Bioactive food and beverage compositions and methods

Abstract

The present invention provides bioactive foods, beverages, compositions, and ingredients comprising beta-endorphin (BND) peptide in a bioactive form, as well as methods and compositions useful for producing beta-endorphin (BND) peptide in a bioactive form, and such bioactive foods, beverages, compositions, and ingredients. The present invention also provides methods and compositions useful for producing beta-endorphin (BND) peptide in a bioactive form in cells, tissues, and plants, as well as cells, tissues, and plants that produce beta-endorphin (BND) peptide in a bioactive form. The present invention further provides methods and compositions for inducing, enhancing, or maintaining relaxation, comprising administering to a subject in need thereof the bioactive foods, beverages, compositions, or ingredients described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS The contents of Australian Provisional Patent Application No. 2022904029, filed December 24, 2022, are incorporated herein by reference in their entirety.

[0002] Technical Field The present invention is in the field of bioactive foods and beverages, as well as compositions and methods for making same. [Background technology]

[0003] background Many benefits of relaxation have been reported: improved concentration, improved digestion, increased blood flow, reduced anger and frustration, lowered blood pressure, reduced risk of stroke, promoted emotional well-being, reduced fatigue, reduced inflammation, and slower heart rate.

[0004] The consumption of certain foods and beverages is associated with increased relaxation. However, some such foods and beverages, such as alcoholic beverages, while promoting some of the relaxation benefits described above, have deleterious effects on other health factors. Furthermore, beverages such as alcoholic beverages can be addictive, which can lead to additional well-documented problems.

[0005] It would therefore be desirable to provide compositions and methods useful for inducing or increasing relaxation that overcome one or more of the disadvantages of existing foods and beverages with the goal of inducing relaxation.

[0006] Beta-endorphin, or β-endorphin or BND, is an endogenous opioid neuropeptide and peptide hormone. BND precursors are formed in the pituitary gland and subsequently processed into BND. BND acts through various mechanisms in both the central and peripheral nervous systems when bound to its cognate μ-opioid receptor.

[0007] The functions of BND are known to be related to hunger, arousal, pain, maternal care, sexual behavior, and reward cognition. In the broadest sense, BND is primarily utilized by the body to reduce stress and maintain homeostasis. In behavioral studies, studies have shown that BND is released via volume conduction within the ventricular system in response to various stimuli.

[0008] Although BND may be recognized as a potential bioactive substance useful in managing relaxation, research has shown that it and many other bioactive peptides are unlikely to exert biological effects in the body when taken orally in an unprotected form for a variety of reasons, including the harsh environment of the gastrointestinal tract, as discussed further below.

[0009] Therefore, despite limited success, efforts to elicit the beneficial effects of BND have focused on stimulants and bioactive substances that induce the endogenous production of BND.

[0010] The issue of absorption is paramount in the emerging science of orally ingested bioactive substances. However, previous studies have revealed little clear evidence that dietary bioactive peptides, other than di- and tripeptides, can cross the intestinal wall intact and enter the hepatic portal system at physiologically relevant concentrations (Miner-Williams et al., 2014, Nutr Res Rev, 27(2):308-29).

[0011] Miner-Williams et al. (2014) reported that two sources secrete proteolytic enzymes into the lumen of the digestive tract: The stomach secretes pepsinogen, which is converted by the action of acid into the active protease pepsin. The pancreas secretes a group of potent proteases, primarily trypsin, chymotrypsin, and carboxypeptidase.

[0012] Other studies have reached similar conclusions, leading to the general consensus that dietary proteins, with very few exceptions, are not absorbed in their unchanged form: rather, they must first be digested into amino acids or di- and tripeptides.

[0013] By the action of these gastric and pancreatic proteases, dietary proteins are hydrolyzed in the lumen of the small intestine, primarily into medium- and small-molecular peptides (oligopeptides).

[0014] The brush border of the small intestine contains a family of peptidases, including lactase and maltase. These peptidases are integral membrane proteins, rather than soluble enzymes. They function for the further hydrolysis of intraluminal peptides, converting them into free amino acids and very small peptides. These end products of digestion, formed on the surface of enterocytes, are ready to be absorbed.

[0015] Absorption of peptides longer than four amino acids has rarely been demonstrated. However, di- and tripeptides are absorbed in large amounts in the small intestine. These small peptides are absorbed into the small intestinal epithelial cells by co-transport with H+ via a transporter called PepT1.

[0016] Once inside the intestinal cells, the large bulk of absorbed di- and tripeptides is digested into amino acids by cytoplasmic peptidases and transported from the cells into the blood. Very few of these small peptides enter the blood intact.

[0017] As emphasized, absorption of intact proteins occurs only in a few circumstances: first, because most proteins do not pass through the chokepoint of soluble and membrane-bound proteases, and second, because "normal" enterocytes lack transporters to carry proteins across the plasma membrane and cannot reliably penetrate tight junctions.

[0018] One important exception to these general statements is that newborns have the ability to absorb intact proteins only during the first few days of life, an ability that is rapidly lost and is important because it allows newborn animals to acquire passive immunity through the absorption of immunoglobulins in colostrum.

[0019] With particular regard to opioid peptides, Asvadi et al., 2014 (Front Pharmacol. 2014;5:18, and references therein) reported that it is well known that peptides, such as opioid peptides, are susceptible to rapid enzymatic degradation. The major peptidases involved in the degradation of opioid peptides are aminopeptidases, angiotensin-converting enzyme (ACE), insulin-degrading enzyme, serine peptidases, and dipeptidyl peptidase III and IV (DPP III, DPP IV).

[0020] Peptides are also reported to be susceptible to degradation by high temperatures, which can cause protein denaturation and aggregation during temperature changes from 60 to 90 °C, resulting in clusters of high molecular weight peptides, which can result in reduced or lost biological activity (Bloom et al. 2015).

[0021] Taken together, these studies suggest that BND, which exerts its effects in the PNS and CNS, is unlikely to be used as a bioactive substance to manage relaxation via oral ingestion, or at least without intestinal protection. This problem may be exacerbated if BND is subjected to high temperatures before ingestion.

[0022] It is an object of the present invention to provide compositions and methods that induce relaxation and / or provide a useful choice to the public. Summary of the Invention [Means for solving the problem]

[0023] Summary of the Invention Applicants have surprisingly shown, for the first time, and contrary to expectations from the prior art discussed in the Background section above, that the native, unprotected, BND peptide can exert a biological effect via oral ingestion and therefore can be used to manage relaxation via oral ingestion.

[0024] Applicants have also surprisingly shown that BND peptides can be used in food and beverage compositions and can maintain their observed biological effects even after being subjected to the relatively high temperatures used in the manufacture of some such food and beverage compositions.

[0025] Thus, the present invention provides novel bioactive food and beverage compositions comprising BND, methods for their preparation, and uses in managing relaxation. The present invention further contemplates the recombinant expression of BND in biological organisms, such as plants, and the use of biological materials containing or expressing the biological materials, to produce bioactive food and beverage compositions in accordance with the present invention.

[0026] Bioactive food or beverage compositions or ingredients In one aspect, the present invention provides a bioactive food or beverage composition or ingredient comprising beta endorphin (BND) peptide in a bioactive form.

[0027] In one embodiment, the biological activity of the food or beverage composition or ingredient is conferred by a BND peptide.

[0028] In one embodiment, the BND peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0029] In a preferred embodiment, the BND peptide is not enteric coated.

[0030] In one embodiment, the BND peptide is recombinantly produced.

[0031] In a preferred embodiment, recombinantly produced BND is more biologically active than synthetic BND.

[0032] In a further embodiment, the recombinantly produced, purified BND is more pure than synthetic BND.

[0033] In a further embodiment, recombinantly produced, purified BND is less expensive to produce than synthetic BND.

[0034] In one embodiment, the BND peptide is recombinantly produced in a cell, tissue, or organism.

[0035] In one embodiment, recombinantly produced BND is purified from a cell, tissue, or organism and added to a bioactive food or beverage composition or ingredient.

[0036] In a further embodiment, the bioactive food or beverage composition or ingredient comprises a cell, tissue, organism, or part thereof in which BND has been recombinantly produced.

[0037] In further embodiments, the cell, tissue, or organism is a plant cell, plant tissue, or plant, respectively.

[0038] In a further embodiment, the cell, tissue, organism, plant cell, plant tissue, or plant is transgenic for a polynucleotide encoding a BND peptide.

[0039] In one embodiment, the polynucleotide encodes an endoplasmic reticulum (ER) targeting or chloroplast targeting signal peptide operably linked to a BND peptide.

[0040] In one embodiment, the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide operably linked to a BND peptide.

[0041] In one embodiment, the polynucleotide is ER targeting signal peptide, and BND peptide is an expression cassette encoding a polypeptide cassette comprising:

[0042] In a further embodiment, the polypeptide cassette further comprises: flexible linkers, ·First cutting site ·Second cutting site, detection / purification tags, and ER retention sequence It includes at least one of the following:

[0043] In a further embodiment, the polypeptide cassette further comprises, from N-terminal to C-terminal: ER targeting signal peptide, flexible linkers, ·1st cutting site, BND peptide ·Second cutting site, detection / purification tags, and ER retention sequence Includes:

[0044] In one embodiment, the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0045] In a preferred embodiment, the first cleavage site is an enterokinase cleavage site.

[0046] In one embodiment, the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0047] In a preferred embodiment, the second cleavage site is a thrombin cleavage site.

[0048] In a preferred embodiment, the first and second cleavage sites are different from each other.

[0049] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5 His tag.

[0050] In one embodiment, the BND peptide is multimerized.

[0051] In one embodiment, the BND peptide is repeated at least in tandem.

[0052] In one embodiment, the polynucleotide encodes a chloroplast targeting signal peptide operably linked to a BND peptide.

[0053] In one embodiment, the polynucleotide is chloroplast targeting signal peptide, and BND peptide is an expression cassette encoding a polypeptide cassette comprising:

[0054] In a further embodiment, the polypeptide cassette further comprises: flexible linkers, ·First cutting site a second cleavage site, and detection / purification tags, It includes at least one of the following:

[0055] In a further embodiment, the polypeptide cassette comprises, from N-terminal to C-terminal: ·Chloroplast targeting signal peptide, flexible linkers, ·1st cutting site, BND peptide, a second cleavage site, and Detection / purification tags Includes:

[0056] In one embodiment, the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0057] In a preferred embodiment, the first cleavage site is an enterokinase cleavage site. In one embodiment, the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0058] In a preferred embodiment, the second cleavage site is a thrombin cleavage site.

[0059] In a preferred embodiment, the first and second cleavage sites are different from each other.

[0060] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5 His tag.

[0061] In one embodiment, the BND peptide is multimerized.

[0062] In one embodiment, the BND peptide is repeated at least in tandem.

[0063] In a further embodiment, the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to a BND peptide; a first flexible linker, detection / purification tags, a second flexible linker, and ·Cut site and at least one of:

[0064] In a further embodiment, the polypeptide cassette comprises, from N-terminal to C-terminal: ·Chloroplast targeting signal peptide, a first flexible linker, detection / purification tags, a second flexible linker, cleavage site, and BND peptide Includes:

[0065] In one embodiment, the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0066] In a preferred embodiment, the cleavage site is an enterokinase cleavage site.

[0067] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5 His tag.

[0068] In one embodiment, the BND peptide is multimerized.

[0069] In one embodiment, the BND peptide is repeated at least in tandem.

[0070] In a further embodiment, the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to a BND peptide; ·Cut site, flexible linkers, and Detection / purification tags and at least one of:

[0071] In a further embodiment, the polypeptide cassette comprises, from N-terminal to C-terminal: ·Chloroplast targeting signal peptide, BND peptide, ·Cut site, a first flexible linker, and Detection / purification tags Includes:

[0072] In one embodiment, the cleavage site is cleaved by the corresponding protease just N-terminal to the cleavage site.

[0073] In one embodiment, the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0074] In one embodiment, the cleavage site is an enterokinase cleavage site.

[0075] In one embodiment, the cleavage site is a thrombin cleavage site.

