cultivated walnut tissue is a walnut embryo, preferably a somatic walnut embryo. In some implementations the walnut embryo may be disposed in a growing medium and exposed to hypoxic conditions. In some implementations the walnut embryos may be submerged in water. In some implementations the walnut embryos may be submerged in water for a period of 0.1- 200 hours, for example 1-6 hours, 1-36 hours, 1-72 hours, 1-100 hours, 1-150 hours, 1-200 hours, 6-36 hours, 6-72 hours, 6-100 hours, 36-72 hours, 36-100 hours, 72-100 hours, 72-150 hours, or 100-200 hours. In some implementations the oxygen concentration of the water can be no greater than 10 12Attorney Docket No. 11716-008WO1 mg / L, no greater than 5 mg / ml, no greater than 2.5 mg / L, or no greater than 1 mg / L. In some implementations, the walnut embryos may be taken directly from the hypoxic environment and subjected to further processing, for example extractive processes to isolate lipids, or dehydrating and grinding to produce a supplement / food composition. In other implementations the walnut embryos may be removed from the hypoxic conditions and exposed to a non-stress environment (i.e., regular cultivation conditions) including regular atmosphere for a period of at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 30 hours, at least 40 hours, or at least 50 hours prior to further processing.

[0071] Also disclosed herein are compounds of Formula (1): [Formula (1)], whereinR1is a C6-C12 alkylene, C6-C12 alkenylene, or C6-C12 alkynylene; R2is a C6-C12 alkyl, C6-C12 alkenyl, or C6-C12 alkynyl; R1Nis a C1-6hydroxyalkyl or C1-6carboxyalkyl; R2Nis H, C1-6alkyl, C1-6hydroxyalkyl, or C1-6carboxyalkyl; or R1Nand R2Ntogether form a 3-8 membered ring having at least one hydroxy group or carboxy group; and one of the following applies: Rc1is H or C(=O)lipid; Rc2is H, and ^ is absent; Rc1and Rc2together form a bond, and ^ is absent; or Rc1is absent, Rc2is H, and ^ is a single bond; wherein lipid is a C6-24alkyl, C6-24alkenyl, or C6-24alkynyl.

[0072] The hydroxyalkylamides of oxidized fatty acids are not naturally occurring in wild-type walnut tissues.

[0073] In some implementations R1Nis CH2CH2OH or CH2COOH, and R2Nis H.

[0074] In some implementations R1is a C6-C12 alkylene, C6-C10 alkylene, C7-C9 alkylene, or C8alkylene. In other implementations R1is a C6-C12 alkenylene, C6-C10 alkenylene, C7-C9 alkenylene, or C8alkenylene. In certain implementations R2is a C6-C12 alkyl, C6-C10 alkyl, C7-C9 alkyl, or C8alkyl.

[0075] In some implementations, RC1and RC2are both H. In some implementations, RC2is H and RC1is C(=O)lipid. In further implementations, lipid is C6-24alkenyl having one, two, three, four, five, or six carbon-carbon double bonds. In some implementations, lipid is derived from a fatty acid. In some implementations, lipid is derived from crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, 13Attorney Docket No. 11716-008WO1 oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, tetra-unsaturated fatty acids, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosanolpentaenoic acid, docosahexaenoic acid, herring acid, petroselinic acid, paullinic acid, gondoic acid, erucic acid, brassidic acid, nervonic acid, or arachidonic acid.

[0076] In some implementations, the compound of Formula (1) is a compound of Formula (1a): [Formula (1a)], wherein:Raand Ra’are in each case all hydrogen, or two of Raand Ra’are hydrogen and the other two of Raand Ra’form a double bond, or all of Raand Ra’together form a triple bond; Rband Rb’are in each case all hydrogen, or two of Rband Rb’are hydrogen and the other two of Rband Rb’form a double bond, or all of Rband Rb’together form a triple bond.

[0077] In certain implementations the compound of Formula (1) is a compound of Formula (1b): [Formula (1b)].

[0078] In certain ia compound of Formula (1b*): [Formula (1b*)], wherein the carburity of at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%.

[0079] In certain implementations the compound of Formula (1) is a compound of Formula (1b**): *)],Attorney Docket No. 11716-008WO1 wherein the carbon marked with an asterisk has an enantiomeric purity of at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%.

[0080] In certain implementations of the compound of Formula (1b), (1b*) and (1b**), Rc1is hydrogen.

[0081] In certain implementations of the compound of Formula (1b) ), (1b*) and (1b**), Raand Ra’are in each case hydrogen, Rb’is in case hydrogen, and both Rbtogether form a double bond.

[0082] In certain implementations of the compound of Formula (1b) ), (1b*) and (1b**), both Ratogether form a double bond, Ra’is in each case hydrogen, Rb’is in case hydrogen, and both Rbtogether form a double bond.

[0083] In some implementations, the compound of Formula (1) is a compound having the formula: , , , H , H , H , ,Attorney Docket No. 11716-008WO1 or

[0084] Hydroxyalkylamides of oxidized fatty acids can be obtained by reacting a hydroxyalkylamine with an oxidized fatty acid. The hydroxyl group may or may not be protected as known in the art depending on the conditions used to form the amide bond. In other embodiments, an amide bond may be formed between the hydroxylalkylamine and a unsaturated fatty acid. The resulting compound may be then oxidized either stereoselectively or non-stereoselectively. In the case of non-stereoselective oxidations, the mixture may be resolved into single enantiomers and diastereomers using chiral chromatography, kinetic resolution for example with enzymatic acylation and / or diacylation, or reaction with an enatiopure compound followed by separation of the diastereomeric products. An exemplary synthetic process is depicted in Figure 14. In other embodiments, the racemic 10-hydroxy derivative of oleolylethanolamide can be resolved via enzyme-catalyzed acylation or deacylation for example using Novozym 435.

[0085] Also disclosed herein are compositions including the enriched walnut tissues described herein and / or one or more compounds of Formula (1). In some implementations the cultivated walnut embryos described herein may be dried and processed to produce a powder. The powder may be loaded into an orally administrable dosage form, e.g., a capsule and used as a nutritional supplement.

[0086] Also disclosed are dried walnut compositions having the following ingredients: 1-15 wt.% fat; 20-50 wt.% protein; and 40-80% carbohydrates.

[0087] In some implementations, the dried walnut composition can include fat in an amount from 1-10 wt.%, 1-5 wt.%, 5-10 wt.%, or 2.5-7.5 wt.%.

[0088] In some implementations, the dried walnut composition can include protein in an amount from 20-40 wt.%, 20-30 wt.%, 30-40 wt.%, or 25-35 wt.%.

[0089] In some implementations, the dried walnut composition can include carbohydrates in an amount from 50-80 wt.%, from 50-70 wt.%, or from 60-70 wt.%.. 16Attorney Docket No. 11716-008WO1 EXAMPLES

[0090] The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention in any manner whatsoever. Example 1 – Extraction of walnut tissue

[0091] Physiologically mature walnut fruits were harvested from three independent trees (Juglans regia cv. ‘Vina’) at the University of California, Davis, courtesy of the Walnut Improvement Program. Trees one and two were physically adjacent to one another in a single block (38°32'19.6"N 121°47'45.4"W), while tree 3 was in the separate but nearby block (38°32'39.8"N 121°47'39.6"W). Fruit maturity was assessed by monitoring Packing Tissue Brown (PTB) and hull dehiscence. The trees were harvested by shaking once PTB was achieved, indicating full maturation of the kernels, and when greater than 50% of the canopy exhibited partial to complete dehiscence of the hull (harvest maturity stages M2 “Hull Split” and M3 “Hull Bloom”, respectively), which facilitates harvest of the in-shell nuts. Bulk samples were collected spatially around the trees in the four cardinal directions, with each quadrant bounded by the nearest ordinal directions (i.e., the “North” quadrant spanned from North-West to North-East). Nuts were collected on tarps, and immature fruit (harvest maturity stage M1 “Hull Intact”) were discarded. Fruit exhibiting hull dehiscence were manually hulled, inspected for insect or pathogen damage, and then bagged and gently air-dried at room temperature (~21°C).

[0092] Following a brief in-shell storage period at ambient conditions, the nuts were cracked open carefully using a small hammer, needle-nose pliers, and a metal scoopula. The two kernel halves from any given nut were retained as a pair to prevent mixing kernel halves and loss of pooled sample independence. Phenotypically uniform kernels that met the DFA of California visual grading standards 1 and 2 ("Extra Light" and "Light", respectively) were set aside. Between two and three whole kernels, comprised of two kernel halves each, were used to compose pooled replicates. Each quadrant produced one pooled replicate, yielding 12 total independent biological (pooled) replicates, with four from each mother tree. The samples were snap-frozen in liquid nitrogen and stored at -80°C to preserve their integrity for downstream processing.

[0093] Isolation of the pellicle from whole kernel samples was achieved through a combined method of cryogens and mechanical disruption. Whole kernels were placed into liquid nitrogen pre-chilled mortars (kept cool over a bed of dry ice) and snap-frozen with further additions of liquid nitrogen. Gentle mechanical disruption of the kernels with a pestle caused fragmentation of the frozen meat and clean separation of the pellicle from the meat. For pellicles that did not separate easily, notably around the connection point with the maternal vascular bundle (funiculus), removal was facilitated by further fragmentation and / or prying with forceps or a scalpel. Following isolation, the pellicle samples were lyophilized overnight in a food-grade freeze drier (Harvest Right; Model HR3000-AL). The lyophilized 17Attorney Docket No. 11716-008WO1 pellicle samples were split evenly between 22-mL Eppendorf microcentrifuge tubes and homogenized in a laboratory mixer mill with a single 5-mm stainless steel bead for 1 minute at 30 Hz (Retsch; Model MM-400). This resulted in a fine powder ultimately used for the subsequent exploratory untargeted metabolomic analysis.

[0094] Broad metabolomic profiles of the pellicle samples were generated by West Coast Metabolomics Center (WCMC) at UC Davis across three provided platforms. These included: 1) complex lipids by BEH C18-QTOF MS / MS; 2) phenolics and fatty acids by BEH C18-Q Exactive MS / MS; and 3) primary metabolites by GC-TOF MS.10-mg of lyophilized tissue was supplied per sample and was sufficient for analysis on all three platforms.

[0095] Samples were extracted using the Matyash extraction procedure, including MTBE, MeOH, and H2O. The organic (upper) phase was dried down and submitted for resuspension and injection onto the LC, while the aqueous (bottom) phase was dried down and submitted to derivatization for GC. They are resuspended with 110 uL of a solution of 9:1 methanol: toluene and 50 ng / mL CUDA. This is then shaken for 20 seconds, sonicated for 5 minutes at room temperature, and then centrifuged for 2 minutes at 16100 rcf. The samples are then aliquoted into three parts.33 uL are aliquoted into a vial with a 50 uL glass insert for positive and negative mode lipidomics. The last part is aliquoted into an eppendorf tube to be used as a pool.