[0076] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5 His tag.

[0077] In one embodiment, the BND peptide is multimerized.

[0078] In one embodiment, the BND peptide is repeated at least in tandem.

[0079] In one embodiment, the BND peptide accumulates in the ER or chloroplast of a cell, tissue, organism, plant cell, plant tissue, or plant.

[0080] In one embodiment, the BND peptide accumulates in the ER of a cell, tissue, organism, plant cell, plant tissue, or plant.

[0081] In one embodiment, the BND peptide accumulates in the chloroplasts of a cell, tissue, organism, plant cell, plant tissue, or plant.

[0082] In a preferred embodiment, the BND peptide has the true N-terminus of native BND.

[0083] In one embodiment, the BND comprises at least one, preferably at least two, more preferably at least three, more preferably at least four, and more preferably all of the first five N-terminal amino acids of SEQ ID NO: 1 or 2.

[0084] In one embodiment, the truncated BND has no more than 5, preferably no more than 4, more preferably no more than 3, more preferably no more than 2, more preferably no more than 1 additional amino acid beyond the N-terminus of SEQ ID NO: 1 or 2, and more preferably no additional amino acids.

[0085] In one embodiment, the truncated BND is at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% correct cleavage to generate an N-terminus as defined above.

[0086] In one embodiment, BND accumulates in the cell, tissue, organism, plant cell, plant tissue or plant at a level of at least 0.1% of total soluble protein.

[0087] Preferably, BND represents at least 0.2%, more preferably at least 0.3%, more preferably at least 0.4%, more preferably at least 0.5%, more preferably at least 0.6%, more preferably at least 0.7%, more preferably at least 0.8%, more preferably at least 0.9%, more preferably at least 1.0%, more preferably at least 1.2%, more preferably at least 1.3%, more preferably at least 1.4%, more preferably at least 1.5%, more preferably at least 1.6%, more preferably at least 1.7%, more preferably at least 1.8%, more preferably at least 1.9%, more preferably at least 2.0% of the total soluble protein in the cell, tissue, organism, plant cell, plant tissue or plant. , more preferably at least 2.1%, more preferably at least 2.2%, more preferably at least 2.3%, more preferably at least 2.5%, more preferably at least 2.5%, more preferably at least 2.6%, more preferably at least 2.7%, more preferably at least 2.8%, more preferably at least 2.9%, more preferably at least 3.0%, more preferably at least 3.1%, more preferably at least 3.2%, more preferably at least 3.3%, more preferably at least 3.4%, more preferably at least 3.5%, more preferably at least 3.6%, more preferably at least 3.7%, more preferably at least 3.8%, more preferably at least 3.8%, more preferably at least 4.0%.

[0088] In one embodiment, the plant cell, plant tissue or plant is suitable for human consumption.

[0089] In a further embodiment, the plant cell, plant tissue or plant is suitable for consumption by an animal.

[0090] In a further embodiment, the plant cell, plant tissue or plant is a forage plant.

[0091] In further embodiments, the plant cell, plant tissue or plant is an alfalfa plant, a rice plant, a wheat plant, a barley plant, a corn plant, a coffee plant, a cocoa plant or a tobacco plant.

[0092] In one embodiment, the food or beverage composition is an alcoholic beverage.

[0093] In one embodiment, the food or beverage composition comprises: a) a hot beverage selected from coffee, tea, or cocoa / hot chocolate beverages; b) ingredients used to prepare hot beverages, and c) Ingredients used as additives in hot beverages is selected from.

[0094] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a coffee plant, a tea plant, or a cocoa plant.

[0095] In a further embodiment, the plant cell or plant tissue is derived from or is part of a coffee bean, tea leaf or cocoa bean.

[0096] In a further embodiment, the BND is added to or infused into the ingredient, plant organ, plant tissue, coffee beans, tea leaves, or cocoa beans.

[0097] In one embodiment, BND is produced recombinantly as described herein and purified prior to injection.

[0098] In one embodiment, the coffee beans are roasted and the biological activity of the BND peptide survives roasting of the coffee beans or portions thereof.

[0099] In one embodiment, the coffee beans or portions thereof have been roasted at a temperature of at least 150°C for at least 10 minutes.

[0100] Preferably, the BND has been roasted at a temperature of at least 155°C, more preferably at least 160°C, more preferably at least 165°C, more preferably at least 170°C, more preferably at least 175°C, more preferably at least 180°C, more preferably at least 185°C, more preferably at least 190°C, more preferably at least 195°C, more preferably at least 200°C, more preferably at least 210°C, more preferably at least 220°C, more preferably at least 230°C, more preferably at least 240°C, more preferably at least 250°C, and the biological activity of the BND peptide survives roasting of the coffee bean or portion thereof.

[0101] Preferably, the BND is roasted at the above temperatures for at least 15 minutes, more preferably at least 20 minutes, more preferably at least 25 minutes, more preferably at least 30 minutes, more preferably at least 35 minutes, more preferably at least 40 minutes, more preferably at least 45 minutes, more preferably at least 50 minutes, more preferably at least 55 minutes, more preferably at least 60 minutes, and the biological activity of the BND peptide survives roasting of the coffee bean or portion thereof.

[0102] In a further embodiment, the food or beverage composition is in a liquid state and the biological activity of the BND survives heating up to at least 50°C.

[0103] Preferably, the BND withstands heating to at least 55°C, more preferably at least 60°C, more preferably at least 65°C, more preferably at least 70°C, more preferably at least 75°C, more preferably at least 80°C, more preferably at least 85°C, more preferably at least 90°C, more preferably at least 95°C, more preferably at least 100°C.

[0104] Expression cassette In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: ER targeting signal peptide, and BND peptide An expression cassette is provided that encodes a polypeptide cassette comprising:

[0105] In a further embodiment, the polypeptide cassette further comprises: flexible linkers, ·1st cutting site, ·Second cutting site, detection / purification tags, and ER retention sequence It includes at least one of the following:

[0106] In a further embodiment, the polypeptide cassette further comprises, from N-terminal to C-terminal: ER targeting signal peptide, flexible linkers, ·1st cutting site, BND peptide, ·Second cutting site, detection / purification tags, and ER retention sequence Includes:

[0107] In one embodiment, the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0108] In a preferred embodiment, the first cleavage site is an enterokinase cleavage site.

[0109] In one embodiment, the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0110] In a preferred embodiment, the second cleavage site is a thrombin cleavage site.

[0111] In a preferred embodiment, the first and second cleavage sites are different from each other.

[0112] In one embodiment, the purification tag is a His tag. In a further embodiment, the His tag is a V-5 His tag.

[0113] In one embodiment, the BND peptide is multimerized.

[0114] In one embodiment, the BND peptide is repeated at least in tandem.

[0115] In one embodiment, the polynucleotide encodes a chloroplast targeting signal peptide operably linked to a BND peptide.

[0116] In a further aspect, the present invention provides a method for producing a composition comprising: chloroplast targeting signal peptide, and BND peptide An expression cassette is provided that encodes a polypeptide cassette comprising:

[0117] In a further embodiment, the polypeptide cassette further comprises: flexible linkers, ·1st cutting site, a second cleavage site, and Detection / purification tags It includes at least one of the following:

[0118] In a further embodiment, the polypeptide cassette further comprises, from N-terminal to C-terminal: ·Chloroplast targeting signal peptide, flexible linkers, ·1st cutting site, BND peptide, a second cleavage site, and Detection / purification tags Includes:

[0119] In one embodiment, the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0120] In a preferred embodiment, the first cleavage site is an enterokinase cleavage site.

[0121] In one embodiment, the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0122] In a preferred embodiment, the second cleavage site is a thrombin cleavage site.

[0123] In a preferred embodiment, the first and second cleavage sites are different from each other.

[0124] In one embodiment, the purification tag is a His tag. In a further embodiment, the His tag is a V-5 His tag.

[0125] In one embodiment, the BND peptide is multimerized.

[0126] In one embodiment, the BND peptide is repeated at least in tandem.

[0127] In a further embodiment, the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to a BND peptide; a first flexible linker, detection / purification tags, a second flexible linker, and ·Cut site and at least one of:

[0128] In a further embodiment, the polypeptide cassette comprises, from N-terminal to C-terminal: ·Chloroplast targeting signal peptide, a first flexible linker, detection / purification tags, a second flexible linker, cleavage site, and BND peptide Includes:

[0129] In one embodiment, the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0130] In a preferred embodiment, the cleavage site is an enterokinase cleavage site.

[0131] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5 His tag.

[0132] In one embodiment, the BND peptide is multimerized.

[0133] In one embodiment, the BND peptide is repeated at least in tandem.

[0134] In a further embodiment, the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to a BND peptide; ·Cut site, flexible linkers, and detection / purification tags, and at least one of:

[0135] In a further embodiment, the polypeptide cassette comprises, from N-terminal to C-terminal: ·Chloroplast targeting signal peptide, BND peptide, ·Cut site, a first flexible linker, and Detection / purification tags Includes:

[0136] In one embodiment, the cleavage site is cleaved by the corresponding protease just N-terminal to the cleavage site.

[0137] In one embodiment, the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0138] In one embodiment, the cleavage site is an enterokinase cleavage site.

[0139] In one embodiment, the cleavage site is a thrombin cleavage site.

[0140] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5 His tag.

[0141] In one embodiment, the BND peptide is multimerized.

[0142] In one embodiment, the BND peptide is repeated at least in tandem.

[0143] plant cells or plants In one aspect, the present invention provides a plant cell, plant tissue, plant, or part thereof that has been genetically engineered to express a BND peptide in a biologically active form.

[0144] In a further aspect, the present invention provides a plant cell, plant tissue, plant or part thereof comprising an expression cassette encoding a polypeptide cassette or a polypeptide cassette of the present invention.

[0145] In one embodiment, the plant cell, plant tissue, plant, or part thereof that has been genetically modified to express a BND peptide in a biologically active form comprises an expression cassette encoding a polypeptide cassette, or a polypeptide cassette of the invention.

[0146] In one embodiment, the BND peptide has the true N-terminus of native BND.

[0147] Preferably, the BND comprises at least the first five nucleotides of SEQ ID NO:1.

[0148] In one embodiment, the plant cell, plant tissue or plant is transgenic for a polynucleotide encoding BND.

[0149] In one embodiment, the polynucleotide encodes an endoplasmic reticulum (ER) targeting or chloroplast targeting signal peptide operably linked to a BND peptide.

[0150] In one embodiment, the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide operably linked to a BND peptide.

[0151] In one embodiment, the polynucleotide encodes a chloroplast targeting signal peptide operably linked to a BND peptide.

[0152] In further embodiments, the BND peptide accumulates in the ER or chloroplast of a cell, tissue, organism, plant cell, plant tissue, or plant.

[0153] In one embodiment, the BND peptide accumulates in the ER of a cell, tissue, organism, plant cell, plant tissue, or plant.

[0154] In a further embodiment, the BND peptide accumulates in the chloroplasts of a cell, tissue, organism, plant cell, plant tissue, or plant.

[0155] In one embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant suitable for human consumption.

[0156] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant that is suitable for consumption by an animal.

[0157] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a forage plant.

[0158] In further embodiments, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of an alfalfa plant, a rice plant, a wheat plant, a barley plant, a corn plant, a coffee plant, a cocoa plant or a tobacco plant.

[0159] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a coffee plant, tea plant or cocoa plant.

[0160] In one embodiment, the plant cell, plant tissue or plant or part thereof is selected from coffee beans, tea leaves, and cocoa beans.

[0161] A method for producing a bioactive food or beverage composition comprising BND. In a further aspect, the present invention provides a method for producing a bioactive food or beverage composition comprising beta endorphin (BND) peptide in a bioactive form.

[0162] In one embodiment, the BND peptide comprises a sequence having at least 90% identity to SEQ ID NO:1.

[0163] In a preferred embodiment, the BND peptide is not enterically coated.

[0164] In one embodiment, the method includes providing a cell, tissue, or organism that includes a BND peptide.

[0165] In a further embodiment, the BND peptide is produced in a cell, tissue or organism, or portion thereof.

[0166] In one embodiment, the cell, tissue or organism, or part thereof, is a plant cell, plant tissue or plant, respectively, or part thereof.

[0167] In one embodiment, the food or beverage composition comprises: a) a hot beverage selected from coffee, tea, or cocoa / hot chocolate beverages; b) ingredients used to prepare hot beverages; c) Ingredients used as additives in hot beverages is selected from.

[0168] In one embodiment, the plant cell, plant tissue or plant or part thereof is a plant cell, plant tissue or plant or part thereof of a coffee plant, a tea plant, or a cocoa plant.

[0169] In one embodiment, the plant cell or plant tissue is derived from, or is part of, a coffee bean, tea leaf, or cocoa bean.

[0170] In one embodiment, the coffee beans are roasted and the biological activity of the BND peptide survives roasting of the coffee beans.

[0171] In one embodiment, the coffee beans have been roasted at a temperature of at least 150°C for at least 10 minutes.

[0172] Preferably, the BND has been roasted at a temperature of at least 155°C, more preferably at least 160°C, more preferably at least 165°C, more preferably at least 170°C, more preferably at least 175°C, more preferably at least 180°C, more preferably at least 185°C, more preferably at least 190°C, more preferably at least 195°C, more preferably at least 200°C, and the biological activity of the BND peptide survives roasting of the coffee bean or portion thereof.

[0173] Preferably, the BND is roasted at the above temperatures for at least 15 minutes, more preferably at least 20 minutes, more preferably at least 25 minutes, more preferably at least 30 minutes, more preferably at least 35 minutes, more preferably at least 40 minutes, more preferably at least 45 minutes, more preferably at least 50 minutes, more preferably at least 55 minutes, more preferably at least 60 minutes, and the biological activity of the BND peptide survives roasting of the coffee bean or portion thereof.

[0174] In a further embodiment, the food or beverage composition is in a liquid state and the biological activity of the BND survives heating up to at least 50°C.

[0175] Preferably, the BND withstands heating to at least 55°C, more preferably at least 60°C, more preferably at least 65°C, more preferably at least 70°C, more preferably at least 75°C, more preferably at least 80°C, more preferably at least 85°C, more preferably at least 90°C, more preferably at least 95°C, more preferably at least 100°C.

[0176] Use of BND peptides in the manufacture of a food or beverage composition for managing relaxation In a further aspect, the present invention provides the use of a BND peptide in the manufacture of a food or beverage or ingredient composition for managing relaxation.

[0177] In one embodiment, the BND peptide is produced in a plant cell, plant tissue, plant or part thereof of the invention or by a method of the invention.

[0178] In one embodiment, a food or beverage composition is produced by processing a plant cell, plant tissue, plant or part thereof of the present invention.

[0179] How to Manage Relaxation In a further aspect, the present invention provides a method of inducing, enhancing or maintaining relaxation, comprising administering to a subject in need thereof a food, beverage or ingredient composition of the present invention, or a food, beverage or ingredient composition produced by a method of the present invention.

[0180] In one embodiment, the subject is a mammal.

[0181] In one embodiment, the mammal is selected from the group consisting of humans, dogs, cats, horses, pigs, cattle, and sheep.

[0182] In a further embodiment, the subject is a human.

[0183] In one embodiment, the subject is an avian species.

[0184] In one embodiment, the bird is selected from the group consisting of chickens and turkeys.

[0185] Detailed Description of the Invention Where patent specifications, other external documents, or other sources of information are referenced herein, this is generally for the purpose of providing a context for discussing features of the present invention. Unless specifically stated otherwise, the reference to such external documents should not be construed as an admission that such documents or sources of information are prior art in any jurisdiction or form part of the common general knowledge in the art.

[0186] As used herein, the term "comprise" means "consisting at least in part of." When interpreting each description herein that includes the term "comprise," features other than those preceded by that term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted similarly.

[0187] BND Human beta-endorphin (BND) is a 31 amino acid linear peptide.

[0188] Amino acid residues 1-25 share 100% sequence identity with BND from other species, such as sheep, horse, cattle, rat, and camel. In embodiments, the BND is human BND. In embodiments, the human BND comprises the amino acid sequence of SEQ ID NO: 2 (below), or a functional variant thereof having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity thereto.

[0189] Previous studies have shown that amino acids 28-31 confer the antigenic properties of BND, and that the chain of amino acids 1-27 has activity equivalent to full-length BND when tested in vitro in the guinea pig ileum assay (Yeung et al., 1978, Int J Pept Protein Res, 12(1), 42-6).

[0190] In a further embodiment, the human BND comprises the amino acid sequence of SEQ ID NO: 1 (below), or a functional variant thereof having at least 70% sequence identity thereto.

[0191] In a further embodiment, the human BND comprises the amino acid sequence of SEQ ID NO: 2 (below), or a functional variant thereof having at least 70% sequence identity thereto.

[0192] Polypeptide variants Polypeptide sequence identity can be determined as follows: A subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs version 2.2.5 [Nov 2002]) in bl2seq, publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ).

[0193] Polypeptide sequence identity can also be calculated over the entire length of overlap between a candidate polynucleotide sequence and a subject polynucleotide sequence using a global sequence alignment program. EMBOSS-needle (available at http: / / www.ebi.ac.uk / emboss / align / ) and the above-discussed GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[0194] A preferred method for calculating the % sequence identity of polypeptides is based on aligning the sequences being compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.).

[0195] Conservative substitutions of one or more amino acids of the described polypeptide sequences that do not significantly alter its biological activity are also encompassed by the present invention. Those skilled in the art will recognize methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0196] Methods for making constructs and vectors Genetic constructs of the present invention comprise one or more polynucleotide sequences of the present invention and / or polynucleotides encoding a polypeptide of the present invention and may be useful, for example, for transforming bacterial, fungal, insect, mammalian or plant organisms.

[0197] Methods for making and using genetic constructs and vectors are well known in the art and are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).

[0198] Methods for Producing Host Cells Containing Polynucleotides, Constructs, or Vectors The present invention provides a host cell comprising a genetic construct or vector of the invention.

[0199] Host cells containing genetic constructs, such as the expression constructs of the invention, are useful in methods well known in the art for the recombinant production of polypeptides of the invention (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987). Such methods may include culturing host cells in an appropriate medium under conditions suitable for or conducive to expression of the peptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells, or culture medium by methods well known in the art (e.g., Deutscher, Ed., 1990, Methods in Enzymology, Vol. 182, Guide to Protein Purification).

[0200] Methods for producing plant cells and plants containing constructs and vectors The present invention further provides plant cells comprising a genetic construct for expressing BND in accordance with the present invention, as well as plant cells modified to alter expression of a polynucleotide or polypeptide of the present invention or used in a method of the present invention. Plants comprising such cells also form an aspect of the present invention.

[0201] Methods for transforming plant cells, plants, and parts thereof with polypeptides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual. Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transformed plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.

[0202] Methods for genetically manipulating plants Many plant transformation strategies are available (e.g., Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297; Hellens RP, et al (2000) Plant Mol Biol 42:819-32; Hellens R et al Plant Meth 1:13). For example, strategies can be designed to increase expression of a polynucleotide / polypeptide in a plant cell, organ, and / or at a particular developmental stage where / when it is normally expressed, or to ectopically express a polynucleotide / polypeptide in a cell, tissue, organ, and / or at a particular developmental stage where / when it is not normally expressed.

[0203] signal peptide Signal peptides are well known to those skilled in the art and can be used to direct the accumulation of recombinant proteins and peptides to intracellular locations in plants.

[0204] ER targeting signal peptide Endoplasmic reticulum (ER) targeting signal peptides are known to those skilled in the art and are described, for example, in Kim and Hwang, 2013, Traffic; 14:613-621.

[0205] In one embodiment, the ER targeting signal peptide used in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to the sequence of SEQ ID NO: 7.

[0206] Chloroplast targeting signal peptide Chloroplast targeting signal peptides are known to those skilled in the art and are described, for example, in Bruce et al., 2000, Trends Cell Biol; 10(10):440-7.

[0207] In one embodiment, the chloroplast targeting signal peptide used in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a sequence selected from SEQ ID NOs: 31 and 39.

[0208] Expression cassette As used herein, expression cassette refers to a polynucleotide sequence having elements that encode a polypeptide to be expressed.

[0209] An expression cassette usually contains a promoter operably linked to the sequence encoding the polypeptide to be expressed. An expression cassette usually also contains a terminator operably linked to the polypeptide to be expressed.

[0210] Polypeptide Cassette As used herein, a polypeptide cassette refers to a polypeptide sequence encoded by an expression cassette. In addition to a target peptide, in this case a BND or a multimer of BND, a polypeptide cassette may include multiple additional peptide elements, such as those described below.

[0211] Purification / Detection Tags A purification or detection tag is preferably included in the polypeptide cassette to facilitate detection or purification of the polypeptide cassette. Such tags are known to those skilled in the art and include, for example: FLAG tag, His tag, V%-His tag, Myc tag, Strep tag, TC tag, and HA tag. Examples of such sequences are shown in Table 1 below.

[0212] [Table 1]

[0213] In one embodiment, a purification / detection tag used in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a purification / detection tag sequence disclosed herein.

[0214] The purification / detection tag can facilitate detection of the polypeptide cassette or the tag itself, for example, by Western blotting.

[0215] The purification / detection tag can facilitate purification of the polypeptide cassette by use of an affinity column as known to those skilled in the art.

[0216] Cutting site Cleavage sites may be included to facilitate cleavage and separation of elements on either side of the cleavage site. Such cleavage sites are known to those of skill in the art and include, for example, enterokinase cleavage sites, thrombin cleavage sites, and examples of such sequences are shown in Table 2 below.

[0217] [Table 2]

[0218] In one embodiment, the cleavage site used in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a cleavage site sequence selected from the sequences disclosed herein.

[0219] ER retention sequence ER retention sequences can be used to retain a target peptide, in this case BND, in the endoplasmic reticulum of a plant cell. Examples of such ER retention sequences include KDEL and HDEL sequences. Examples of such sequences are shown in Table 3 below.

[0220] [Table 3]

[0221] In one embodiment, the ER retention sequence used in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to an ER retention sequence disclosed herein.

[0222] Flexible Linker In a preferred embodiment, the flexible peptide linker is soluble.

[0223] In one embodiment, the flexible peptide linker comprises the sequence (GGGS)n or (Gly-Gly-Gly-Ser)n, where n is a number from 1 to 5. Examples of such sequences are shown in Table 4 below.

[0224] [Table 4]

[0225] In one embodiment, the ER retention sequence used in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to an ER retention sequence disclosed herein.