[0096] The samples are then loaded up on an Agilent 1290 Infinity LC stack. The positive mode was run on an Agilent 6546 with a scan range of m / z 120-1200 Da with an acquisition speed of 2 spectra / s. The other sample aliquot was run in negative mode, which was run on Agilent 1290 Infinity LC stack. The acquisition rate was 2 spectra / s with a scan range of m / z 60-1200 Da. The mass resolution for the Agilent 6546 is 10,000 for ESI (+) and 30,000 for ESI (-) for the Agilent 6550.

[0097] Chromatographic conditions: separations were performed using Acquity Premier BEH C181.7 µm, 2.1 x 50 mm Column in both polarities. The gradient used for both polarities is 0 min 15% (B), 0.75 min 30% (B), 0.98 min 48% (B), 4.00 min 82% (B), 4.13-4.50 min 99% (B), 4.58-5.50 min 15% (B) with a flow rate of 0.8 mL / min. ESI (+) Mobile phase A: 60:40 v / v acetonitrile:water + 10 mM ammonium formate + 0.1% formic acid Mobile phase B: 90:10 v / v isopropanol:acetonitrile + 10 mM ammonium formate + 0.1% formic acid. Chromatographic parameters for ESI (-): Mobile phase A: 60:40 v / v acetonitrile:water + 10 mM ammonium acetate Mobile phase B: 90:10 v / v isopropanol:acetonitrile + 10 mM ammonium acetate. For both operating modes: column temperature was set constant at 65°C. The acquisition rate was 2 spectra / s with a scan range of m / z 60-1200 Da. The mass resolution for the Agilent 6546 is 10,000 for ESI (+) and 30,000 for ESI (-) for the Agilent 6550.

[0098] The general workflow for data processing is using MS-DIAL, followed by a blank subtraction in Microsoft Excel and cleanup of data using MS-FLO. The first step is to converted files using the Abf 18Attorney Docket No. 11716-008WO1 Converter. Default parameters are used for the processing of MS-Dial data, except for minimum peak height and width which is adjusted for the instrument where the samples ran. Once the results have been exported from MS-DIAL, a blank reduction is done based on the max peak height relative to blank average height, the average of all non-zero peak heights for samples, and if the feature is found in at least one sample. Next using MS-FLO, potential duplicates and isotopes are checked and deleted if confirmed. Then MS / MS spectra were checked before combining adducts. Peaks are annotated in manual comparison of MS / MS spectra and accurate masses of the precursor ion to spectra given in the Fiehn laboratory’s LipidBlast spectral library. Additional peaks were found by manual curation of sample chromatograms on a scan-by-scan basis. MassHunter Quant software was then used to verify peak candidates based on peak shape, peak height reproducibility and retention time reproducibility in replicate samples. Valid and reproducible peaks were analyzed by targeted MS / MS with the aim of increasing overall peak annotations in both positive and negative modes.

[0099] The samples were then injected onto a Waters Acquity Premier BEH C181.7 µm, 2.1 x 50 mm column. The gradient used was 0 min 1% (B), 0.50 min 1% (B), 7.50 min 99% (B), 9.00 min 99% (B), 9.20 min 1% (B), 10.00 min 1% (B), with a flow rate of 0.6 mL / min. Mobile phase A was 100% LC / MS grade water + 0.1% Formic Acid and mobile phase B was 100% ACN + 0.1% Formic Acid. Injection volume varies by study and ranges between 0.1 uL and 5 uL. Vanquish UHPLC system (ThermoFisher Scientific) was used. A Thermo Q-Exactive HF Orbitrap MS instrument was used in both positive and negative ESI modes to acquire LC-MS / MS data with the following parameters: mass range 80−1200 m / z; full scan MS1 mass resolving power 60,000, data-dependent MSMS (dd-MSMS) 2 scans per cycle (4 scans per cycle for pooled MSMS injections), normalized collision energy at 20%, 30%, and 40%, dd-MSMS mass resolving power 15,000.

[0100] Samples were extracted using 1 mL of 80:20 MeOH:H2O. Samples were vortexed and centrifuged.450 uL of the supernatant was dried for analysis. Dried samples were resuspended with 100 uL of a solution 75:25 H2O:ACN containing internal standards (CUDA, D3-L-Carnitine, Val-Tyr-Val, D4- Daidzein, D9-Reserpine, and D5-Hippuric Acid). Samples were then vortexed for 10 seconds, sonicated for 5 minutes at room temperature, and then centrifuged for 2 minutes at 16,000 rcf.60 uL supernatant from each samples was transferred into a LC-MS vial containing a glass microinsert.30 uL supernatant from each sample was then transferred into an eppendorf tube and vortexed for use as a pool.

[0101] The samples were then injected onto a Waters Acquity Premier BEH C181.7 µm, 2.1 x 50 mm column. The gradient used was 0 min 1% (B), 0.50 min 1% (B), 7.50 min 99% (B), 9.00 min 99% (B), 9.20 min 1% (B), 10.00 min 1% (B), with a flow rate of 0.6 mL / min. Mobile phase A was 100% LC / MS grade water + 0.1% Formic Acid and mobile phase B was 100% ACN + 0.1% Formic Acid. Injection volume varies by study and ranges between 0.1 uL and 5 uL. Vanquish UHPLC system (ThermoFisher Scientific) 19Attorney Docket No. 11716-008WO1 was used. A Thermo Q-Exactive HF Orbitrap MS instrument was used in both positive and negative ESI modes to acquire LC-MS / MS data with the following parameters: mass range 80−1200 m / z; full scan MS1 mass resolving power 60,000, data-dependent MSMS (dd-MSMS) 2 scans per cycle (4 scans per cycle for pooled MSMS injections), normalized collision energy at 20%, 30%, and 40%, dd-MSMS mass resolving power 15,000.

[0102] Samples extracted using Matyash extraction procedure which includes MTBE, MeOH, and H2O. The organic (upper) phase was dried down and submitted for resuspension and injection onto the LC while the aqueous (bottom) phase was dried down and submitted to derivatization for GC. They are shaken at 30C for 1.5 hours. Then 91 uL of MSTFA + FAMEs to each sample and they are shaken at 37C for 0.5 hours to finish derivatization. Samples are then vialed, capped, and injected onto the instrument.

[0103] We use a 7890A GC coupled with a LECO TOF.0.5 uL of derivatized sample is injected using a splitless method onto a RESTEK RTX-5SIL MS column with an Intergra-Guard at 275°C with a helium flow of 1 mL / min. The GC oven is set to hold at 50°C for 1 min then ramp to 20°C / min to 330°C and then hold for 5 min. The transferline is set to 280°C while the EI ion source is set to 250°C. The Mass spec parameters collect data from 85m / z to 500m / z at an acquisition rate of 17 spectra / sec.

[0104] Raw data files are preprocessed directly after data acquisition and stored as ChromaTOF-specific *.peg files, as generic *.txt result files and additionally as generic ANDI MS *.cdf files. ChromaTOF vs. 2.32 is used for data preprocessing without smoothing, 3 s peak width, baseline subtraction just above the noise level, and automatic mass spectral deconvolution and peak detection at signal / noise levels of 5:1 throughout the chromatogram. Apex masses are reported for use in the BinBase algorithm. Result *.txt files are exported to a data server with absolute spectra intensities and further processed by a filtering algorithm implemented in the metabolomics BinBase database. The BinBase algorithm (rtx5) used the settings: validity of chromatogram (<10 peaks with intensity >10^7 counts s-1), unbiased retention index marker detection (MS similarity>800, validity of intensity range for high m / z marker ions), retention index calculation by 5th order polynomial regression. Spectra are cut to 5% base peak abundance and matched to database entries from most to least abundant spectra using the following matching filters: retention index window ±2,000 units (equivalent to about ±2 s retention time), validation of unique ions and apex masses (unique ion must be included in apexingmasses and present at >3% of base peak abundance), mass spectrum similarity must fit criteria dependent on peak purity and signal / noise ratios and a final isomer filter. Failed spectra are automatically entered as new database entries if s / n >25, purity <1.0 and presence in the biological study design class was >80%. All thresholds reflect settings for ChromaTOF v.2.32. Quantification is reported as peak height using the unique ion as default, unless a different quantification ion is manually set in the BinBase administration software BinView. A quantification report table is produced for all database entries that are positively 20Attorney Docket No. 11716-008WO1 detected in more than 10% of the samples of a study design class (as defined in the miniX database) for unidentified metabolites. A subsequent post-processing module is employed to automatically replace missing values from the *.cdf files. Replaced values are labeled as ‘low confidence’ by color coding, and for each metabolite, the number of high-confidence peak detections is recorded as well as the ratio of the average height of replaced values to high-confidence peak detections. These ratios and numbers are used for manual curation of automatic report data sets to data sets released for submission index.

[0105] The analytical approaches described in section 1.4 enabled the detection of 1595 features, 5274 features, and 534 features for the three platforms, respectively. However, the number of known, identified metabolites was of 216 features (60 in negative mode and 156 in positive mode), 221 (88 in negative mode and 133 in positive mode), and 156 features in each dataset from the three platforms, respectively. Each feature was investigated and manually annotated from the provided lists of identified metabolites. Querying publicly accessible information from KEGG (Kyoto Encyclopedia of Genes and Genomes), NIH-PubChem (National Institute of Health), and HMDB (Human Metabolome Database) resources with international chemical identifier (InChI) keys facilitated the acquisition of database identifiers, biochemical pathway information, and molecular structure information for each metabolite. Each metabolite was annotated with KEGG and PubChem identifiers, a “superpathway” and related “subpathway(s)” based on their known involvement in biological pathways, and biochemical classifications and related subclassifications based on molecular structure and functional groups.

[0106] Pellicle-enriched waste by-products from the mechanized industrial processing of walnuts were kindly provided by Carriere Family Farms. These by-products consisted of two different waste streams: “blower fluff” (BF) and “sorting room meal” (SRM), which come from earlier and later processing stages, respectively. The BF waste stream is generated at the end of the shelling process and is comprised of small shell pieces, dried packing tissue, nutmeat pieces, and pellicle fragments (Figure 2). The BF waste stream is generated at the end of the shelling process and is comprised of tiny shell pieces plus dried packing tissue, nutmeat pieces, and pellicle fragments (Figure 2). By weight, these components made up approximately 59%, 30%, and 11%, respectively. The SRM waste stream comes from the subsequent sorting of shelled kernels. It is composed almost exclusively of nutmeat pieces and pellicle fragments, with by-weight percentages of approximately 67% and 33%, respectively. Though both waste classes are composed of other walnut tissues besides the pellicle, these are the two most pellicle-enriched wastes from the mechanized processing of walnuts in California. The bulk samples, sealed in air-tight plastic bags, were immediately stored at –80 °C to maintain their integrity and were submitted for metabolomic analysis within one month of acquisition.

[0107] For each waste stream, three (technical) replicates were taken from each bulk sample and used to fuel a targeted metabolomics analysis. This 127-member panel included oxylipins (92), N- 21Attorney Docket No. 11716-008WO1 acylethanolamines (NAEs) (30), and polyunsaturated fatty acids (PUFAs) (5). No further processing of the samples was performed before LC-MS analysis as the type of wastes selected for investigation were already commercially dried and in the form of a coarse powder.

[0108] Complex lipids were extracted with 8:10:11 isopropanol / cyclohexane / ammonium acetate, followed by alkaline hydrolysis and isolation by solid phase extraction using modifications of previously reported methods. Briefly, ~50 mg ground waste byproduct was mixed with 5µL 0.2 mg / mL butylated hydroxytoluene / EDTA and homogenized in 410 µL isopropanol on a vertical ball mill (GenoGrinder 2010, SPEX SamplePrep, Metuchen, NJ, U.S.A.). Samples were mixed with 520 µL cyclohexane by vortexing for 3 min. The homogenate was then transferred to a 2-mL polypropylene 96-well plate. The homogenate was mixed with 570 µL of 0.1 M ammonium acetate, vortexed for 3 min, and centrifuged for 5 min at 2.3 g and 4 °C. The organic phase was transferred to a new plate, and the aqueous phase was re-extracted with 520 µL cyclohexane. The combined organic phases were evaporated under vacuum, and the residue was dissolved in 100 µL 1:1 methanol / toluene (v / v).

[0109] These total lipid extracts were then enriched with a suite of deuterated oxylipin free acids, incubated with 100 µL of 0.5 M sodium methoxide for 50 min at 50 °C, mixed with 100 µL water, and returned to 50 °C for 50 min. Samples were neutralized with 10 µL 20% glacial acetic acid and then diluted with 1 mL 0.1% acetic acid / 5% methanol, and oxylipins were trapped on a 10 mg Oasis HLB solid phase extraction column (Waters Corp, Milford Mass). After washing with 2 mL 0.1% acetic acid / 30% MeOH, analytes were eluted in 250 µL methanol with 1% acetic acid followed by 1 mL ethyl acetate and collected into plate wells containing 10 µL pf 20% glycerol in methanol. Solvents were removed by vacuum evaporation, and residues were reconstituted in 125 µL of 1-cyclohexyl ureido 3-dodecanoic acid (CUDA) and 1-phenyl ureido 3-hexanoic acid (PUHA) (Cayman Chemical; Ann Arbor, MI) at 100 nM in 1:1 methanol:acetonitrile. Samples were chilled at -20 °C for 15 min, filtered with 0.2 µm PVDF 96- well plates (Agilent Technologies; Santa Clara, CA), and then stored at -20 °C until analysis within 48 h.

[0110] Residues in extracts were separated on a 2.1 mm x 150 mm, 1.7 µm Acquity BEH C18 column (Waters, Milford, MA) and detected by electrospray ionization with scheduled multiple reaction monitoring on 6500 QTRAP (Sciex; Redwood City, CA). A binary gradient elution program, based on water with 0.1% Acetic Acid (solvent A) and 90% Acetonitrile / 10% isopropanol (solvent B), was adopted. The flow rate was always set at 0.5 mL / min and the column temperature at 60 °C. Analytes were quantified using isotope dilution and internal standard ratio-response methodologies against a minimum 7 pt calibration curve bracketing reported concentrations. F2-isoprostanes (F2isoPs) were detected as a complex cluster of analytes with the same mass transition as and surrounding PGF2a. F2- IsoP concentrations were estimated using the response of prostaglandin F2a (353.3 > 193.2 m / z. 22Attorney Docket No. 11716-008WO1

[0111] Analytical results generally met quality control criteria with respect to surrogate recoveries and replicate precision. Method performance was assessed through the routine analysis of blanks using analytical methods corrected for the performance of analytical surrogates. While apparent surrogate recoveries were ~20–50%, lower than generally observed for animal tissues, they were deemed acceptable. Experimental replicates appeared reasonable. Results are expressed in nmol / g tissue (i.e. µM) with the exception of a subset of analytes, including polyunsaturated fatty acids and six alpha- linolenic acid-derived oxylipins for which commercially available standards were not available: These analytes (denoted as screens) are reported as relative abundance across all measured samples (i.e. the sum of each metabolite across all samples is set to 100%). Out of 127 analytes panel, 45% were present in the samples and fulfilled all QC requirements, whereas 30% of the analytes were not detected.9- HODE and 13-KODE were above the highest calibration standard, and results were estimated by linear extrapolation of the calibration curve and should, therefore, only be used qualitatively.

[0112] All analysis and visualizations were performed in GraphPad PRISM (Version 10.1.1).

[0113] The curated annotations were used to explore the composition of major biochemical classes and subclasses within the untargeted metabolome of the walnut pellicle. Summarization of the data included counting feature membership in various biochemical classes, subclasses, and structurally related features withing a class or subclass. Each biochemical class or subclass was represented as relative percentage over the total number of the identified features in the dataset or in a specific class or subclass.

[0114] For each dataset (representing independent analytical platforms), the relative intensity of each metabolite was expressed as percentages of the total intensity for their respective platform. Using all of the data per platform, including unidentified features, the total un-normalized intensity was calculated. The sum total intensity for each sample was determined, and then the average of these total intensities (n = 12) was calculated as the global average intensity. For every metabolite, the average intensity across all samples (n = 12) was calculated and then divided by the global average intensity. These results were multiplied by 100 to express as percentages. To understand the relative intensity of multiple metabolites with respect to the other members within their family, the total dataset was filtered down to just members of the oxylipins, NAEs, and dcFAs. The same calculations as described above were performed to determine the percentage intensity of each query metabolite among the narrower groups.

[0115] With regard to the targeted panel, Welch’s t-test (p < 0.05) was used to determine statistical differences in the bioactive lipid concentrations between the two waste byproducts. No corrections for multiple comparisons were employed due to the small number of technical replicates for each class.

[0116] The raw data of the untargeted metabolomic profile are given as peak heights for the quantification ion (mz value) at the specific retention time (rt value). 23Attorney Docket No. 11716-008WO1

[0117] Exploration of the global metabolic profile of the walnut pellicle was enabled by a triple- platform analytical pipeline as described in the Materials and Methods section. A grand total of 7403 features were detected; with 1595 features identified with Platform 1 (“Complex Lipid Analysis”), 5274 with Platform 2 (“Polyphenol and Fatty Acid Analysis”), and 534 with Platform 3 (“GC-MS Analysis for primary Metabolism”). From this total number of detected features, 565 unique metabolites (216, 221, and 156 from Platform 1, 2, and 3, respectively) were successfully identified.

[0118] Annotations for each metabolite, including biochemical pathway membership and classification by molecular structure, were used to summarize the metabolomic profile of the pellicle. As shown in Figure 3, 56.28% (318) of the identified metabolites were classified as lipids, followed by carbohydrates at 17.17% (97), phenolics at 15.04% (85), peptides and amino acids at 5.66% (32), alkaloids at 1.77% (10) and lastly nucleic acids at 1.24% (7). Seed coat metabolomic studies have generally been interested in the phenolics fraction due to their role in barrier function, contributing antioxidant and antimicrobial properties. This has also largely been the case for walnut, with pellicle (and shell and hull) metabolomics focused primarily on the diversity of phenolics contributing to antioxidant capacity. To the best of our knowledge, this investigation is the first deep metabolomic profiling of the walnut pellicle, revealing a vast repertoire of diverse lipids and lipid-derived compounds.

[0119] The lipid fraction accounted for a total of 318 unique metabolites, which were identified as lipid molecules using their biochemical class annotations. The footprint of each major lipid class, represented as metabolite counts and relative percentage of the overall lipids, can be seen in Figure 4A. These major classes encompass glycerolipids, fatty acids (and fatty acyls), phospholipids, terpenoids, and sphingolipids.

[0120] The glycerolipid (GL) class was the most abundant of the lipid classes, accounting for 40.57% (129) of the total lipid features. This class includes triacylglycerols (TG), diacylglycerols (DG), and monoacylglycerols (MAG), which were represented at 84.50% (109), 10.08% (13), and 5.43% (7) of the total GL class, respectively (Figure 4B). Within the DGs, multiple species of glycolipids were detected and comprised 3.1% and 1.26% of the GLs and total lipid features, respectively. Of particular note are glycerolipids containing a glucuronic acid moiety, specifically two diacylglyceryl glucuronide molecules, named DGGA 34:2 (16:0 / 18:2) and DGGA 36:3 (18:1 / 18:2). This peculiar subclass of GLs has, to the best of our knowledge, never been reported in the walnut pellicle.

[0121] Fatty acids, belonging to the “Fatty Acids and Acyls” class, were the second most frequently identified group in the walnut seed coat, representing 21.70% (69) of the total lipid fraction expressed in counts (Figure 4A). A rich and highly diverse array of free fatty acids (FFAs) were identified, accounting for 63.77% (44) of the total fatty acids (Figure 4C). FFAs diversity was explored by grouping metabolites according to their structural characteristics including carbon chain length, chemical modification of the 24Attorney Docket No. 11716-008WO1 carbon chain, and degree of unsaturation. The majority of FFAs, up to 68.18% (30) of the total fatty acid counts, were long chain fatty acids, with chain lengths between 13-21 carbon atoms, followed by medium chain 11.36% (5) with chain lengths of 6-12 carbon atoms, and very long chain fatty acids 18.18% (8) with chain lengths ≥ 22 carbon atoms (Figure 5C). Unsaturation degree of the carbon chain was nearly equally represented in the FFAs population; 34.09% (15), 31.81% (14), and 31.81% (14) accounted for by polyunsaturated FFAs (PUFAs), monounsaturated FFAs (MUFAs), and saturated FFAs (SFAs), respectively (Fig.3B). Notably, almost half of the FFA, 47.73% (21), possessed oxygenated carbon chains. These included mono- and poly-hydroxy FFAs along with mono-hydroxyperoxy and epoxy FFAs (Figure 5D). Common long chain monosaturated fatty acids (LC-MUFAs) and long chain polyunsaturated fatty acids (LC-PUFAs), which include oleic, linoleic and linolenic acids, accounted together for just 9% (4) of the total FFAs; similarly, low count was found also for simple long chain saturated fatty acids (LC- SFAs) at 4.55% (2) of the total FFAs identified (Figure 5A). This larger class of fatty acids also encompassed more unusual fatty acids belonging to the classes of dicarboxylic fatty acids (DC-FAs) at 11.59% (9), fatty acid amides (FAAs) at 11.59% (8), lactones at 7.25% (5), fatty acid esters (FAEs) at 2.90% (2), and lastly fatty alcohols (FOHs) at 1.45% (1) (Figure 4C). To the best of our knowledge, this is the first time these subclasses of fatty acids, particularly the DC-FAs and lactones, along with a substantial percentage of oxylipins, were revealed in walnut-derived tissue. Phospholipids (PLs), terpenoids / isoprenoids, and sphingolipids (SLs) represented 16.35% (52), 11.01% (35), and 10.38% (33) of the total number of lipid features, respectively (Figure 4A). With regard to the PLs, phosphatidylcholines (PCs) were the most represented subclass with 69.77% (30) of the total PL’s population. PCs were followed by phosphatidylinositols (PIs) at 18.75% (9), phosphatidylethanolamines (PEs) at 18.60% (8), phosphatidic acids (PAs) at 9.30% (4), and finally phosphatidylethanols (Peths) at 2.33% (1) (Fig.2D). A noteworthy subclass of PLs, the lyso-phospholipids (lyso-PLs), were also identified in this study primarily contributed by the PCs class; in total five Lyso-PCs were identified counting for the 16.67% of the total PC features and 9.67% of the total PL population. Concerning the class of terpenoids / isoprenoids, membership was mainly represented by tetraterpenoids which reached up to 42.86% (15), followed by monoterpenoids at 22.86% (8), diterpenoids at 14.29% (5), sesquiterpenoids at 11.43% (4), and sterols at 8.57% (3) (Figure 4E). Lastly, the SL class was accounted mainly by simple sphingolipids (sphingoid bases and ceramides) comprising 69.70% (23) of the total SL population, while complex sphingolipids (sphingomyelins and glycosphingolipids) accounted for the remaining 30.30% (10) of the total SL features (Figure 4F).