[0226] Plant transformation protocol Representative publications that disclose genetic transformation protocols that can be used to genetically transform the following plant species are listed below: coffee (Mishra MK and Slater A, 2012, Biotechnol Res Int. 2012; 580857); tea (Sandal I. et al., 2007, Plant Cell Rep; 26(2): 169-76); cocoa (Sain SL et al., 1994, Plant Cell, Tissue and Organ Culture volume 37, pages 243-251); rice (Alam et al., 1999, Plant Cell Rep. 18, 572); alfalfa / Medicago (Wand et al., 2016, Protein Pept Lett., 23(5): 495-502); apple (Yao et al., 1995, Plant Cell Reports 14,407-412); corn (U.S. Patent Nos. 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Patent No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9, :821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227,1229); cotton (U.S. Pat. No. 5,846,797 and U.S. Pat. No. 5,004,863); grasses (U.S. Pat. No. 5,187,073 and U.S. Pat. No. 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci. 104, 183); carawe (Krens et al.,1997,Plant Cell Rep,17,39); バナナ(US Patent No. 5,792,935); ダイズ(US Patent No. 5,416,011; US ​​Patent No. 5,569,834; US Patent No. 5,82 No. 4,877; U.S. Patent No. 5,563,04455 and U.S. Patent No. 5,968,830); パイナップル (U.S. Patent No. 5,952,543); ポプラ (U.S. Patent No. 5,952,543). No. 4,795,855); Common cotyledonous plants (U.S. Patent No. 5,591,616 and U.S. Patent No. 6,037,522); Brassica (U.S. Patent No. 5,188,958; U.S. Patent No. 5,463,174 and U.S. Patent No. 5,750,871); Cereals (U.S. Patent No. 6,074,877); Matsuda et al., 2005, Plant Cell Rep. 24 (1): 45-51); Prunus (Ramesh et al., 2006 Plant Cell Rep. 25 (8): 821-8; Song and Sink 2005 Plant Cell Rep. 2006; 25 (2): 117-23; Gonzalez Padilla et al., 2003 Plant Cell Rep.22(1):38-45);イチゴ(Oosumi et al.,2006 Planta.223(6):1219-30;Folta et al.,2006 Planta Apr 14;PMID:16614818),バラ(Li et al. al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol.1995;44:129-33), Tomat (Dan et al., 2006, Plant Cell Reports V25:432-441), Rinko (Yao et al., 1995, Plant Cell Rep. 14, 407-412), Brassica napus L (Cardoza and Stewart, 2006 Methods Mol Biol.343:257-66), safflower (Orlikowska et al., 1995, Plant Cell Tissue and Organ Culture 40:85-91), rye (Altpeter et al., 2004 Developments in Plant Breeding 11(7):255-250), rice (Christou et al., 1991 Nature Biotech. 9:957-962), maize (Wang et al., 2009 In: Handbook of Maize pp. 609-639), and kiwifruit (Actinidia eriantha) (Wang et al., 2006, Plant Cell Rep. 25, 5:425-31). Transformation of other species is also contemplated by the present invention. Suitable methods and protocols are available in the scientific literature.

[0227] plant Plant cells, plant tissues, plants and parts thereof in which BND is produced according to the present invention or which contain the constructs of the present invention may be derived from any plant species.

[0228] In one embodiment, the plant cells, plant tissues, plants and parts thereof are derived from a gymnosperm species.

[0229] In a further embodiment, they are derived from angiosperm species.

[0230] In a further embodiment, they are derived from a dicotyledonous plant species.

[0231] In a further embodiment, they are derived from monocotyledonous plant species.

[0232] In one embodiment, they are derived from commercial crop species.

[0233] Preferred dicotyledonous genera include: Amygdalus, Anacardium, Arachis, Brassica, Cajanus, Cannabis, Carthamus, Carya, Ceiba, Cicer, Cocos, Coriander ndrum, Coronilla, Cossypium, Crotalaria, Dolichos, Elaeis, Glycine, Gossypium, Helianthus, Lathyrus, Lens, Lespedeza, Linum usitatissimum inum, Lotus, Lupinus, Macadamia, Medicago, Melilotus, Mucuna, Olea, Onobrychis, Ornithopus, Papaver, Phaseolus, Date palm Phoenix, Pistacia, Pisum, Prunus, Pueraria, Ribes, Ricinus, Sesamum, Theobroma, Trifolium, Trigonella, Vicia and Vigna.

[0234] Preferred dicotyledonous species include: almond (Amygdalus communis), cashew (Anacardium occidental), peanut (Arachis hypogaea), rapeseed (Brassica napus), black mustard (Brassica nigra), Brassica campestris, pigeon pea (Cajanus cajan), Cajanus indicus, cannabis sativa, safflower (Carthamus tinctorius), pecan (Carya illinoinensis), kapok (Ceiba pentandra), chickpea (Cicer arietinum), coconut (Cocos nucifera), coriander (Coriandrum sativum), and wisteria japonica (Coronilla varia, Gossypium hirsutum, Crotalaria juncea, Dolichos lablab, Elaeis guineensis, Gossypium arboreum, Gossypium nanking, Gossypium barbadense, Gossypium herbaceum, Gossypium hirsutum, Glycine max, Glycine ussuriensis, Glycine gracilis, Sunflower (Helianthus annus, Lathyrus angustifolius, Lathyrus luteus, Lathyrus mutabilis, Lathyrus sericea, Lathyrus striatastriata, Lathyrus uliginosus, Lathyrus sativus, Lentil (Lens culinaris), Marbled laurel (Lespedeza stipulacea), Flax (Linum usitatissimum), Lotus corniculatus, Lupinus albus, Medicago arabica, Medicago arborea, Medicago falcata, Medicago hispida, Medicago officinalis, Medicago sativa, Medicago tribuloides, Macadamia nuts integrifolia, Melilotus albus, Mucuna pruriens, Olive (Olea europaea), Onobrychis viciifolia, Ornithopus sativus, Mung bean (Phaseolus aureus), Phaseolus aureus cerasifera, Phaseolus aureus cerasus, Phaseolus aureus coccineus, Phaseolus aureus domestica, Phaseolus aureus lunatus lunatus), Phaseolus aureus maheleb, Phaseolus aureus mungo, Phaseolus aureus persicapersica, Phaseolus aureus pseudocerasus, Phaseolus aureus vulgaris, poppy (Papaver somniferum), tepary bean (Phaseolus acutifolius), date palm (Phoenix dactylifera), pistachio (Pistacia vera), pea (Pisum sativum), almond (Prunus amygdalus), apricot (Prunus armeniaca), kudzu (Pueraria thunbergiana), blackcurrant (Ribes nigrum), redcurrant (Ribes rubrum), goosecurrant (Ribes grossularia), castor bean (Ricinus communis), sesame (Sesamum indicum, Trifolium augustifolium, Trifolium diffusum, Trifolium hybridum, Red clover (Trifolium incarnatum), Trifolium nigressens, Red clover (Trifolium pratense), White clover (Trifolium repens), Trifolium resupinatum, Trifolium subterraneum, Theobroma cacao, Trifolium alexandrinum, Fenugreek (Trigonella foenumgraecum), Vigna angustifolia angustifolia, Vigna atropurpurea, Vigna calcarata, Vigna dasycarpa, Vigna erviliaervilia, Vigna oxycoccos, Vigna pannonica, Yard pea (Vigna sesquipedalis), Vigna sinensis, Vigna villosa, Broad bean (Viciafaba), Wild pea (Vicia sative), and Adzuki bean (Vigna angularis).

[0235] Preferred monocotyledonous genera include: Agropyron, Allium, Alopecurus, Andropogon, Arrhenatherum, Asparagus, Avena, Bambusa, Bothrichloa, Bouteloua, Bromus, Cenchrus, Chloris, Cymbopogon, Cynodon, Dactylis, Dichanthium, and Digitaria. ), Eleusine, Elymus, Eragrostis, Fagopyrum, Festuca, Hordeum, Lolium, Oryza, Panicum, Paspalum, Pennisetum, Phalaris, Phleum, Poa, Saccharum, Secale, Setaria, Sorghastrum, Sorghum, Triticum, Vanilla, Triticale Triticosecale and Zea.

[0236] Preferred monocotyledonous plant species include: false wheat (Agropyron desertorum), Agropyron elongatum, Agropyron spicatum, Agropyron trachycaulum, Agropyron trichophorum, leek species (Allium fistulosum), garlic (Allium sativum), Alopecurus pratensis, big bluestem (Andropogon gerardi), giant crabapple (Arrhenatherum elatius), asparagus officinalis, oat (Avena sativa), bamboograss (Bambusa vulgaris, Bothrichloa barbinodis, Bothrichloa ischaemum, Bouteloua curipendula, Bouteloua gracilis, Bromus erectus, Cenchrus ciliaris, African sedge grass (Chloris gayana), Ceylon citronella (Cymbopogon nardus), Corngrass (Cynodon dactylon), Orchard grass (Dactylis glomerata), Dichanthium annulatum, Digitaria decumbens, Finger millet (Eleusine coracan, Elymus angustus, Eragrostis curvula, Eragrostis tef, buckwheat (Fagopyrum esculentum), Tartary buckwheat (Fagopyrum tataricum), Tall fescue (Festucaarundinacea, two-rowed barley (Hordeum distichum), barley (Hordeum vulgare), ryegrass (Lolium perenne), ryegrass (Lolium multiflorum), rice (Oryza sativa), Panicum italicium, ryegrass (Panicum maxymum), common millet (Panicum miliaceum), striped buckthorn (Paspalum dilatatum), kikuyu grass (Pennisetum clandestinum), pearl millet (Pennisetum glaucum), reed canary grass (Phalaris arundinacea), Phleum bertolinii, Poa fendleriana, and strawberry vine (Poa nemoralis, robustum sugarcane (Saccharum robustum), oat sugarcane (Saccharum sinense), rye (Secale cereale), golden foxtail (Setaria sphacelata), Indian grass (Sorghastrum nutans), corn sorghum (Sorghum dochna), corn sorghum (Sorghum halepense), sorghum (Sorghum bicolor), wheat (Triticum aestivum), emmer wheat (Triticum dicoccum), triticale (X. triticosecale), corn (Zea mays), fairway wheatgrass (Agropyron cristatum), Agropyron intermedium, Western wheatgrass (Agropyron smithii), shallot (Allium ascalonicum), onion (Allium cepa), Chinese radish (Allium chinense), leek (Allium porrum), chives (Allium schoenoprasum), naked oats (Avena nuda), bamboo shoots (Bambusavulgaris, Bothrichloa saccharoides, Bouteloua eriopoda, Bromus inermis, Bromus riparius, Dactylis aristatum, Dactylis sericeum, Digitaria smutsii, Elymus junceus, Festuca ovina, Festuca pratensis, Festuca rubra, Panicum purpurascens, Switchgrass virgatum, American paspalum (Paspalum notatum), napier grass (Pennisetum purpureum), pearl millet (Pennisetum spicatum), timothy grass (Phleum pratense), longgrass (Poa pratensis), sugarcane (Saccharum officinarum), wasehana (Saccharum spontaneum), Sudangrass (Sorghum sudanense), durum wheat (Triticum durum), einkorn (Triticum monococcum), vanilla (Vanilla fragrans), and corn (Zea mays).

[0237] Preferred plants are those of the genera Lolium and Trifolium, particularly preferred are the species Lolium perenne and Trifolium repens.

[0238] Particularly preferred monocotyledonous plant species are ryegrass (Lolium perenne) and rice (Oryza sativa).

[0239] A preferred genus is Coffea. A preferred Coffea species is Coffea arabica.

[0240] A further preferred genus is Oryza. A preferred rice species is Oryza sativa.

[0241] Another preferred genus is Medicago. Preferred Medicago species include Medicago sativa and Medicago truncatula. A particularly preferred Medicago species is Medicago sativa, commonly known as alfalfa.

[0242] Another preferred genus is Glycine. Preferred Glycine species include Glycine max and Glycine wightii (also known as Neonotonia wightii). A particularly preferred Glycine species is Glycine max, commonly known as soybean. A particularly preferred Glycine species is Glycine wightii, commonly known as perennial soybean.

[0243] Plant organs, propagules and offspring The term "plant" is intended to include the whole plant, any part of the plant, the seeds, fruits, seeds and progeny of the plant.

[0244] The term "seed" means any part of a plant that can be used in reproduction or propagation, either sexually or asexually, such as seeds and cuttings.

[0245] Plants of the invention may be grown on their own or in crosses with different plant lines, and the resulting progeny containing the polynucleotide or construct of the invention and / or expressing the BND sequence / construct also form part of the invention.

[0246] Preferably, the plants, plant organs, offspring and progeny according to the invention comprise the polynucleotide or construct of the invention and / or express the BND sequence / construct.

[0247] Methods for producing food and beverage compositions Methods for producing food and beverage compositions, including the incorporation of bioactive ingredients, are known in the art and are described, for example, in WO2017037263, WO2019045576; WO2011146140 and WO2017124075.