[0122] As a general conclusion, the qualitative compositional lipid profile the walnut pellicle followed the order GL > FA > PL > Terpenoids / Isoprenoids > SL. GL and FA were the lipid classes most abundant in feature counts, with the TG and FFA subclasses contributing the highest counts of molecular species, 25Attorney Docket No. 11716-008WO1 respectively. Above all, the fatty acid profile of walnut seed coat showed more insightful peculiarities compared to the commonly detected and extensively characterized fatty acids in whole walnut kernel, like linoleic and linolenic acids.

[0123] The analytical pipeline unveiled the detection of oxygenated fatty acids, i.e., oxylipins, up to 43.47% (30) of the total fatty acids. As shown in Figure 6A, the majority of oxylipins were detected in the FFA’s class, followed by the lactone and DC-FA classes. Compositionally, oxygenated FFA accounted for nearly 50% of the total FFA features (Figure 5D). As mentioned in the previous section, the type of oxygenation forms included mono-, di-, and poly-hydroxylation, mono-hydroperoxidation and mono- epoxidation (Figure 6B). To summarize, the majority of detected FFA oxylipins had long carbon chains, one or more unsaturations, and one or more oxygenations (Figure 6E,F,G). Looking at the combinations of these structural hallmarks, the majority of indentified FFA oxylipins were long chain polyhydroxy monounsaturated fatty acids (LC-PH-MUFA), followed by long chain monohydroxy polyunsaturated fatty acids (LC-MH-PUFA), and long chain monohydroperoxy polyunsaturated fatty acids (LC-MHp-PUFA). The list of metabolites belonging to the LC-PH-MUFA subclass is reported in Table 1 along with each metabolites’ relative abundance (%) to the global normalized intensities.

[0124] The second major subclass of fatty acids detected in this study were the DC-FAs (Figure 4C), which were also structurally classified based on chain length, degree of saturation, and chemical modification. DC-FAs were detected mainly as medium chain with similar frequency of mono- and poly- unsaturations (Figure 7A,C,D). While the majority were non-oxygenated, several oxylipins of DC-FAs were detected, making up37% of the total DC-FAs features. These results are seen in Table 2 which reports a list of the identified DC-FAs in the walnut pellicle along with their relative abundance (%). Interestingly, methyl esters of DC-FAs were also identified and classified as under the fatty acid ester (FAE) class.

[0125] The third most abundant subclass of the fatty acids were the fatty acid amides (FAAs). FAAs were classified as fatty acid primary amides (FAPAs), N-acylethanolamines (NAEs), and ethylamides (FA- EtAs), which accounted for 50% (4), 37% (3), and 12.50% (1) of the total FAA profile, respectively. Of the identified FAAs, possessed chain lengths of C16 and C18; including pamitoleoyl ethanolamide, palmitamide, stearamide, oleoylethanolamide, and oleamide. Erucamide was the only FAA with a C22 carbon chain. The list of these species and their relative abundances (%) are reported in Table 3.

[0126] Subsequent to their detection in the untargeted metabolomics analysis, oxylipins and N- acylethanolamines (NAEs) were quantified in two industrial waste streams from handling processes of walnuts in California, named “Sorting Meal Room (SRM)” and “Blower Fluff (BF)”. While not the only walnut processing waste streams, SRM and BL were selected for being enriched in pellicle and generally considered as an abundant, renewable, and low-value agricultural waste. These types of wastes were of 26Attorney Docket No. 11716-008WO1 particular interest for potential re-utilization and valorization into high-value products, due to their array of potent bioactive lipids with relevance to plant and human health.

[0127] Oxylipins derived from free fatty acids and NAEs, also called Acyl-ethanolamides (Acyl-EAs), were quantified in SRM and BF wastes along with ketones, PUFAs, Acyl-Amino Acids (Acyl-AAs), and prostaglandins (PGs). The 74-member panel of metabolites were identified and quantified by targeted metabolomic. As for percentages, oxylipins composed 74.68% of the panel, followed by Acyl-EAs (11.39%), PUFAs (6.33%), Acyl-AAs, and PGs (3.8%). Within the oxylipins group, metabolites were distinguished by oxygenation type (Figure 9A): including epoxides of fatty acids (21.52%), monohydroxy fatty acids (R-OH) (20.25%), vicinal diols (vic-Diols) (21.52%), ketones (R=O) (7.59%), triol fatty acids (2.53%), and hydroperoxy fatty acids (R-OOH) (1.27%).

[0128] Concentrations of monohydroxy fatty acids, including 13-, 9-, 12-, and 10-HODE, were the highest along with triols, like 9,12,13-TriHOME (Figure 9B and E), in both waste streams; reaching concentrations of 80-100 uM. Comparing the two waste streams, BF was more enriched in vicinal diols like 15,16-, 12,13-, and 9,10-DiHODE (Figure 9D). These subsets of metabolites are reported as relative abundance across all measured samples (i.e. the sum of each metabolite across all samples is set to 100%) rather than concentrations in uM. Along similar lines, 9,10-DiHO, as well as 12,13- and 9,19- DiHOME, were also more abundant in BF and had lower concentration compared to triols and R-OH (Figure 9C). Epoxides of fatty acids were enriched in both wastes: 9(10)EpO, 12(13)EpOME and 9(10)EpOME were detected in concentrations of 10-35 uM (Figure 9F).

[0129] A rich profile of NAEs were detected and quantified (Figure 9G). Linoleoylethanolamide (N- C18:2n6_EA) was the most abundant NAE detected in both waste streams with an average value of 5.7 uM, followed by oleoylethanolamide (N-C18:1n9_EA) at 1.2 uM , linolenoylethanolamide (N- C18:3N3_EA) at 0.9 uM, palmitoylethanolamide (N-C16:0_EA) at 0.69 uM, and finally stearoylethanolamide (N-C18:0_EA) at 0.5 uM.

[0130] The establishment of a successful process for pellicle isolation from nutmeat enabled the identification of metabolism specifically occurring in the pellicle. An untargeted metabolomic analysis enabled a comprehensive qualitative description of pellicle lipid composition. The proportion and composition of major lipid classes, expressed as percentages of total feature counts, were largely consistent between isolated pellicle and previously studied kernel (which includes the nutmeat and the pellicle in the majority of published studies).

[0131] Free fatty acid (FFA) species were also found to represent a considerable portion of the pellicle lipid population. Shown extensively in the literature, walnut kernels possess a high content of polyunsaturated fatty acids (PUFAs), especially linoleic acid, and the lowest saturated fatty acids: total fatty acids ratio and greatest content of PUFAs compared with other nuts. Accumulation of long chain 27Attorney Docket No. 11716-008WO1 (LC) FFAs like linoleic, oleic, and linolenic acids have also been shown in dried walnut shells. LC-MUFAs and LC-PUFAs, including oleic, linoleic and linolenic acids, accounted for 27.3% and 34.1% of the total fatty acid counts and roughly 4% and 5% of the total identified lipid features in the pellicle. Also revealed in this study was the presence of very long chain (VLC) FAs, with carbon chain lengths containing 22-30 carbon atoms, which are rare and in low abundance within kernel. In the pellicle, these species accounted for 18.18% (8) of the total FFA counts. As a minor class of the FFAs, medium chain (MC) FAs were identified at similar abundance to kernel (Yan et al.2021); making up 11.36% of the FFA counts (Figure 5A,C). However, this analysis also revealed the pellicle FFA profile to be compositionally distinct from the reported kernel FFA profile; with the pellicle containing many uncommon metabolites (Figure 4C & 6C).

[0132] The reported walnut kernel lipid profile generally places glycerolipids (GLs) as the predominant lipid class which was mirrored in the pellicle, with triacylglycerols (TGs) being the most abundant subclass followed by diacylglycerols (DGs) and monoacylglycerols (MGs). Interestingly, galactolipids falling under the larger class of DGs were detected and consistent with prior reporting. These include digalactosyldiacylglycerol (DGDG), monogalactosyldiacylglycerol (MGDG) and sulfoquinovosyl diacylglycerol (SQDG); with DGDG and MGDG accounting for less than the 5% and 2% of the total GLs and lipid classes respectively. The levels of these compounds are influenced by drying and may be accumulated as the pellicle matures and desiccates, as well as from commercial drying processes.

[0133] This agreement between pellicle and kernel composition was also largely true for phospholipids (PLs), sphingolipids (SLs), and terpenoids / isoprenoids. With respect to the PLs, PCs as the most accounted for subclass followed by phosphatidylinositols (PIs), phosphatidylethanols (PEs), and phosphatidic acids (PAs). Furthermore, high frequency of lyso-PCs, up to 24% and 26% of the total PC population and 5% and 6% of the total PL population, was found in the walnut kernel in other studies. These species, which arise from enzymatically-mediated PC hydrolysis by the action of phospholipases, like PLA2, can be highly dependent on genotype, tissue maturity, and abiotic stresses imposed by management practices such as drying and storage conditions. In terms of SLs, both the walnut kernel and isolated pellicle are more enriched in simple sphingolipids, like ceramides, than complex sphingolipids, like HexCer. The SL population makes up roughly 10% of the total lipid metabolites for the pellicle, similar to the case of kernel. Similar to the PLs, the increase in SLs were reported in the kernel to be an outcome of the drying process. As for terpenoids / isoprenoid class, phytosterols (i.e. steroidal alcohols) belonging to the group of triterpenoids, were mainly found in nuts including whole walnuts and walnut oil, while a more complex profile for other terpenoid subclasses was found for walnut leaves by VOC analysis. Unlike the kernel, the pellicle was revealed to possess a more diverse repertoire terpenoid subclasses including mono-, di-, tetra-, and sesquiterpenoids (in addition to the 28Attorney Docket No. 11716-008WO1 aforementioned phytosterols). While monoterpenoids were previously identified in walnut kernels by supercritical fluid extraction coupled to GC-MS analysis, detection of further subclasses were not reported. Overall, these results are largely consistent with the published data for walnut kernel obtained with analytical pipeline similar to one adopted for this study.