[0248] BRIEF DESCRIPTION OF THE DRAWINGS The invention will now be described with reference to the following non-limiting drawings. [Brief explanation of the drawings]

[0249] [Figure 1] 1 shows a graph demonstrating the relaxation effect on subjects of orally ingested BND dissolved in normal saline compared to a normal saline control. [Figure 2] 1 shows a graph demonstrating the relaxation effect on subjects of orally ingested coffee infused with BND compared to a coffee control. [Figure 3] 1 shows a graph demonstrating the relaxation effect on subjects of orally ingested BND samples subjected to various temperature regimes prior to ingestion. [Figure 4] 1 shows a graph demonstrating the relaxation effect on subjects of orally ingested coffee samples made from beans roasted at various temperatures and infused with BND prior to production and consumption of the coffee samples. [Figure 5]1 shows a graph demonstrating the effect on subjects of orally ingesting beer infused with BND compared to a beer control. [Figure 6] 1 shows the output from TargetP analysis of the complete peptide sequence of the ER targeting cassette. [Figure 7] The output from NetGene2 is shown. The sequence from SEQ ID NO: 32 was analyzed by NetGene2 to predict splicing. [Figure 8] Output from TargetP analysis of the chloroplast targeting cassette complete peptide sequence is shown. [Figure 9] The output from NetGene2 is shown. The sequence from SEQ ID NO: 35 was analyzed by NetGene2 to predict splicing. [Figure 10] Shown is the output from TargetP analysis of the full-length ER-targeting, tandem repeat BND. [Figure 11] Output from TargetP analysis of full-length chloroplast targeting, tandem repeat sequence BND, is shown. [Figure 12] Immunoblot analysis of soluble protein extracts from N. benthamiana leaves transiently expressing BND fusion constructs. Replicate leaf extracts were subjected to SDS-PAGE before immunoblotting and probing with anti-V5 antibody. The top and bottom panels represent different exposure times of the immunoblot (10 and 30 seconds, respectively). Panel A shows extracts from plants expressing a single BND construct targeted to the ER and chloroplasts. Panel B shows extracts from plants expressing tandem BND constructs targeted to the ER and chloroplasts. VC = vector control. [Figure 13] ELISA results for leaves expressing single β-endorphin constructs are shown. [Figure 14]Immunoblot analysis of soluble proteins extracted from N. benthamiana leaves transiently expressing BND fusion constructs in a buffer containing 1 M urea and 1% Triton X-100. The white arrow indicates the 12.6 kDa chloroplast signal truncated tandem repeat endorphin cassette. The black arrow indicates the 8.7 kDa ER signal truncated tandem repeat endorphin cassette. [Figure 15] Quantification of crude recombinant BND accumulation in N. benthamiana leaves. A) Dilution series from leaf extracts is shown in parallel with dilution series of 2, 5, 10, 20, and 40 ng of V5 fusion protein standard. White arrows indicate the 8.7 kDa and 12.9 kDa signal-cleaved single and tandem repeat endorphin cassettes, respectively. B) Standard curves generated by Image Lab 5.2.1 software after scanning the dilution series results in panel A are shown. C) Average BND production was calculated as FW (μg / g) of transiently expressing leaves. [Figure 16] Immunoblot analysis of endorphin purification using Ni-affinity chromatography. The upper and lower panel images show the unstained gel and immunoblot membrane, respectively. [Figure 17] Immunoblot analysis of endorphin purification using Ni affinity binding and peptide concentration by ultrafiltration using an Amicon® 3 kDa cutoff filter. The upper and lower panel images show unstained gels and immunoblot membranes, respectively. Arrows indicate ER-targeting BND dimers and oligomers. DETAILED DESCRIPTION OF THE INVENTION

[0250] Example The invention will now be described with reference to the following non-limiting examples.

[0251] Example 1 - Bioactive Effects of Orally Administered BND Peptides Materials and Methods The BND peptide used in the present study contained amino acids 1 to 27, a 27-mer of the human β-endorphin peptide sequence. The BND 27-mer was synthesized by Leon Biological Technology Co Ltd, Nanjing, China.

[0252] Ten mg of BND peptide was dissolved in 200 ml of normal saline, and this BND test sample was ingested by subject A (an adult male), and the relaxation index was monitored over a period of 4 to 6 hours. A control test was conducted under the same conditions with the same subject using a control test sample of 200 ml of normal saline (no BND).

[0253] The relaxation index was self-assessed by the subjects immediately after ingesting the test and control samples as shown below. The degree of relaxation (shown in Table 5 below) was recorded at short intervals during the test period. Physical data such as pulse, blood pressure, and other physical parameters were recorded and closely aligned with the self-assessed relaxation index.

[0254] [Table 5]

[0255] result The results are shown in Figure 1.

[0256] This data unexpectedly demonstrates, for the first time to the Applicant's knowledge, that BND can produce bioactive effects when taken orally. Existing literature suggests that BND, like many other bioactive peptides, is unlikely to exert biological effects in the body when taken orally in an unprotected form for a variety of reasons, including the harsh environment of the gastrointestinal tract, as discussed in the Background of the Invention section.

[0257] FIG. 1 shows that orally ingested BND produces a net relaxing effect that lasts up to 4 hours after ingestion relative to controls.

[0258] Example 2 - Bioactive Effects of BND Infused into Pre-Made Hot Coffee Materials and Methods The BND peptide used and the relaxation index assessment were as described in Example 1.

[0259] 200 ml of coffee brewed at 60°C was infused with 10 mg of BND peptide after 5 minutes at room temperature, and the BND-infused coffee was consumed by the test subject over 5 minutes. A control test was conducted on a different day under the same conditions with the same subject using a 200 ml control test sample of coffee (without BND). Stress index assessment began immediately after consumption of each coffee sample.

[0260] result The results, shown in Figure 2, indicate that the BND-infused coffee produced a significantly stronger relaxing effect than the control coffee, an effect that lasted for up to four hours after ingestion.

[0261] These results demonstrate that the unexpected bioactive effects of BND demonstrated in Example 1 are surprisingly maintained even when BND is added to coffee at 60°C, indicating that neither the presence of coffee nor heating to 60°C has a detrimental effect on the efficacy of BND to induce relaxation in test subjects.

[0262] Example 3 - Bioactive effects of BND subjected to various temperatures, including conditions mimicking coffee roasting The purpose of these studies was to evaluate the effect of elevated temperature on the bioactive efficacy of BND as demonstrated in the examples above, and to determine whether efficacy is maintained after exposure to conditions equivalent to roasting coffee beans.

[0263] Materials and Methods The BND peptide used and the assessment of the relaxation index were as described in Example 1.

[0264] 10 mg of BND sample was placed on a filter paper and subjected to the following temperature regime: 14 minutes at room temperature (20°C) 14 minutes at 160°C in a fan-forced oven 14 minutes at 230°C in a fan-forced oven were applied separately.

[0265] Non-ambient temperatures (160°C and 230°C) and heating times were chosen because these parameters are often used in coffee roasting processes.

[0266] Each test sample was then dissolved in 200 ml of normal saline (20°C) for 5 minutes and ingested by the test subject, followed by a relaxation index assessment.

[0267] result The results are shown in Figure 3 and demonstrate that the BND efficacy produced after each temperature regimen was approximately equal, and surprisingly, the BND peptide maintains its BND efficacy even after being subjected to relatively harsh environments (160°C and 230°C) equivalent to the conditions used in coffee roasting.

[0268] Example 4 - Bioactive effects of BND in coffee beans containing BND after roasting the beans The purpose of this example was to evaluate the effect of roasted beans containing BND on BND and to test the hypothesis that plant material expressing BND could be processed into a beverage that would provide the same bioactive effects. To this end, raw coffee beans were infused with BND and then subjected to conditions equivalent to coffee roasting.

[0269] Materials and Methods The BND peptide used and the assessment of the relaxation index were as described in Example 1.

[0270] Raw (unroasted) coffee beans were infused with BND peptide by soaking the beans for 12 hours in a 1 mg / ml solution of BND dissolved in normal saline.

[0271] Samples of unheated BND-infused beans were then roasted in separate trials in a fan-forced oven for 14 minutes at 160°C and 230°C. Pre-roasted and post-infused beans served as controls.

[0272] The roasted (BND infused) test beans and the control beans were then ground and used separately to brew coffee.

[0273] 200 ml of each test coffee (prepared at each roasting temperature) and control coffee were consumed by the test subjects after 5 minutes, followed by a relaxation index assessment.

[0274] result The results are shown in Figure 4.

[0275] As shown, the BND effect on subject relaxation was surprisingly demonstrated in coffee brewed with BND-infused beans subjected to both roasting temperatures, an effect similar to that exhibited by coffee brewed with pre-roasted BND-infused beans.

[0276] This indicates that if BND can be recombinantly expressed in biological material (e.g., plant material) in the correct form, the biological material can be processed under relatively harsh processing conditions to produce food or beverage compositions that retain the demonstrated BND bioactivity.

[0277] Example 5 - BND in alcoholic beverages Materials and Methods The BND peptide used and the assessment of the relaxation index were as described in Example 1.

[0278] 10 mg of BND was infused into 200 ml of Stella Artois beer at 20° C. for 5 minutes, then ingested by the test subject and assessed for a relaxation index.

[0279] A control beer test was conducted on a different day under the same conditions using a control beer (without BND).

[0280] result The results are shown in Figure 5.

[0281] This data indicates that when combined with beer, BND has a relaxing effect that is greater than that demonstrated by beer alone and lasts for up to 4 hours after ingestion.

[0282] This data demonstrates that beer and / or the alcohol content of beer did not adversely affect the efficacy of BND in inducing a relaxed state.

[0283] Furthermore, the relaxing effect of beer and BND combined was more pronounced in test subjects than the relaxing effect of beer alone.

[0284] Example 6 - Expression of BND in plants background To the best of applicant's knowledge, BND has never been expressed in plants, let alone in an active form or at commercially significant levels.

[0285] The N-terminus of BND has been reported to be important for activity. Therefore, it may be important or essential to express a peptide with the correct N-terminal sequence for the production of active BND in plants. The sequence of the mature peptide fragment (31 residues) is highly conserved: YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE (SEQ ID NO: 2).

[0286] Proteins, polypeptides, and even peptides have been expressed in plants, but the level of production of any given peptide is unpredictable. Signal peptides are used to target proteins, polypeptides, and peptides to organelles for increased accumulation, but these are also unpredictable.

[0287] Cleavage of targeting signal sequences is also unpredictable and can be influenced by factors such as sequence differences between the downstream peptide and the signal sequence (e.g., hydrophobicity, charge, size, etc.) Various software applications exist to predict where cleavage will occur, but these are far from guaranteed to be accurate.

[0288] Thus, there are several unforeseen challenges to overcome before BND can be produced in plants at reasonable levels in an active / correctly cleaved form.

[0289] Materials and Methods Structure Design Constructs were generated in the model plant Nicotiana benthamiana to express and target BND to the endoplasmic reticulum (ER) and chloroplasts using a signal peptide sequence (described further below). A C-terminal V-5 His tag was also included for detection / quantification and / or purification.

[0290] Expression cassette characteristics Both cassettes were designed using the same GATEWAY™ Recombination directional cloning process to place them into a binary vector, with the BND fusion peptide under the control of the CaMV35s promoter (constitutive) and NOS terminator. Both cassettes were optimized for expression in Nicotiana benthamiana; they contain introns with appropriate predicted splicing sites and efficiencies; contain Kozak sequences; delete polyadenylation signal sequences; delete mRNA instability sequences; and contain double stop codons and tetranucleotides (Scott et al., 2010, Plant Biotechnology Journal. 8:912-927). It will be understood by those skilled in the art that these optimizations, while not required, may be beneficial. The sequences used in the constructs are shown in the sequence listing below.

[0291] Endoplasmic reticulum (ER) targeting of single BNDs To target BND to the ER, the Arabidopsis thaliana Purple Acid Phosphatase signal peptide sequence (SEQ ID NO: 7) was used. The expression cassette was based on Winichayakul et al. (2009).

[0292] The full peptide sequence expressed is: ER targeting signal peptide (SEQ ID NO: 7) Flexible linker (SEQ ID NO: 18) Enterokinase cleavage site (SEQ ID NO: 13) BND peptide (SEQ ID NO: 2) Thrombin cleavage site (SEQ ID NO: 16) V-5 His tag (SEQ ID NO: 20) ER retention sequence (SEQ ID NO: 22) It included.