[0134] As previously mentioned, fatty acid species comprised 21.70% of the total lipids detected in the walnut pellicle, more than twice the number detected in the kernel in earlier reports. The FFAs as a subclass constituted 67.77% of the FA profile, and similar to the kernel, were mostly LC-MUFAs and LC- PUFAs (Figure 5A,B,C). However, these groups contained many unusual members aside from the conventional FAs (i.e., C18:1 oleic, C18:2 linoleic, and C18:3 linoleic acid) in the pellicle. Nearly half of the total FFAs (47.73%) were oxygenated in the form of hydroxy-, peroxy-, or epoxy- fatty acids which fall into the umbrella term of “oxylipins” (Figure 5D & 6B). These FFA-derived oxylipins are also involved in the production of related molecular families including jasmonic acid and certain gamma-lactones that may contribute to the barrier function of the pellicle. Independent of the oxylipins, the second and third major subclasses of detected FFAs were the dicarboxylic FAs (DC-FAs) and the fatty acids amides (FAAs). To the best of our knowledge, this is the first reported detection of DC-FAs a walnut tissue, as prior metabolomic studies of walnut did not report this unique subclass of FFAs. Regarding the FAAs, of which N-acylethanolamines (NAEs) are a subgroup, detection and quantification of NAEs in whole walnut kernel has been reported previously.

[0135] In general, oxylipins have been implicated in various critical functions related with plant defense. The species identified within the walnut pellicle were largely long chain hydroxy FAs, which are a main component of cutin, the insoluble lipophilic polyester that makes up the plant cuticle framework (along with intracuticular waxes made of VLC-FAs like those detected in this study). This waxy layer on the outer surface of the plant epidermis creates a hydrophobic skin around plant tissue; controlling the exchange of moisture and gasses with the environment, as well as providing protection against would- be pathogens. Degradation of the cuticle by pathogen-secreted cutinases produce bioactive oxylipins, namely monohydroxy-, polyhydroxy-, and epoxy FAs, that serve as damage associated molecular patterns (DAMPs) that trigger the plant immune response. These same compounds also act directly as antimicrobials, with several hydroxy FAs known to possess antifungal properties. Among the oxylipins detected in the pellicle, and further validated by quantification in pellicle-enriched waste streams, the polyhydroxy FAs 9,10,11 and 9,12,13-trihydroxy C18:1 have demonstrated antifungal activity against aggressive oomycete pathogens like Phytophthora. The monohydroxy fatty acids 9- and 13-OH C18:3 are also known to have antifungal effect against Alternaria which can cause mold in walnut kernels. Similar antifungal activity has been observed in saturated and unsaturated FAs (i.e., palmitic and linoleic acids) and other mono- and di-hydroxy FAs against Fusarium and Phomopsis that cause molding in walnuts. 29Attorney Docket No. 11716-008WO1 From an evolutionary standpoint, the development of a waxy layer with potent antimicrobial properties can be considered an enabling structure for the colonization of land by plants and expansion into increasingly harsh environments. This is all the more critical for seed coats, like the walnut pellicle, that serve the essential role of protecting a plant’s progeny against abiotic and biotic stressors that could compromise survivability. The detection of VLC-FAs and oxylipins in the pellicle is evidence of the wax and cutin layers, respectively, which protect the susceptible oil-rich seed from abiotic and biotic stress.

[0136] Aside from being produced as part of the cuticular barrier, oxylipins accumulate in plant tissues through two other noteworthy pathways: synthesis within oil bodies (OBs) and intracellular oxygenation of membrane-derived PUFAs. OBs (i.e., lipid micelles) function as subcellular factories for antifungal oxylipins and are enriched in FA-containing TGs and oxylipin biosynthetic enzymes like phospholipases (PLA2), alpha-dioxygenases (alpha-DOX), and 9- and 13-lipoxygenases. Accumulation of OBs occurs in plant embryos and oil-rich seeds, as well as in senescent plant tissues where lipids are actively recycled from membranes and organelles. In senescent tissues, these OBs serve as peroxisomal energy sources for succinate production to fuel gluconeogenesis. As the walnut pellicle achieves maturity it enters senescence and is likely consequently enriched in OBs, which is consistent with the molecular hallmarks of this process (like 9- and 13- monohydroxy fatty acids and abundant TGs) observed in this study. Another route leading to the accumulation of oxylipins is intracellular PUFA oxygenation following the liberation of FFAs from membrane lipids. This conserved cellular metabolism is fundamental to induce signaling cascades and immune responses, like in the case of jasmonic acid, where the initial synthesis starts from the oxygenation of liberated linolenic acid. As tissue senescence is an oxidative stress-driven process, this may activate the molecular mechanisms for defense such as membrane-derived PUFA oxidation for bolstering cellular chemical defenses.

[0137] Revealed in this study was an unexpected presence of DC-FAs. This family of FFAs are produced in both plants and mammals through omega-oxidation, an alternative route of lipid catabolism that occurs in the endoplasmic reticulum and microsomal membranes. This family may be considered hallmarks of stress and mitochondrial dysfunction, as omega-oxidation is the “rescue plan” for when beta-oxidation is compromised. The produced DC-FAs may be catabolized in the peroxisome to maintain energy homeostasis, transferred out of the cell along with other oxylipins as precursors to the biopolymers cutin and suberin, and / or serve as intracellular molecular signals to induced defense responses. In plants this occurs in various organs under particular conditions, such as geminating cotyledons and senescing leaves, while in mammals, the production of DC-FAs is associated with starvation and severe metabolic dysfunction. Among the DC-FAs identified in the pellicle, azelaic acid is an important regulator of plant immune response. Stress-induced production of reactive oxygen species (ROS), which serve as mediators for rapid cellular signaling in a cascade, lead to breakage and oxidation 30Attorney Docket No. 11716-008WO1 of plastid FAs into azelaic acid. The azelaic acid in turn primes the immune response through the induction of SAR-inducer glycerol 3 phosphate and salicylic acid. With regard to the pellicle, oxidative stress is imposed during development, maturation, and desiccation. Taken together, the synthesis of DC- FAs might be considered a molecular biosignature of mitochondrial dysfunction and cell energy impairment during pellicle senescence and desiccation.

[0138] Maturation and senescence processes in seeds is marked by significant oxidative stress and signaling by abcisic acid (ABA) in preparation for desiccation. Both ROS and ABA induce the expression of phospholipase D (PLD) as a regulator of key signaling networks during these programmed cellular processes as well as during biotic and abiotic stress. PLD is responsible for hydrolyzing N- acylphosphatydilethanolamines (NAPEs), a subset of the PLs located in cellular membranes, leading to the synthesis of NAEs. These NAEs are negative molecular regulators of plant growth through the inhibition of plastid development and function. Inhibition of plastid function is detrimental to energy homeostasis due to its complex relationship with the mitochondria (mediated by a variety of signals including ROS). Down-regulation or functional impairment of either of these two organelles can lead to extensive cellular damage and even to programmed cell death (PCD) in a cooperative manner. Indeed, typical plastids in nuts are called elaioplast and are crucial for the synthesis of fatty acids used as energy substrates by mitochondria. Another molecular sign of mitochondrial stress is the presence of fatty acid primary amides (FAPAs), like oleamide, which have been detected in this study. In mammals, oleamide can modulate intracellular calcium (Ca2+) level which in turn causes mitochondrial stress and the release of cytochrome-c therefore triggering PCD mediated by apoptosis. A similar function can be hypothesized to occur in the case of plants. NAEs-induced stress in plastids can trigger caspase-like activity following the release of cytochrome-f from plastids. Senescence-dependent PCD driven by the down-regulation of plastid function and mitochondrional apoptosis is a plausible mechanism of organelle disruption during senescence and desiccation processes. This is supported by having also detected ceramides in this study, SLs that are known regulators of PCD. Given that the walnut pellicle at the late stages of maturity is a dead tissue, as evidenced by the distinct disappearance of organelles, the release of NAEs may be a regulated process to interrupt maturation and shift into senescence and desiccation.

[0139] Overall, these various classes of bioactive lipids identified in this study provide an elegant explanation of the molecular processes intrinsically related to stress resilience and the physiological processes associated with programmed desiccation of the walnut pellicle (Figure 10).

[0140] Intriguingly, the same classes of bioactive lipids identified in this study show potent bioactivities in mammals (notably humans) and are pivotal modulators of various physiological processes related with health and wellbeing. As previously mentioned, bioactive lipids are can have peculiar structural 31Attorney Docket No. 11716-008WO1 features including oxygenations of the carbon chain, conjugation to other bioactive molecules (like amino acids), or unusual degree of unsaturation.

[0141] Oxylipins are bioactive lipids resulting from the oxygenation of PUFAs. This is a highly conserved molecular pathway among plants and mammals, as oxylipins are the backbone of numerous signaling molecules that function as homeostatic regulators for immunity, inflammation, and other physiological processes preventing serious diseases. In mammals, ω6- and ω 3-PUFA metabolism is mediated by the activity of cyclooxygenases and lipoxygenases, producing both pro- and anti-inflammatory oxylipins, respectively. Additionally, cytochrome P450 (CYP450) enzymes mediate the epoxidation of PUFAs at existing unsaturations. The epoxy-PUFAs have been implicated in reduction of pain, blocking of inflammation, and the regulation of the immune system, cardiovascular function, skin barrier functions, and various metabolic dysfunctions (like obesity). A notable role of the CYP450s is the epoxidation of endocannabinoids, like anandamide, that have been identified in the brain, heart, and liver. These compounds have important bioactivities but are easily degraded into less bioactive FA diols by soluble epoxide hydrolases, mitigating their benefits. Epoxy fatty acids (EpFAs), like 9,10- and 12,13-EpOMe that were identified in walnut pellicle-enriched waste streams, are important negative regulators of inflammation and pain. Aside from the EpFAs, this study identified multiple hydroxy FAs that have their own relevance for human health. This group of compounds exhibit antimicrobial properties, providing protection against infections, and also serve as master regulators of energy metabolism, specifically through the enhancement of mitochondrial and peroxisomal FA metabolism by PPAR-α activation (Yokoi et al.2010). This makes this particular class of oxylipins promising therapeutic candidates for diseases driven by metabolic dysfunction. Hydroxy FA activation of PPAR-α in the brain has been shown to exert neuroprotective effects and improve memory, while derivatives of linoleic acid (like 9- and 13-HODE) are ligands for GPR132 (G2A) receptors in the skin, discouraging damaged cells from proliferating while stimulating the growth of keratinocytes. Considering the clinical outcomes from both EpFAs and hydroxy FAs, natural sources of these compounds for applications in health and wellbeing represent an exciting opportunity to improve quality of life. Walnuts have a great potential to be a rich source of oxylipins; interestingly walnut fermentation showed to enrich oxylipin profile. Quantification of both groups of metabolites in walnut industrial wastes was on the order of µM and can be consequently considered as good sources of potent oxylipins, specifically the Ep- and hydroxy- FAs.