[0293] The entire peptide sequence (theoretical 11.92 kDa) encoded by the ER targeting cassette (SEQ ID NO: 26) was processed by the signal prediction software TargetP. The results are shown in Figure 6 and indicate that the signal peptide is cleaved to leave the true N-terminal sequence of the BND peptide.

[0294] The ER targeting peptide coding sequence was optimized, engineered, and custom synthesized for tobacco by GenScript. The complete GATEWAY™ flanked ER targeting BND-V-5 His with intron nucleic acid sequence is shown in SEQ ID NO:26.

[0295] After using Geneious Prime GATEWAY subcloning, the sequence determined from the TATATAA box to the double stop codon (TAATGA) of the CaMV35S promoter (SEQ ID NO: 32) was analyzed by NetGene2 to predict splicing (Figure 7). These results indicate that the intron is predicted to be correctly spliced.

[0296] Chloroplast targeting of single BNDs A truncated Nicotiana tabacum NtRBCs rubisco signal peptide (SEQ ID NO: 31) was used to target BND to the chloroplast (Eseverrie et al., 2020). The chloroplast targeting cassette is shown in SEQ ID NO: 27.

[0297] The full peptide sequence expressed (SEQ ID NO:27) is: Chloroplast targeting signal peptide (SEQ ID NO: 31) Flexible linker (SEQ ID NO: 18) Enterokinase cleavage site (SEQ ID NO: 13) BND peptide (SEQ ID NO: 2) Thrombin cleavage site (SEQ ID NO: 16) V-5 His tag (SEQ ID NO: 20) It included.

[0298] The entire peptide sequence encoded by the chloroplast targeting cassette (theoretical 13.47 kDa) was processed by the signal prediction software TargetP. The results are shown in Figure 8 and indicate that the signal peptide is cleaved to leave the true N-terminal sequence of the BND peptide.

[0299] The chloroplast targeting peptide coding sequence was optimized for expression in Nicotiana benthamiana to generate the sequence shown in SEQ ID NO:33.

[0300] The optimized chloroplast targeting sequence has attL1, 5'UTR, AtDGAT1 intron 3, a double stop codon, attL2, and an added sequence (NB T changed from A to remove an mRNA instability sequence, which changes the CCA codon to CCT, both of which encode proline) as shown in SEQ ID NO:34.

[0301] After using Geneious Prime GATEWAY subcloning, the sequence from the TATATAA box to the (TAATGA) double stop codon of the CaMV35S promoter is shown in SEQ ID NO: 35. The sequence was analyzed by NetGene2 and splicing was predicted (Figure 9).

[0302] Tandem repeat BND construct A construct was also made to express BND as a tandemly repeated peptide sequence separated by a flexible linker, as shown in SEQ ID NO: 36. A sequence containing an enterokinase cleavage site, a tandemly repeated BND sequence, a thrombin cleavage site, and engineered cloning restriction sites is shown in SEQ ID NO: 37.

[0303] The nucleotide sequence was optimized for expression in Nicotiana benthamiana; it contains an intron with appropriate predicted splicing sites and efficiency; a Kozak sequence, the polyadenylation signal sequence was deleted, and the mRNA instability sequence was deleted. The nucleic acid and peptide sequences of the tandem repeat fragments are shown in the sequence listing.

[0304] The tandem repeat BND cassette was then subcloned into the ER and chloroplast targeting cassettes described above.

[0305] The peptide sequence of the ER-targeting tandem repeat sequence BND (SEQ ID NO: 29) (predicted to be 16.08 kDa) was processed with the signal sequence recognition software TargetP (FIG. 10).

[0306] For the chloroplast-targeting tandem repeat BND expression cassette, a modified version of the chloroplast transit peptide (SEQ ID NO: 39) was used.

[0307] The peptide sequence of the chloroplast targeting tandem repeat BND expression cassette (SEQ ID NO: 38) (predicted to be 22.35 kDa) was also processed with TargetP software (FIG. 11).

[0308] Cloning and transformation Cloning of plant organelle-targeting BND Both the designed ER- and chloroplast-targeting BND cassettes were individually cloned into the binary vector pRSh1 using Gateway™ LR Clonase™ reactions (Scott et al., 2010). The conformation of the plasmid constructs was confirmed by restriction enzyme mapping and sequencing.

[0309] Cloning of plant organelle-targeting tandem repeat sequence BND The tandem 2x repeat BND fragment was cloned into the 056488 pPCR Script-ER targeting cassette and the 056486 pPCR Script-CHL targeting cassette. The plasmid constructs 056488-ER-2xEdph and 056486-CHL-2xEdph were selected and confirmed by restriction enzyme mapping. Both the ER- and chloroplast-targeted 2xEdph were individually cloned into the binary vector pRSh1 using Gateway™ LR Clonase™ reactions (Scott et al., 2010). The conformation of the plasmid constructs was confirmed by restriction enzyme mapping and sequencing.

[0310] Transformation of the binary vector containing the BND expression cassette into Agrobacterium cells Plasmid DNAs of pRSh1-ER-Edph (ER-targeted single BND), pRSh1-CHL-Edph (chloroplast-targeted single BND), pRSh1-ER-2xEdph (ER-targeted tandem BND), and pRSh1-CHL-2xEdph (chloroplast-targeted tandem BND) were transformed into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method and selected on appropriate antibiotic-containing medium. A. tumefaciens cells containing the plasmid DNA were selected and confirmed by PCR using the pRSh1 forward and reverse primers.

[0311] Agrobacterium-mediated transient expression of BND-containing cassettes in tobacco Agrobacterium cells containing the BND expression cassette were infiltrated into tobacco (Nicotina benthamiana) leaves. Leaf samples were collected, soluble proteins were extracted, and BND expression was analyzed by SDS-PAGE immunoblot (Figure 12) and ELISA (Figure 13).

[0312] result Immunoblotting Immunoblot results for ER-targeted BND (ER-Edph) and tandem repeat BND (ER-2xEdph) showed that both accumulated to detectable levels (Figure 12). However, no peptide was observed for chloroplast-targeted BND (CHL-Edph), and only a small amount of peptide was observed for chloroplast-targeted tandem repeat BND (CHL-2xEdph), which could not be predicted by an experienced person skilled in the art. Transient expression in N. benthamiana using Agrobacterium cells containing the vector control (VC) did not reveal any immunoblot signal.

[0313] ELISA Enzyme-linked immunosorbent assay (ELISA) is a technique used for quantification and typically has a wider dynamic range than gel scanning. Alternatively, V5::6xHis-tagged BND contained in a matrix of total leaf soluble protein extract was absorbed onto a nickel-coated plate (Pierce™) and subsequently immunologically detected with an anti-V5 antibody (Figure 13). ELISA results also confirmed the accumulation of ER-targeted BND (ER-Edph). No color development was observed with the none (no protein coating), vector control (VC), and chloroplast-targeted BND (CHL-Edph) soluble protein extracts. The reaction was kinetically incubated at 37°C for 10 minutes, and the optical density change at 405 nm was recorded.

[0314] Transient expression of the chloroplast-targeted BND cassette was detected in N. benthamiana leaves only after using an alternative extraction buffer.

[0315] The lack of recombinant protein from the chloroplast targeting cassette was surprising; to see if it could be due to poor extraction efficiency (chloroplasts are small, discreet organelles with multiple membranes), total protein extraction was repeated using a different buffer, which contained 1 M urea and 1% Triton x-100 (which solubilized bilayer membrane proteins).

[0316] Immunoblotting showed relatively strong accumulation of the chloroplast-targeting tandem repeat BND(CHL-2xEdph) peptide, but not the chloroplast-targeting single BND(CHL-Edph) peptide (Figure 14). The lack of accumulation in chloroplasts could be due to the tandem repeat sequence compared to the single peptide. However, unpublished results suggest that the use of different chloroplast targeting sequences in the two constructs is more likely to be responsible. While a previously tested targeting sequence (Winichayakul et al., 2009) was used in the tandem construct, a recently published truncated version was used for the monomer cassette (Eseverri et al., 2020).

[0317] The hand formation patterns of the immunoblots suggest that the ER and chloroplast targeting peptides are correctly processed for signal cleavage.

[0318] The predicted size of the uncleaved ER-Edph peptide is 11.9 kDa, and the predicted size of the signal cleavage peptide is 8.7 kDa. Therefore, it appears that the majority of the detected ER-Edph peptides are properly cleaved (Figure 12, panel A and Figure 14). The relatively faint, tall band could represent either an incorrectly cleaved peptide or a dimerization of the cleaved peptide; given the size difference, it is more likely to be a dimer.

[0319] The predicted size of the uncleaved ER-2xEdph peptide is 16.1 kDa, and the signal-cleaved form is 12.9 kDa; however, immunoblots (Figure 12, panel B) showed a banding pattern very similar to that of the single ER-Edph peptide (Figure 12, panel A). While cleavage of the ER signal sequence and the first BND would explain the banding pattern, this is unlikely given the absence of specific peptidase sites located between the BND repeats. Given the relatively small size of the peptides, it is more likely that they were cleaved and subsequently dimerized, causing them to migrate faster than expected on this particular 4-15% gradient acrylamide gel. This can be confirmed by running ER-Edph and ER-2xEdph extracts side-by-side on the same gel and / or using a higher % polyacrylamide gel with a different running buffer, such as Tris-tricline, to separate smaller protein sizes (<10 kDa).

[0320] The predicted size of uncleaved CHL-2XEdph is 22 kDa, and the cleaved form is 12.6 kDa (or 18.5 kDa if cleaved at repeated transit peptide cleavage sites). The immunoblot in Figure 14 suggests that the 12.6 kDa form is present, indicating that the first transit peptide cleavage site is recognized. The larger immunoreactive band could represent either a miscleaved peptide or dimerization of the cleaved peptide.

[0321] Quantification of ER-targeted BND from crude extracts The amount of BND expressed in the transient leaf expression system was determined as follows: 2, 5, 10, 20, and 40 ng of PEAPOD-V5 fusion protein standard (prepared in another project by expression in bacterial cells and purification by affinity gel binding, Figure 15, Panel A) were immunoblotted on the same gel with leaf extracts from ER-targeting BND (ER-Edph) and tandem repeat BND (ER-2xEdph). The band intensities of the standards were scanned and plotted as a standard curve using Image Lab 5.2.1 software (Figure 15, Panel B). From the curve, the average accumulation of BND (µg / gFW) in N. benthamiana leaves could be calculated (Figure 15, Panel C).

[0322] The levels of total recombinant protein, as well as the levels of accumulated recombinant BND, were converted to % of total soluble protein and are shown in Table 6.

[0323] [Table 6]

[0324] Purification and requantification of ER-targeting BND Although the recombinant protein appears to accumulate to relatively low levels in the transient expression system, it can be purified and concentrated to produce sufficient quantities for further studies, such as cleavage and mass spectrometry conformation of the appropriate sequence, bioassays, etc. In this proof-of-concept study, we added a C-terminal V-5 His tag to the BND fragment to aid in purification and concentration.

[0325] Seventy-two hours after Agrobacterium infiltration, leaves transiently expressing ER-targeted BND were collected (FW 15.87 g) and homogenized in 10 mL of 2.5x ice-cold extraction buffer (pH 7.4) containing 50 mM sodium phosphate buffer and 2.5% Triton X-100. The crude extract was centrifuged at 10,000 x g for 5 min at 4°C to remove leaf debris. The protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1 mM, and the final volume of the soluble extract was adjusted to 25 mL with cold, sterile Milli-Q water.

[0326] Initially, the soluble extract precipitated after the addition of imidazole (5-15 mM), and most of the histidine-tagged material did not bind to the Ni column. This was likely due to unknown compounds in the extract from N. benthamiana leaves. Subsequently, the pre-diluted crude extract was passed through a 3 kDa cutoff filtration membrane (which retained the recombinant peptide) before adding imidazole and loading the extract onto the column (Figure 16).

[0327] In Figure 16, immunoblots showed that C-terminally tagged ER-targeting BND was present in both the soluble extract (lane 2) and the filtered extract (lane 4). After loading the filtered extract onto the column, the peptide was eluted with a range of imidazole concentrations (80–400 mM). The disappearance of the monomer (a smaller band approximately 8.7 kDa) and the appearance of a larger band (previously thought to be a dimer or uncleaved signal BND) were observed. This may be due to the use of a 3 kDa cutoff filter, which served to concentrate the recombinant protein, leading to an increased degree of oligomerization.

[0328] In elutions 1, 2, and 3, we were unable to detect any protein on a stain-free gel; consequently, they were pooled and concentrated (Supplementary Protocol 2). Protein samples were subsequently analyzed by immunoblot as shown in Figure 17 (lower panel, lane 14).

[0329] Immunoblotting revealed that the concentrated soluble eluate contained ER-targeted BND, present both as a dimer and as a larger oligomer (Figure 27, lane 14). This allowed us to recalculate the level of recombinant protein in the total soluble leaf protein extract and found that the level of recombinant BND (as a percentage of total soluble leaf protein) was significantly higher than originally determined (Table 6 vs. Table 7).

[0330] [Table 7]

[0331] Conclusions and further experiments We demonstrated for the first time that the mature BND peptide can be recombinantly synthesized and accumulated in plants. This was achieved by targeting the peptide (as a cleavable signal sequence) to the endoplasmic reticulum (ER) or chloroplasts. In the latter case, a truncated form of the chloroplast transit peptide sequence did not result in detectable BND accumulation. Immunoblot analysis suggested that the ER-targeting signal sequence was efficiently cleaved, and the uncleaved chloroplast transit peptide was likely eliminated approximately 50% of the time.

[0332] Purification of the recombinant protein (by both size exclusion and affinity chromatography) allowed for more precise quantification of the level of accumulated BND; when targeted to the ER, this amounted to approximately 2% of total soluble protein. The ability to purify and concentrate BND should allow further characterization of the peptide.

[0333] A first step may involve further determination of the efficiency and precision of signal cleavage. This may be achievable by excising gel slices from appropriate migration points and subjecting them to trypsin digestion and mass spectrometry. Similarly, the efficiency and precision of β-enterokinase cleavage just upstream of the mature BND peptide may be achieved by first subjecting the gel slices to enterokinase prior to trypsin digestion, followed by mass spectrometry. These analyses may also allow for confirmation of the correct sequence from examination of the C-terminus.

[0334] The chloroplast-targeted fragment can be efficiently cleaved of its N-terminal targeting sequence, leaving the correct N-terminal residue of the mature BND peptide. The use of a β-enterokinase site can facilitate this. An advantage of chloroplast targeting is that it is possible to produce peptides with the correct C-terminus, since retention in the chloroplast (unlike the ER) does not require additional sequences.

[0335] Example 7 - Further confirmation of the N-terminus and activity of recombinantly expressed BND Mass spectrometry can be performed on the current recombinant peptide and subsequent work with the mature peptide alone (untagged) can be facilitated using commercially available antibodies against the mature peptide.

[0336] For example, mass spectrometry can be used to confirm the sequence of the recombinant peptide (with and without enterokinase treatment) from the chloroplast-targeting endorphin tandem repeat cassette. This can indicate between which residues the chloroplast-targeting peptide is cleaved, as well as the percentage of the peptide that is cleaved. Similarly, this can demonstrate that enterokinase cleaves at the appropriate site, the percentage of the peptide that is cleaved, and that the cleavage leaves the expected N-terminal residue of β-endorphin. The synthetic BND described in Example 1 can be used as a standard for mass spectrometry analysis.

[0337] Alternatively, purification and quantification of the recombinant mature peptide may be possible without the use of anti-BND antibodies. Alternatively, a V-5 His tag could be placed between the chloroplast signal sequence and the β-enterokinase site upstream of the BND peptide. This would allow purification and concentration from chloroplasts, as well as removal of the N-terminal signal and tag.

[0338] Assuming that the sequence is revealed by mass spectrometry and that the enterokinase cleavage is correct, the following: An expression cassette containing a chloroplast transit peptide (Winichayakul et al 2009)::internal His tag::enterokinase cleavage site::single BND peptide can be produced.

[0339] As an example, the amino acid sequence of such an expression cassette is shown in SEQ ID NO:40.

[0340] The full peptide sequence expressed (SEQ ID NO: 40) is: Chloroplast targeting signal peptide (SEQ ID NO: 31) Flexible linker (SEQ ID NO: 18) V-5 His tag (SEQ ID NO: 20) Flexible linker (SEQ ID NO: 18) Enterokinase cleavage site (SEQ ID NO: 13) BND peptide (SEQ ID NO: 1) Includes:

[0341] As an example, a polynucleotide sequence encoding such a cassette is shown in SEQ ID NO:41.

[0342] This cassette can be ligated by standard procedures into an expression cassette having the CaMV35S promoter and terminator to produce the sequence shown in SEQ ID NO:42.

[0343] The entire expression cassette can be used to transform Agrobacterium by standard procedures and / or as described above, before being introduced into plants by transient or stable expression as described herein.

[0344] below: Further alternative expression cassettes can be produced containing chloroplast transit peptide (Winichayakul et al 2009)::single BND peptide::enterokinase cleavage site::His tag.

[0345] As an example, the amino acid sequence of such an expression cassette is shown in SEQ ID NO:51.

[0346] The full peptide sequence expressed (SEQ ID NO: 51) is: Chloroplast targeting signal peptide (SEQ ID NO: 31) BND peptide (SEQ ID NO: 1) Enterokinase cleavage site (SEQ ID NO: 13) Flexible linker (SEQ ID NO: 18) V-5 His tag (SEQ ID NO: 20) Includes:

[0347] As an example, a polynucleotide sequence encoding such a cassette is shown in SEQ ID NO:52.

[0348] This cassette can be ligated by standard procedures to an expression cassette containing the CaMV35S promoter and terminator, as described above.

[0349] The entire expression cassette can be used to transform Agrobacterium by standard procedures and / or as described above, before being introduced into plants by transient or stable expression as described herein.

[0350] The polynucleotide coding sequence may be codon optimized for the species being transformed.

[0351] Bioassays to determine the activity of recombinant BND compared to chemically synthesized peptides can be performed after production and purification of recombinantly expressed BND peptides (see, e.g., Examples 1-5 herein).

[0352] Example 8 - Stable transformation of plants to recombinantly express active BND The expression cassettes described in Examples 6 and 7 can be cloned into vectors suitable for stable (as well as transient) transformation of plants.

[0353] For example, the expression cassettes described in Examples 6 and 7 can be cloned into pRSh1 (Scott et al 2010) replacing the constitutive promoter cauliflower mosaic virus 35S (CaMV35Sp) driven GATEWAY® adapted expression cassette to generate a binary vector, or can be cloned from pDONR™221 into pBR2 by GATEWAY® LR cloning (Thermo Fisher Scientific).

[0354] Alfalfa Alfalfa can be stably transformed, for example, as described in Wand et al., 2016, Protein Pept Lett., 23(5):495-502.

[0355] Rice Rice can be stably transformed as described, for example, in Alam et al., 1999, Plant Cell Rep. 18, 572.

[0356] wheat Wheat can be stably transformed as described, for example, in Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877.

[0357] Barley Barley can be stably transformed, for example, as described in Lazzeri, P. 1995, Methods Mol Biol: 49:95-106.

[0358] corn Maize can be stably transformed as described, for example, in US Pat. No. 5,177,010 and US Pat. No. 5,981,840.

[0359] tobacco Tobacco can be stably transformed, for example, as described in Horsch et al., 1985, Science 227, 1229.

[0360] coffee Coffee can be stably transformed, for example, as described in Ribas et al., BMC Plant Biol. 2011;11:92.

[0361] tea Tea can be stably transformed as described, for example, in Chen et al., 2022 Front. Plant Sci., Sec. Plant Systematics and Evolution Volume 13.

[0362] cocoa Cocoa can be stably transformed as described in Sain SL et al., 1994, Plant cell, Tissue and Organ Culture volume 37, pages 243-251, and Maximova et al., 2003, Plant cell Rep 21, 872-883.

[0363] Other plant species can be stably transformed as described herein and using other protocols known in the art.

[0364] Example 9 - Analysis of BND expressed by stable transformation Recombinantly expressed BND produced by stable transformation (as well as transient expression) can be purified and analyzed as described in Examples 6 and 7 above.

[0365] Example 10 - Further confirmation of the activity of synthetic and recombinantly expressed BND in animal studies With respect to Examples 1-5 above, Applicants have surprisingly first shown that, contrary to expectations from the prior art discussed in the Background section above, native unprotected BND peptides can exert biological effects upon oral ingestion and therefore can be used to administer relaxation upon oral ingestion.

[0366] To further confirm and explore the biological activity of BND (which may be produced synthetically or recombinantly), larger-scale studies in which BND is ingested by rats can be conducted, for example, at The Howard Florey Institute of Neuroscience & Mental Health (Floor 2, 161 Barry Street, Carlton, Victoria 3053, Australia) using the protocol described below.

[0367] the purpose The purpose of the study was to test the relaxation effects of human beta-endorphin (BND) administered orally to groups of SD rats in a double-blind, controlled study over a six-week period. This study can be used to test the effects of synthetically and recombinantly produced BND as described herein and to confirm the biological activity of recombinantly produced BND.

[0368] The purpose of this study is to test whether the orally administered BND group shows demonstrable signs of enhanced relaxation relative to the control group.

[0369] Group composition Each of the four treatments consisted of 15 male (or female) SD rats caged in groups of four in separate areas and randomly assigned in a blinded manner.

[0370] test The groups are as follows: 1. SD rats, n=15, orally treated with vehicle, unrestrained (control for impact of stress) 2. SD rats, n=15, orally treated with test recombinant compound, unrestrained (Control for Impact of Compound) 3. SD rats, n=15, orally administered with vehicle, restrained (Impact of Stress) 4. SD rats, n=15, orally administered synthetic test compounds, restrained (Impact of Compound on Stress)

[0371] In an alternative study, rats from each treatment can be divided into three groups for replication. Optionally, either all male or all female rats can be used.

[0372] End of exam At week 6, all groups are sacrificed.

[0373] Postmortem studies of organs are carried out, cardiac puncture is performed, and specimens for hematological and biochemical studies are taken.

[0374] Cortisol test Serum cortisol tests are performed on all test subjects at time zero, 2 weeks, 4 weeks, and the 6 week sacrifice.

[0375] Blood (0.5 ml) is collected by venipuncture from the tail. Care is taken to acclimate the rodent to this procedure, and a local anesthetic is applied to the tail venipuncture to minimize trauma.

[0376] BND Dosage Regimen The equivalent daily dose (pro rata by weight) corresponds to a 20 mg dose in a 70 kg human. The doses of synthetically and recombinantly produced BND described in the examples above are used.

[0377] Alternatively: dose equivalents (proportional to body weight) are equivalent to a 20 mg dose in a 70 kg human. Doses of synthetically and recombinantly produced BND as described in the examples above can be used during weeks 1-2, then during week 5, to test multiple phases of stress reduction.

[0378] Stressor Planning Restraint stress of 2 hours per day for 3 weeks may be used. Restraint times may be rotated so that animals have a different restraint time on each successive day.

[0379] After completing the three weeks with or without restraint, all rats are tested using the following four tests, which are performed for a further three weeks:

[0380] Elevated Plus Maze (Anxiety Test) The elevated plus maze is custom-made from light-colored Perspex, consisting of two open arms (10 cm x 44 cm for rats) and two closed arms (10 cm x 44 cm x 10 cm for rats) extending from a central platform (12 x 12 cm for rats). It is mounted on a base 40 cm above the floor. This is a test of anxiety in rodents, as rodents prefer the safety of the closed arms and are wary when entering the open arms. Rodents exhibit a conflict between their affinity for novel spaces and the opportunities they present, and the potential danger of unprotected areas.