[0142] As previously mentioned, DC-FAs can rescue cellular energy metabolism in case of mitochondrial impairment through the omega-oxidation route. Dysfunction of the mitochondria is a known underlying factor in many metabolic disorders, including increasingly prevalent conditions like diabetes. DC-FAs are important substrates for cellular energetics as intermediates between FAs and sugars (like glucose). Their catabolism within the peroxisome produces succinic acid, feeding the TCA cycle and generating 32Attorney Docket No. 11716-008WO1 ATP in the case of mitochondrial dysfunction. This property is critically important during pathological conditions associated with insulin resistance and type-2 diabetes mellitus. For this reason, dietary supplementation with DC-FAs might be an important tool for individuals suffering with diabetes. Previous studies showed that administration of DC-FAs is safe and well tolerated in humans, although more research is needed to understand the metabolism of DC-FAs within the context of different tissues and organs. Given this potential, natural sources of DC-FAs like the walnut pellicle can be used as inputs for food supplements and / or nutraceuticals targeted to individuals with diabetes or other metabolic conditions.

[0143] Lastly, this study revealed the presence of FAAs in the walnut pellicle and related wastes; in particular, the N-acylethanolamines (NAEs) that have considerable impacts for human health. The NAE family includes oleoylethanolamide (OEA), palmitoylethanolamide (PEA), stearoylethanolamide (SEA), and linoleoylethanolamide (LEA), among other possible analogues. NAEs are classified as para- endocannabinoids, as their structure resembles true endocannabinoids, like anandamide, but their bioactivity is not mediated by interaction with CB1 and CB2 cell surface receptors. Rather, the NAEs exert their action through the activation of nuclear receptors such as (PPAR-α), cell-surface transient receptor potential cation channel vanilloid-1 (TRPV1), and G-protein coupled receptor 119 (GPR119). Activation of GPR119 is especially noteworthy for its relationship with increasing satiety, as it increases GLP-1 secretion from intestinal L-cells, thus enhancing insulin levels and consequently inhibiting glucose- dependent glucagon secretion. OEA especially is a potent satiety factor through the regulation of lipid and sugar metabolism; its supplementation in obese rodent models and human shown to reduce food intake and body weight gain. Further metabolic and pharmacological effects of OEA supplementation include (but are not limited to) neuroprotection and anti-inflammatory action, as well as the amelioration of mood disorders, infertility, and the improvement of cognitive function. Other NAEs like PEA, SEA, and LEA exert biological functions similar to OEA. These outcomes suggest a profound role of NAEs as messengers of the gut-brain axis; being made and / or assimilated in the gut and traveling via the vagal nerve to stimulate the vagal sensory nerves by activating TRPV1 in the brain. Activation of TRPV1, which modulates pain and thermosensation, also contributes to the regulation of energy homeostasis through the control of feeding and energy expenditure. Although endogenously produced in human body, primarily in the small intestine, NAEs can be readily degraded into FFAs and ethanolamine by intracellular FAA hydrolase (FAAH) and NAE-hydrolyzing acid amidase (NAAA) in the intestinal epithelium. NAE catabolism is controlled through the interaction genetic and external factors like stress, disease, and lifestyle that impact the presence and effects in the body. Inhibition of FAAH by a peculiar subclass of the endocannabinoids, the N-acyl amino acids (Acyl-AA) and N-acyl glycines (NAAs), play an important role in the preservation of NAEs. Although in lower concentration than the NAEs themselves, 33Attorney Docket No. 11716-008WO1 the acyl-AAs were identified within the walnut pellicle and waste byproducts in this study. This makes walnuts not only a great source of NAEs but also of the FAAH-inhibiting acyl-AAs. Taken together, this study revealed the walnut pellicle to be enriched in NAEs and the waste byproducts to be promising sources of this precious class of bioactive lipids; thus, paving the way for the development of novel super foods, food supplements, and nutraceuticals to regulate metabolic disorders (like diabetes and obesity), as well as improve cognitive health and energy homeostasis by way of the gut-brain axis.

[0144] Considering the relevance of the discussed bioactive lipids for human clinical outcomes, this broad group of compounds should be considered as “essential bioactive dietary lipids”. Therefore, the exploration of edible dietary sources highly enriched in oxylipins, DC-FAs, and para-endocannabinoids (like NAEs and Acyl-AAs) is a promising avenue for improving incorporation of these compounds into the human diet. The identification of these bioactive lipids in the walnut pellicle paves the way for the identification of molecular features and / or pathways at the origin of the plethora of currently unexplained clinical outcomes (from cohort studies and randomized controlled trials) coming from walnut consumption. These outcomes range from reducing risk of certain cancers, regulation of dysfunctional metabolic conditions like diabetes and obesity, improvement of cognitive function, amelioration of mood disorders and addiction behaviors, support of gut health by modulation of the gut microbiome, amelioration of male fertility, and age-related cognitive decline. While all of these conditions are major societal concerns, the latter are becoming increasingly important as the global population is aging, and fertility rate is declining in the industrialized countries. The consumption of functional foods to improve these various aspects of health and wellbeing is a significant trend among young adult consumers. For this reason, the identification of bioactive lipids and their associated clinical outcomes might encourage walnut consumption in the younger population. Furthermore, the discovery of a natural source of bioactive lipids, in the form of waste byproducts from walnut industrial processing, will pave the way for the development of novel edible supplements, nutraceuticals, or precursors for derma care products and / or pharmaceuticals. This discovery has significant economic impacts as rising walnut production will lead to a commensurate rise in waste by-products from the handling and processing pipelines. What have been classically low-value waste by-products may now be an opportunity for walnut industries to upcycle; encouraging sustainability and creating additional revenue streams to support industry longevity. Finally, walnut improvement programs may leverage breeding or bio-engineering approaches to enhance the production of these various bioactive lipids in the walnut pellicle, or other walnut tissues, to supply greater concentrations of these potent molecules for applications in human health.

[0145] Table 1 34Attorney Docket No. 11716-008WO1

[0146] Table 2

[0147] Table 3 35Attorney Docket No. 11716-008WO1Example 2: Cultivation of somatic walnut embryos

[0148] Materials: DKW powder, a basal salt mixture composed of macro- and micronutrients specific to support J. regia / walnut growth and micropropagation in culture. DKW is commercially available. Sugar to support growth. Gelzan is a polysaccharide gelling agent added to solidify the media.

[0149] One liter of distilled water was added to 5.32 g DKW powder and mixed thoroughly. 500 ml of the DKW water solution was added to a 500 ml bottle with 1.1 of Gelzan. The bottle was sealed and autoclaved at 121° C. for 30 minutes. The solution was cooled and poured into sterile plates and allowed to solidify.

[0150] Embryo development successfully takes place on this media at room temperature in dark; selection of embryos at different developmental stages is accomplished after carefully morphological examination of individual embryos.

[0151] Mature embryos were submerged in deionized (DI) sterilized water in six or twelve well cell culture plates. Alteration of oxygen concentration in each well was measured with a dissolved oxygen meter. At the beginning of the experiment, oxygen concentration was observed to be around 7 mg / L which is considered to be a stressful hypoxic condition. However, after just 10 minutes of exposure the plates the concentration of dissolved oxygen in each well containing water-submerged embryos fell below 5mg / L; at 6 hrs the dissolved oxygen concentration dropped to near 2mg / L. Embryos that were subjected to these sustained stressful hypoxic conditions were harvested at 6, 36 and 72 hrs and quick frozen in liquid nitrogen and the frozen tissues were extracted for metabolomic analysis. Another batch of embryos similarly subjected to hypoxic stress for 6, 36 and 72 hrs were allowed to recover by exposing to air for re-oxygenation for 6, 36 and 72 hrs samples of these re-oxygenated tissues were also 36Attorney Docket No. 11716-008WO1 sampled by first flash freezing in liquid nitrogen and the frozen tissues were extracted for metabolomic analysis.

[0152] Metabolomic analysis of waterlogged walnut somatic embryos (under hypoxia stress and hypoxia stress + reoxygenation) showed an increase in oxylipins and oleoylethanolamide (OEA) compared to controls. Also increase in PUFAs and dicarboxylic fatty acids was also detected. Water Stress 6hrs vs Control (No water stress) Compounds P- FDR Fold_Chan Log2FC Type value ge7Attorney Docket No. 11716-008WO1 9-Hydroxy-13-oxo-10-octadecenoic Acid 0.1 0.1956214 20.326605 4.345297 up 69 9 432 9-Oxo-10,12-Octadecadienoic Acid 0.000 0.0291687 6.4594616 2.691413 up 588 572 925 9-Oxooctadeca-10,12-Dienoic Acid 0.063 0.1525360 37.955474 5.2462 603 33 29 074Hydroperoxylinoleic acid*0.106 0.20471702.0241940 1.017347 up 571 39 51 602 Hydroxypentadecenoic acid glucoside 0.063 0.1525360 96.240868 6.588577 up 603 33 58 758Water Stress 36hrs vs NoessCompounds P-value FDR Fold_Change Log2FC Type 9-Oxo-10,12-Octadecadienoic Acid 0.1 0.21249 78.37936 6.29240 up38Attorney Docket No.11716-008WO1 9,12,13-Trihydroxy-10-octadecenoic acid 0.03085 0.16726 21.57751 4.43145 up 8 2 7 2-hdr x ntd n i id 002764 016215 7496479 290621Attorney Docket No. 11716-008WO1 10-hydroxydecanoicacid10-O-beta-D- 0.00405 0.08082 11.22503 3.48864 up glucopyranoside 9 5 7 (9Z12Z15Z)-O t d tri n i id 000258 007367 7070071 282172Compounds P-value FDR Fold_Change Log2FC Type 9-Oxo-10,12-Octadecadienoic Acid 0.01328 0.09356 55.2176 5.78705 up40Attorney Docket No.11716-008WO1 9,10,13-TriHOME 0.00710 0.07212 20.25044 4.33988 up 6 1 Hdr x riin li id 003140 013773 2009051 432844Attorney Docket No. 11716-008WO1 10-hydroxydecanoicacid10-O-beta-D- 0.00974 0.08173 12.30212 3.62083 up glucopyranoside 3 4 5 (9Z12Z15Z)-O t d tri n i id 000847 007786 869075 311948 [01y y p y d in walnut somatic embryos at different developmental stages with the aim to identify a stage of the development particularly enriched in these bioactive fatty acids. Initial stages showed embryos with a globular, heart and torpedo shape along with cotyledonary translucent somatic embryos; middle stage includes large translucent cotyledonary somatic embryos; the last stage includes somatic embryos ready initiate germination which usually are opaque cotyledonary embryos. Differences in lipid mediator profiles were observed for the three stages of embryo development as shown below and Figure 3, 4, 5. In particular, the later stages of somatic embryo development in culture were enriched in fatty acid ethanolamines. ANOVATukey's post hoc Y l I i i l Middl L te B B B B B B B BAttorney Docket No. 11716-008WO1 ALA Log[9(10)-EpODE_(screen)] 0.0289 B A B ALA Log[12(13)-EpODE_(screen)] 0.0092 A B B ALA L 910 DiHODE 00001 A B AB B B B B

[0154] Wa, ted to an accredited laboratory recognized by the United States Food and Drug Administration (FDA) fto develop a certified nutritional profile. The results obtained from the analysis of 100 g of fresh walnut somatic embryos are shown below: 43Attorney Docket No. 11716-008WO1 [015t somatic embryos is presented below: Walnut Somatic Embryos Conventional walnuts (100g) ( rojection for dried embr os 100 )

[0156] A synthesis of the 10-hydroxy derivative of oleolylethanolamide was performed as depicted in Figure 14. Each of the separated isomers had greater than 98% enantiomeric excess. 44Attorney Docket No. 11716-008WO1 Additional embodiments A method obtaining a lipid from a walnut tissue composition, comprising extracting the walnut tissue composition to separate the lipid from the walnut tissue composition. The method according to a preceding embodiment, wherein the lipid comprises a hydroxyalkyl amide of a fatty acid, a carboxyalkyl amide of a fatty acid, or a combination thereof. The method according to a preceding embodiment, wherein the lipid comprises a 2-ethanolamide of a fatty acid. The method according to a preceding embodiment, wherein the lipid comprises a 2-ethanolamide of a fatty acid, wherein the fatty acid comprises a saturated fatty acid, mono-unsaturated fatty acid, di- unsaturated fatty, tri-unsaturated fatty acid, tetra-unsaturated fatty acid, or a combination thereof. The method according to a preceding embodiment, wherein the fatty acid comprises an omega-3 fatty acid, a hydroxyalkyl amide or carboxyalkyl amide of an omega-3 fatty acid, an omega-6 fatty acid, a 2- ethanolamide of an omega-6 fatty acid, or a combination thereof. The method according to a preceding embodiment, wherein the lipid comprises a 2-ethanolamide of a fatty acid, wherein the fatty acid comprises a hydroxylated fatty acid, an epoxidized fatty acid, a keto- fatty acid, or a combination thereof. The method according to a preceding embodiment, wherein the lipid comprises a 2-ethanolamide of a fatty acid, wherein the fatty acid comprises crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, dihomo-γ-linolenic acid, eicosatrienoic acid, tetra-unsaturated fatty acids, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosanolpentaenoic acid, docosahexaenoic acid, herring acid, petroselinic acid, paullinic acid, gondoic acid, erucic acid, brassidic acid, nervonic acid, arachidonic acid, or a combination thereof. The method according to a preceding embodiment, wherein the lipid comprises oleoylethanolamide. The method according to a preceding embodiment, wherein the walnut tissue composition comprises water in an amount of 25 wt.% or less, 20 wt.% or less, 15 wt.% or less, 10 wt.% or less, 5 wt.% or less, 1 wt.% or less, 0.5 wt.% or less, or 0.1 wt.% or less. The method according to any preceding embodiment, wherein the extraction comprises pressing the walnut tissue, optionally in the presence of a solvent. The method according to a preceding embodiment, wherein the extraction comprises contacting the walnut tissue composition with a solvent and separating the solvent from the walnut tissue to provide a lipid-enriched solvent composition. The method according to a preceding embodiment, wherein the extraction comprises contacting the walnut tissue composition with a solvent, separating the solvent from the walnut tissue to provide a lipid-enriched solvent composition, and separating the lipid from the lipid-enriched solvent composition. The method according to a preceding embodiment, wherein the solvent comprises a polar, aprotic solvent, a polar, protic solvent, a non-polar solvent, or a combination thereof. 45Attorney Docket No. 11716-008WO1 The method according to a preceding embodiment, wherein the solvent comprises supercritical carbon dioxide. The method according to a preceding embodiment, wherein the walnut tissue is dehydrated prior to the extraction. The method according to a preceding embodiment, wherein the walnut-tissue composition comprises a walnut pellicle, a walnut embryo, a walnut shoot, or a combination thereof. The method according to a preceding embodiment, wherein the walnut-tissue composition comprises a somatic walnut embryo. The method according to a preceding embodiment, wherein the walnut-tissue composition comprises a lipid-enriched walnut composition. The method according to a preceding embodiment, wherein the walnut-tissue composition comprises a lipid-enriched somatic walnut embryo. The method according to a preceding embodiment, comprising cultivating a walnut embryo under conditions to enhance bioproduction of the lipid. The method according to any of the preceding embodiments, wherein the walnut tissue composition comprises a somatic walnut embryo having enhanced biolipid production. The method of any of the embodiments 19-21, wherein the somatic walnut embryo comprises elevated concentrations of fatty acyl ethanolamides as compared wild type somatic walnut embryos. The method of any of the embodiments 19-22, wherein the somatic walnut embryo is grown in liquid media. A walnut embryo having a modified fatty acid composition. The somatic walnut embryo of embodiment 24 comprising elevated concentration of fatty acyl ethanolamides as compared to wild type somatic walnut embryos. A walnut shoot, obtained from the somatic walnut embryo of embodiment 24. A compound having the formula: , whereinR1is a C6-C12 alkylene, C6-C12 alkenylene, or C6-C12 alkynylene; R2is a C6-C12alkylene, C6-C12alkenylene, or C6-C12alkynylene; R1Nis a C1-6hydroxyalkyl; R2Nis H, C1-6alkyl, or C1-6hydroxyalkyl; or R1Nand R2Ntogether form a 3-8 membered ring having at least one hydroxy group; and one of the following applies: 46Attorney Docket No. 11716-008WO1 Rc1is H or C(=O)lipid; Rc2is H, and ^ is absent; Rc1and Rc2together form a bond, and ^ is absent; or Rc1is absent, Rc2is H, and ^ is a single bond; wherein lipid is a C6-24alkyl, C6-24alkenyl, or C6-24alkynyl. The compound of any preceding embodiment, wherein R2Nis H. The compound of any preceding embodiment, wherein R1Nis R1Nis CH2CH2OH or CH2COOH, and R2Nis H. The compound of any preceding embodiment, wherein R1is a C6-C12alkylene, C6-C10alkylene, C7-C9alkylene, or C8alkylene The compound of any preceding embodiment, wherein R2is a C6-C12 alkyl, C6-C10 alkyl, C7-C9 alkyl, or C8alkyl. The compound of any preceding embodiment, wherein R2is a C6-C12 alkenyl, C6-C10 alkenyl, C7-C9 alkenyl, or C8alkenyl. The compound of any preceding embodiment, wherein RC1and RC2are both H. The compound of any preceding embodiment, wherein RC2is H and RC1is C(=O)lipid. The compound of any preceding embodiment, wherein lipid is C6-24alkenyl having one, two, three, four, five, or six carbon-carbon double bonds. The compound of any preceding embodiment, having the formula: , Raand Ra’are in ydrogen and the other two ofRaand Ra’form a double bond, or all of Raand Ra’together form a triple bond; Rband Rb’are in each case all hydrogen, or two of Rband Rb’are hydrogen and the other two of Rband Rb’form a double bond, or all of Rband Rb’together form a triple bond. The compound of any preceding embodiment, wherein both Raare hydrogen and both Rbform a double bond. The compound of any preceding embodiment, wherein both Raform a double bond, and both Rbform a double bond. The compound of any preceding embodiment, having the formula: .Attorney Docket No. 11716-008WO1 The compound of any preceding embodiment, having the formula: , wherein the carbon mark at least 90%, at least 95%, at least 98%, at least 99%, aThe compound of any preceding embodiment, having the formula: , wherein the carbon mark at least 90%, at least 95%, at least 98%, at least 99%, a. , . . The compound of any preceding embodiment, having the formula: , , , H , H ,Attorney Docket No. 11716-008WO1 O OH N or

[0157] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches. 49

Claims

Attorney Docket No. 11716-008WO1 CLAIMS What is claimed is:

1. A method of cultivating a walnut embryo, comprising subjecting the embryo to an environmental stress to produce an embryo having enhanced lipid content.

2. The method of claim 1, wherein the walnut embryo comprises a somatic walnut embryo.

3. The method of claim 1, wherein the walnut embryo comprises an early-stage embryo.

4. The method of claim 1, wherein the walnut embryo comprises mid-stage embryo.

5. The method of claim 1, wherein the walnut embryo comprises a late-stage embryo.

6. The method of claim 1, wherein the environmental stress comprises exposure to increased temperature, exposure to decreased temperature, overexposure to light, under exposure to light, submersion in water, water deprivation, oxygen deprivation, oxygen oversaturation, carbon dioxide deprivation, carbon dioxide oversaturation, treatment with one or more of dopamine, jasmonic acid, auxin, cytokinin, or a combination thereof.

7. The method of claim 1, wherein the environmental stress comprises submersion in water.

8. The method of claim 1, wherein the environmental stress comprises submersion in water for a period of 0.1-200 hours, 1-6 hours, 1-36 hours, 1-72 hours, 1-100 hours, 1-150 hours, 1-200 hours, 6-36 hours, 6-72 hours, 6-100 hours, 36-72 hours, 36-100 hours, 72-100 hours, 72-150 hours, or 100-200 hours.

9. The method of claim 1, wherein the environmental stress comprises submersion in water having an oxygen content no greater than 10 mg / L, no greater than 5 mg / ml, no greater than 2.5 mg / L, or no greater than 1 mg / L.

10. The method of claim 1, wherein the environmental stress comprises cultivation in a medium having an oxygen content no greater than 10 mg / L, no greater than 5 mg / ml, no greater than 2.5 mg / L, or no greater than 1 mg / L.

11. The method of claim 1, wherein the environmental stress comprises cultivation in an atmosphere having an oxygen content no greater than 10 mg / L, no greater than 5 mg / ml, no greater than 2.5 mg / L, or no greater than 1 mg / L.

12. The method of claim 1, wherein the environmental stress comprises cultivation in a medium having a CO2content greater than 1,000 ppm, greater than 1,500 ppm, greater than 2,000 ppm, greater than 2,500 ppm, greater than 3,000 ppm, greater than 4,000 ppm, or greater than 5,000 ppm.

13. The method of claim 1, wherein the environmental stress comprises cultivation in an atmosphere having a CO2 content greater than 1,000 ppm, greater than 1,500 ppm, greater than 2,000 ppm, greater than 2,500 ppm, greater than 3,000 ppm, greater than 4,000 ppm, or greater than 5,000 ppm.

14. The method of claim 1, wherein the environmental stress comprises cultivation in the presence of ethylene.

15. The method of claim 1, wherein the environmental stress comprises cultivation in a medium comprising ethylene at a concentration from 200-10,000 ppm, from 200-5,000 ppm, from 200- 50Attorney Docket No. 11716-008WO1 2,500 ppm, from 500-2,500 ppm, from 500-1,500 ppm, from 500-1,000 ppm, from 1,000-1,500 ppm, or from 750-1,250 ppm.