[0381] At the beginning of the experiment, the rodent is placed in the center of the maze facing the open arm; the following variables: time spent in the open and closed arms and entries are tracked with a Cleversys Topscan tracking system. Time spent in the open and closed arms is expressed as a percentage of the total time in the maze. Open and closed arm entries are defined as the rodent's entry of all four paws into one arm of the maze. Room lighting is approximately 10-20 lux. Testing is conducted in a single 10-minute trial.

[0382] Gait exercise test (motor function test) Rodents are removed from their home cage and placed in the center of a Med Associates locomotor chamber. The system monitors a range of defined parameters, including but not limited to distance traveled, time spent moving, number of movements, and time spent rearing. General locomotor activity, typically measured over a 60-90 minute period, can also be assessed using this system. General locomotor activity, measured using the aforementioned parameters, can provide insight into general phenotypes induced by genetic manipulation or drug administration.

[0383] Light / Dark Test (Anxiety Test) This test can also be performed automatically using a Med Associates locomotor system, which has a black Perspex box insert located in one half of the locomotor chamber, providing a light and a dark area. The black Perspex box has a small opening that allows the rat to move from the dark area to the open, lighted area (450 lux). At the beginning of the test, the rat is placed in the dark area and allowed to explore both areas for a 10-minute trial. A software program records the number of transitions from light to dark, the length of time spent in each area, and the latency to emerge from the dark to light area. Rats that spend a longer time in the dark area relative to the light area may exhibit higher levels of basal anxiety-like behavior when compared to control rats.

[0384] Large open field test (anxiety test) Rodents were removed from their home cages and placed in the center of a square arena (110 cm × 110 cm), exposing them to a brightly lit open-field area. Their movements were tracked by automated tracking software (Cleversys Topscan). The trial lasted 10 minutes, after which the rodents were placed back into their transport box and returned to their home cages. The well-lit (450 lux) large area posed a threat to rodents, which prefer dark conditions and small spaces. This again presented the rat with a conflict between the potential dangers of the large area (i.e., aerial predators) and the novel food source / mating opportunity in a new space. Consequently, this elicited various responses, which could include high levels of exploration, passage through the center of the arena, or remaining still and occupying the sides and corners of the test arena. Rats that spent longer time in the perimeter and corner areas of the arena, as captured and analyzed by our CleverSys rodent tracking software, were considered to have higher levels of anxiety-like behavior.

[0385] The results of these studies will provide insight into the behavioral manifestations of restraint and the potential of test compounds as viable anxiolytic agents.

[0386] References Baulcombe D. (2004) RNA silencing in plants. Nature 431:356-363. Eseverri A,Baysal C,Medina V,Capell T,Christou P,Rubio LM,Caro E.(2020) Transit peptides from photosynthesis-related proteins mediate import of a marker protein into different plastid types and within different species.Frontiers in Plant Science,11:560701. Hosfield T,Lu Q.(1999) Influence of the amino acid residue downstream of (Asp)4Lys on enterokinase cleavage of a fusion protein.Analytical Biochemistry,269:10-16. Mazur BJ,Chui CF (1985) Sequence of a genomic DNA clone for the small subunit of ribulose bis-phosphate carboxylase-oxygenase from tobacco.Nucleic Acids Research,13:2373-2386. Scott RW,Winichayakul S,Roldan M,Cookson R,Willingham M,Castle M,Pueschel R,Peng C,Tzen JTC,Roberts NJ.(2010) Elevation of oil body integrity and emulsion stability by polyoleosins,multiple oleosin units joined in tandem head-to-tail fusions.Plant Biotechnology Journal,8(8):912-927. Skala W, Goettig P, Brandstetter H. (2013) Do-it-yourself histidine tagged bovine enterokinase: A handy member of the protein engineer’s toolbox. Journal of Biotechnology, 168(4):421-425. Terpe K. (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 60:523-533. Winichayakul S, Pernthaner A, Scott R, Vlaming R, Roberts N. (2009) Head-to-tail fusions of camelid antibodies can be expressed in planta and bind in rumen fluid. Biotechnology and Applied Biochemistry, 53(2):111-122.

[0387]

Table 8

[0388]

Table 9

[0389]

Table 10

[0390]

Table 11

[0391] Table 12

Claims

1. A bioactive food or beverage composition or ingredient comprising beta endorphin (BND) peptide in a bioactive form.

2. 10. The bioactive food or beverage composition or ingredient of claim 1, wherein the bioactivity of said food or beverage composition or ingredient is conferred by said BND peptide.

3. 3. The bioactive food or beverage composition or ingredient of claim 1 or 2, wherein the BND peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:1 or SEQ ID NO:

2.

4. The bioactive food or beverage composition or ingredient of any one of claims 1 to 3, wherein the BND peptide is not enterically coated.

5. 5. The bioactive food or beverage composition or ingredient of any one of claims 1 to 4, wherein said BND peptide is produced by recombinant technology.

6. 6. The bioactive food or beverage composition or ingredient of any one of claims 1 to 5, wherein the BND peptide is recombinantly produced in a cell, tissue or organism, or part thereof.

7. 7. The physiologically active food or beverage composition or ingredient of claim 6, wherein the recombinantly produced BND is purified from a cell, tissue or organism, or part thereof, and added to the physiologically active food or beverage composition or ingredient.

8. 7. The bioactive food or beverage composition or ingredient of claim 6, comprising said cell, tissue or organism, or part thereof, in which said BND is recombinantly produced.

9. A physiologically active food or beverage composition or ingredient according to any one of claims 6 to 8, wherein the cell, tissue or organism, or part thereof, is a plant cell, plant tissue or plant, or part thereof, respectively.

10. 10. The food or beverage composition of any one of claims 6 to 9, wherein the cell, tissue, organism, plant cell, plant tissue or plant, or part thereof, is transgenic for a polynucleotide encoding the BND peptide.

11. 11. The food or beverage composition of claim 10, wherein the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide or a chloroplast targeting signal peptide operably linked to the BND peptide.

12. 12. The food or beverage composition of claim 10 or 11, wherein the BND peptide accumulates in the ER or chloroplast of the cell, tissue, organism, plant cell, plant tissue or plant, or part thereof.

13. 13. The food or beverage composition of any one of claims 6 to 12, wherein the BND peptide has the true N-terminus of native BND.

14. 14. The food or beverage composition of any one of claims 1 to 13, wherein the BND accumulates at a level of at least 0.1% of total soluble protein.

15. 15. The food or beverage composition according to any one of claims 1 to 14, wherein the plant cell, plant tissue or plant, or part thereof, is a plant cell, plant tissue or plant, or part thereof, of an alfalfa plant or a rice plant.

16. a) a hot beverage selected from coffee, tea, or cocoa / hot chocolate beverages; b) ingredients used to prepare said hot beverage; c) ingredients used as additives in said hot beverages; 16. The food or beverage composition according to any one of claims 1 to 15,

17. 17. The food or beverage composition according to any one of claims 1 to 16, wherein the plant cell, plant tissue or plant, or part thereof, is a plant cell, plant tissue or plant, or part thereof, of a coffee plant, tea plant, or cocoa plant.

18. 13. A food or beverage composition according to any one of claims 6 to 12, wherein the plant cells or plant tissue are derived from or are part of coffee bean, tea leaf or cocoa bean material.

19. 20. The food or beverage composition of claim 18, wherein the coffee beans are roasted and the biological activity of the BND peptide survives roasting of the coffee beans.

20. 20. The food or beverage composition of claim 18 or 19, wherein the coffee bean material has been roasted at a temperature of at least 150°C for at least 10 minutes.

21. 21. A food or beverage composition according to any one of claims 1 to 20, in a liquid state and wherein the biological activity of the BND withstands heating up to at least 50°C.

22. a. i. an ER targeting signal peptide, and ii. at least one chloroplast targeting signal peptide; b. a BND peptide; An expression cassette encoding a polypeptide cassette comprising:

23. A plant cell, plant tissue or plant that has been genetically engineered to express a BND peptide in a biologically active form.

24. 24. A plant cell, plant tissue or plant according to claim 23, comprising an expression cassette according to claim 22, or a polypeptide cassette encoded by said expression cassette.

25. 25. The plant cell, plant tissue or plant of claim 23 or 24, wherein the BND peptide has the true N-terminus of native BND.

26. 26. The plant cell, plant tissue or plant of any one of claims 23 to 25, which is transgenic for a polynucleotide encoding said BND.

27. 27. The plant cell, plant tissue or plant of claim 26, wherein the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide or a chloroplast targeting signal peptide operably linked to the BND peptide.

28. 28. The plant cell, plant tissue or plant of claim 27, wherein the BND peptide accumulates in the ER or chloroplasts of the cell, tissue, organism, plant cell, plant tissue or plant.

29. 29. The plant cell, plant tissue or plant according to any one of claims 23 to 28, wherein the plant cell, plant tissue, plant or part thereof is a plant cell, plant tissue, plant or part thereof of an alfalfa plant, a rice plant, a wheat plant, a barley plant, a corn plant, a coffee plant, a cocoa plant or a tobacco plant.

30. 30. The plant cell, plant tissue or plant according to any one of claims 23 to 29, wherein the plant cell, plant tissue, plant or part thereof is a plant cell, plant tissue, plant or part thereof of a coffee plant, tea plant or cocoa plant.

31. 31. The plant part according to any one of claims 23 to 30, selected from coffee beans, tea leaves, and cocoa beans.

32. A method for producing a bioactive food or beverage composition that is bioactive and contains a BND peptide.

33. 33. The method of claim 32, wherein the BND peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

34. The method of any one of claims 32-33, wherein the BND peptide is not enterically coated.

35. 35. The method of any one of claims 32-34, wherein the BND peptide has the true N-terminus of native BND.

36. 36. The method of any one of claims 32 to 35, comprising providing a cell, tissue, organism or part thereof comprising said BND peptide.

37. 37. The method of claim 36, wherein the BND peptide is produced in a cell, tissue, organism, or part thereof.

38. 38. The method of claim 36 or 37, wherein the cell, tissue, organism or part thereof is a plant cell, plant tissue, plant or part thereof, respectively.

39. The food or beverage composition comprises: a) a hot beverage selected from coffee, tea or cocoa / hot chocolate beverages; b) ingredients used to prepare said hot beverage; c) ingredients used as additives in said hot beverages; The method according to any one of claims 32 to 38, wherein the compound is selected from the group consisting of:

40. 40. The method of any one of claims 32 to 39, wherein the plant cell, plant tissue, plant or part thereof is a plant cell, plant tissue, plant or part thereof of a coffee plant, tea plant, or cocoa plant.

41. 41. The method of any one of claims 32 to 40, wherein the plant cells, plant tissues are derived from or are part of coffee bean, tea leaf or cocoa bean material.

42. 42. The method of any one of claims 32-41, wherein the coffee beans are roasted and the biological activity of the BND peptide survives the roasting of the coffee beans.

43. 43. The method of any one of claims 32 to 42, wherein the coffee bean material has been roasted at a temperature of at least 150°C for at least 10 minutes.

44. A method according to any one of claims 32 to 43, wherein the food or beverage composition according to any one of claims 1 to 21 is in a liquid state and the biological activity of the BND withstands heating up to at least 50°C.

45. Use of BND peptides in the manufacture of a food or beverage or ingredient composition for managing relaxation.

46. 46. ​​The use according to claim 45, wherein the BND peptide is produced in a plant or plant tissue according to any one of claims 23 to 30 or by a method according to any one of claims 32 to 44.

47. 47. The use according to claim 46, wherein the food or drink composition is produced by processing a plant, plant tissue or plant material according to any one of claims 23 to 30.

48. 10. A method of inducing, enhancing or maintaining relaxation, comprising administering to a subject in need thereof a food or beverage composition according to any one of claims 1 to 21 produced by a method according to any one of claims 32 to 44 or extracted from a cell, plant cell, tissue, plant tissue, organism or plant according to any one of claims 23 to 30.

49. 49. The method of claim 48, wherein the subject is selected from the group consisting of mammals, humans, animals, dogs, cats, horses, pigs, cattle, sheep, and birds.

50. 49. The method of claim 48, wherein the subject is a human.

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