16. The method of claim 1, wherein the environmental stress comprises cultivation in an atmosphere comprising ethylene at a concentration from 200-10,000 ppm, from 200-5,000 ppm, from 200-2,500 ppm, from 500-2,500 ppm, from 500-1,500 ppm, from 500-1,000 ppm, from 1,000-1,500 ppm, or from 750-1,250 ppm.

17. The method of claim 1, wherein the environmental stress comprises cultivation in an environment comprising jasmonic acid.

18. The method of claim 1, wherein the environmental stress comprises cultivation in a medium comprising jasmonic acid at a concentration from 1-100 ppm, from 1-50 ppm, from 10-50 ppm, from 10-25 ppm, or from 25-50 ppm.

19. The method of claim 1, wherein the environmental stress comprises cultivation in an atmosphere comprising jasmonic acid at a concentration from 1-100 ppm, from 1-50 ppm, from 10-50 ppm, from 10-25 ppm, or from 25-50 ppm.

20. The method of claim 1, wherein the environmental stress comprises cultivation in a growing medium comprising a moisture content of less than 10%, less than 8%, less than 6%, less than 4%, less than 2%, or less than 1%.

21. The method of claim 1, wherein the environmental stress comprises cultivation in a desiccator.

22. The method of claim 1, wherein the environmental stress comprises cultivation in a growing medium comprising polyethylene glycol.

23. The method of claim 1, wherein the environmental stress comprises cultivation in a growing medium comprising polyethylene glycol, wherein the polyethylene glycol has a MW from 2,000- 50,000, from 2,000-25,000, from 2,000-15,000, from 2,000-10,000, from 2,000-8,000, from 2,000-6,000, or from 2,000-4,000.

24. The method of claim 1, wherein the environmental stress comprises cultivation in a growing medium comprising polyethylene glycol at a concentration from 20-500 mmol / kg, from 20-400 mmol / kg, from 20-300 mmol / kg, from 20-200 mmol / kg, from 20-100 mmol / kg, from 20-80 mmol / kg, or from 40-80 mmol / kg.

25. The method of claim 1, wherein the environmental stress comprises exposure to a temperature from 30-50 °C. from 35-45 °C., from 35-40 °C., from 40-45 °C., or from 37.5-42.5 °C. for a period of 0.1-200 hours, 1-6 hours, 1-36 hours, 1-72 hours, 1-100 hours, 1-150 hours, 1-200 hours, 6-36 hours, 6-72 hours, 6-100 hours, 36-72 hours, 36-100 hours, 72-100 hours, 72-150 hours, or 100- 200 hours.

26. The method of claim 1, wherein the environmental stress comprises exposure to a temperature from 0-15 °C. from 2-15 °C., from 5-15 °C., from 5-10 °C., from 10-15 °C. or from 7.5-12.5 °C. for a period from 1-14 days, from 3-14 days, from 5-15 days, from 5-10 days, or from 10-15 days.

27. The method of claim 1, wherein the environmental stress comprises exposure to light for a period from 1-10 hours / day, from 3-10 hours / day, from 5-10 hours a day from 3-5 hours days, or from 4-7 hours / day.

28. The method of claim 1, wherein the embryo having enhanced lipid content is directly processed following the environmental stress.

29. The method of claim 1, wherein the embryo having enhanced lipid content is exposed to a non- stress environment following the environmental stress. 51Attorney Docket No. 11716-008WO1 30. The method of claim 1, wherein the embryo having enhanced lipid content is exposed to a non- stress environment following the environmental stress for a period of at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 30 hours, at least 40 hours, or at least 50 hours.

31. The method of claim 1, wherein the embryo having enhanced lipid content is dehydrated, converted to a powder, or a combination thereof.

32. The method of claim 1, wherein the embryo having enhanced lipid content comprises an omega-3 fatty acid, an omega-6 fatty, or combination thereof, in higher concentration that found in conventional walnut embryos.

33. The method of claim 1, wherein the embryo having enhanced lipid content comprises an omega-3 fatty acid, an omega-6 fatty, or combination thereof, in higher concentration that found in conventional walnut embryos, wherein the omega-3 fatty acid, an omega-6 fatty, or combination thereof comprises crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, dihomo-γ- linolenic acid, eicosatrienoic acid, tetra-unsaturated fatty acids, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosanolpentaenoic acid, docosahexaenoic acid, herring acid, petroselinic acid, paullinic acid, gondoic acid, erucic acid, brassidic acid, nervonic acid, arachidonic acid, or a combination thereof 34. A nutritional supplement comprising the embryo having enhanced lipid content according to claim 1.

35. A method of obtaining a lipid from a walnut tissue composition, comprising extracting the walnut tissue composition to separate the lipid from the walnut tissue composition.

36. The method of claim 35, wherein the walnut tissue comprises water in an amount of 25 wt.% or less, 20 wt.% or less, 15 wt.% or less, 10 wt.% or less, 5 wt.% or less, 1 wt.% or less, 0.5 wt.% or less, or 0.1 wt.% or less.

37. The method of any of claims 35-36, wherein the walnut tissue comprises a walnut pellicle, a walnut embryo, a walnut shoot, or a combination thereof.

38. The method of any of claims 35-37, wherein the walnut tissue comprises a walnut embryo.

39. The method of any of claims 35-38, wherein the walnut tissue comprises a somatic walnut embryo.

40. The method of any of claims 35-39, wherein the walnut tissue comprises the embryo with enhanced lipid content obtained by the method of any of claims 1-34.

41. The method of any of claims 35-40, wherein the lipid comprises a 2-ethanolamide of a fatty acid, wherein the fatty acid comprises crotonic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, gadoleic acid, eicosenoic acid, erucic acid, linoleic acid, eicosadienoic acid, docosadienoic acid, linolenic acid, pinolenic acid, eleostearic acid, dihomo-γ- linolenic acid, eicosatrienoic acid, tetra-unsaturated fatty acids, stearidonic acid, arachidonic acid, eicosatetraenoic acid, adrenic acid, bosseopentaenoic acid, eicosapentaenoic acid, ozubondo acid, sardine acid, tetracosanolpentaenoic acid, docosahexaenoic acid, herring acid, petroselinic acid, paullinic acid, gondoic acid, erucic acid, brassidic acid, nervonic acid, arachidonic acid, or a combination thereof.

42. The method of any of claims 35-41, wherein the lipid comprise oleoylethanolamide. 52Attorney Docket No. 11716-008WO1 43. The method of any of claims 35-42, wherein the extraction comprises pressing the walnut tissue, optionally in the presence of a solvent.

44. The method of any of claims 35-43, wherein the extraction comprises contacting the walnut tissue composition with a solvent and separating the solvent from the walnut tissue to provide a lipid-enriched solvent composition.

45. The method of any of claims 35-44, wherein the extraction comprises contacting the walnut tissue composition with a solvent, separating the solvent from the walnut tissue to provide a lipid-enriched solvent composition, and separating the lipid from the lipid-enriched solvent composition.

46. The method of any of claims 35-45, wherein the solvent comprises a polar, aprotic solvent, a polar, protic solvent, a non-polar solvent, or a combination thereof.

47. The method of any of claims 35-46, wherein the solvent comprises supercritical carbon dioxide.

48. The method of any of claims 35-47, wherein the walnut tissue is dehydrated prior to the extraction.

49. A dried walnut composition, comprising: 1-15 wt.% fat; 20-50 wt.% protein; and 40-80% carbohydrate.

50. The composition of claim 49, wherein the dried walnut comprises dried walnut embryos.

51. The composition of claim 49 or 50, wherein the dried walnut comprises dried walnut embryos, wherein the walnut embryos are produced by the process according to any of claims 1-34.

52. The composition any of claims 49-51, comprising fat in an amount from 1-10 wt.%, 1-5 wt.%, 5- 10 wt.%, or 2.5-7.5 wt.%.

53. The composition any of claims 49-52, comprising protein in an amount from 20-40 wt.%, 20-30 wt.%, 30-40 wt.%, or 25-35 wt.%.

54. The composition any of claims 49-53, comprising carbohydrates in an amount from 50-80 wt.%, from 50-70 wt.%, or from 60-70 wt.%.

55. A compound having the formula: , whereinR1is a C6-C12 alkylene, C6-C12 alkenylene, or C6-C12 alkynylene; R2is a C6-C12alkylene, C6-C12alkenylene, or C6-C12alkynylene; R1Nis a C1-6hydroxyalkyl; R2Nis H, C1-6alkyl, or C1-6hydroxyalkyl; or R1Nand R2Ntogether form a 3-8 membered ring having at least one hydroxy group; and one of the following applies: Rc1is H or C(=O)lipid; Rc2is H, and ^ is absent; 53Attorney Docket No. 11716-008WO1 Rc1and Rc2together form a bond, and ^ is absent; or Rc1is absent, Rc2is H, and ^ is a single bond; wherein lipid is a C6-24alkyl, C6-24alkenyl, or C6-24alkynyl.

56. The compound of claim 55, wherein R2Nis H.

57. The compound of any of claims 55-56, wherein R1Nis R1Nis CH2CH2OH or CH2COOH, and R2Nis H.

58. The compound of any of claims 55-57, wherein R1is a C6-C12alkylene, C6-C10alkylene, C7-C9alkylene, or C8alkylene 59. The compound of any of claims 55-58, wherein R2is a C6-C12 alkyl, C6-C10 alkyl, C7-C9 alkyl, or C8alkyl.

60. The compound of any of claims 55-59, wherein R2is a C6-C12 alkenyl, C6-C10 alkenyl, C7-C9 alkenyl, or C8alkenyl.

61. The compound of any of claims 55-60, wherein RC1and RC2are both H.

62. The compound of any of claims 55-61, wherein RC2is H and RC1is C(=O)lipid.

63. The compound of any of claims 55-62, wherein lipid is C6-24alkenyl having one, two, three, four, five, or six carbon-carbon double bonds.

64. The compound of any of claims 55-63, having the formula: , wherein Raand RaRa’are hydrogen and the othertwo of Raand Ra’form a double bond, or all of Raand Ra’together form a triple bond; Rband Rb’are in each case all hydrogen, or two of Rband Rb’are hydrogen and the other two of Rband Rb’form a double bond, or all of Rband Rb’together form a triple bond.

65. The compound of any of claims 55-64, wherein both Raare hydrogen and both Rbform a double bond.

66. The compound of any of claims 55-65, wherein both Raform a double bond, and both Rbform a double bond.

67. The compound of any of claims 55-66, having the formula:

68. The compound of69. The compound of any of claims 55-68, having the formula: 54Attorney Docket No. 11716-008WO1 , wherein the carb purity of at least 90%, at least 95%, at least 98%70. The compound of any of claims 55-69, having the formula: , wherein the carb purity of at least 90%, at least 95%, at least 98%, , . , . .

71. The compound of any of claims 55-70, having the formula: , , , H , H , H ,Attorney Docket No. 11716-008WO1 , , or .56

Images