Bioactive food and beverage compositions and methods

Abstract

The invention provides bioactive foods, beverages, compositions and ingredients comprising a beta-endorphin (BND) peptide in a bioactive form, and methods and compositions useful for producing a beta-endorphin (BND) peptide in a bioactive form, and such bioactive foods, beverages, compositions and ingredients. The invention also provides methods and compositions useful for producing a beta-endorphin (BND) peptide in a bioactive form in cells, tissues and plants, and cells, tissues and plants producing a beta-endorphin (BND) peptide in a bioactive form. The invention further provides methods and compositions for inducing, increasing, or maintaining relaxation, comprising administering to a subject in need thereof a bioactive food, beverage, composition or ingredient as described.

Description

[0001] BIOACTIVE FOOD AND BEVERAGE COMPOSITIONS AND METHODS

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] The contents of Australian provisional patent application number 2022904029, filed 24 December 2022, are incorporated herein by reference in their entirety.

[0004] TECHNICAL FIELD

[0005] The present invention is in the field of bioactive food and beverages and in compositions and methods for their production.

[0006] BACKGROUND

[0007] There are many reported benefits of relaxation including: improved concentration, improved digestion, increased blood flow, reduced anger and frustration, lowered blood pressure, lowering of the risk of stroke, promotion of emotional well-being, reduced fatigue, reduced inflammation, and lowering heart rate.

[0008] Consumption of certain food and beverages is associated with increasing relaxation. However, some such foods and beverages, for example alcoholic beverages, while promoting some of the benefits of relaxation described above, have detrimental effects on other health factors. In addition, beverages such as alcoholic beverages are also addictive, which can lead to further well-documented problems.

[0009] It would therefore be desirable to provide compositions and methods useful for inducing or increasing relaxation that overcome one or more of disadvantages of existing food and beverages taken with the goal of inducing relaxation.

[0010] Beta-Endorphin, or g-Endorphin or BND, is an endogenous opioid neuropeptide and peptide hormone. A BND precursor is formed in the pituitary gland and subsequently processed into BND. BND functions through various mechanisms in both the central and peripheral nervous systems, when bound to its cognate mu-opioid receptor.

[0011] The function of BND is known to be associated with hunger, thrill, pain, maternal care, sexual behaviour, and reward cognition. In the broadest sense, BND is primarily utilized in the body to reduce stress and maintain homeostasis. In behavioural research, studies have shown that BND is released via volume transmission into the ventricular system in response to a variety of stimuli.

[0012] While BND might be seen as a potential bioactive useful for managing relaxation, studies have shown that it, and many other bioactive peptides, are unlikely to exert any biological effect inside the body if orally ingested in an unprotected form, for various reasons including the harsh environment of the gastro-intestinal tract, as discussed further below.

[0013] Hence, efforts to tap into the beneficial effects of BND have focussed on stimulants and bioactives that induce endogenous production of BND, albeit with limited success.

[0014] In the emerging science of orally ingested bioactives, the issue of the absorption is paramount. However, previous studies have found that there is little unequivocal evidence that dietary bioactive peptides, other than di- and tri peptides, can cross the gut wall intact and enter the hepatic portal system in physiologically relevant concentrations (Miner- Williams et al., 2014, Nutr Res Rev, 27(2):308-29).

[0015] Miner-Williams et al. (2014) report that two sources secrete proteolytic enzymes into the lumen of the digestive tube:

[0016] • the stomach secretes pepsinogen, which is converted to the active protease pepsin by the action of acid.

[0017] • the pancreas secretes a group of potent proteases, chief among them trypsin, chymotrypsin and carboxypeptidases.

[0018] Other studies have reached similar conclusions leading to the general view that dietary proteins are, with very few exceptions, not absorbed in an unaltered form. Rather, they must be first digested into amino acids or di- and tripeptides.

[0019] Through the action of these gastric and pancreatic proteases, dietary proteins are hydrolyzed within the lumen of the small intestine predominantly into medium and small peptides (oligopeptides).

[0020] The brush border of the small intestine is equipped with a family of peptidases, such as lactase and maltase. These peptidases are integral membrane proteins rather than soluble enzymes. They function to further the hydrolysis of lumenal peptides, converting them to free amino acids and very small peptides. These end products of digestion, formed on the surface of the enterocyte, are ready for absorption. Virtually no absorption of peptides longer than four amino acids has been documented previously. However, there is abundant absorption of di- and tripeptides in the small intestine. These small peptides are absorbed into the small intestinal epithelial cell by cotransport with H+ ions via a transporter called PepTl.

[0021] Once inside the enterocyte, the vast bulk of absorbed di- and tripeptides are digested into amino acids by cytoplasmic peptidases and exported from the cell into blood. Only a very small number of these small peptides enter blood intact.

[0022] As emphasized, absorption of intact proteins occurs only in a few circumstances. First because very few proteins get through the gauntlet of soluble and membrane-bound proteases intact. Second, because "normal" enterocytes do not have transporters to carry proteins across the plasma membrane and they certainly cannot permeate tight junctions.

[0023] One important exception to these general statements is that for a very few days after birth, neonates have the ability to absorb intact proteins. This ability, which is rapidly lost, is of importance because it allows the newborn animal to acquire passive immunity by absorbing immunoglobulins in colostral milk.

[0024] Specifically with respect to opioid peptides, Asvadi et al., 2014, (Front Pharmacol. 2014; 5: 18, and references within) report that it is well-known that peptides including 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, dipeptidyl peptidase III and IV (DPP III, DPP IV).

[0025] Peptides are also reported to be susceptible to degradation by high temperate. Heat causes protein denaturation and aggregation during temperature changes from 60 to 90 °C, which may cause high molecular mass peptides form clusters and, as a result, bioactivity may diminish or be lost (Bloom et al. 2015).

[0026] Together, these studies indicate that it is highly unlikely that BND, which exerts its effects in the PNS and CNS, could be used as a bioactive to manage relaxation via oral ingestion, or at least not without being enterica lly protected. This problem may be exacerbated if the BND is subjected to high temperatures prior to ingestion.

[0027] It is an object of the invention to provide compositions and methods for inducing relaxation and / or to provide the public with a useful choice. SUMMARY OF THE INVENTION

[0028] The applicants have surprisingly shown for the first time, contrary to what is expected from the prior art as discussed in the Background section above, that native, unprotected, BND peptide can exert a biological effect via oral ingestion, and can thus be used to manage relaxation via oral ingestion.

[0029] The applicants have also surprisingly shown that the BND peptide can be used in food and beverage compositions, and that the observed biological effects can be maintained even after the BND peptide has been subjected to relatively extreme temperatures used in producing some of such food and beverage compositions.

[0030] The invention therefore provides novel bioactive food and beverage compositions comprising BND, methods for their production, and use in managing relaxation. The invention further contemplates recombinant expression of BND in biological organisms such as plants, and use of biological material comprising or expressing biological material to produce bioactive food and beverage compositions in accordance with the invention.

[0031] Bioactive food or beverage composition or ingredient

[0032] In one aspect the invention provides a bioactive food or beverage composition or ingredient comprising a beta-endorphin (BND) peptide in a bioactive form.

[0033] In one embodiment the bioactivity of the food or beverage composition or ingredient is conferred by the BND peptide.

[0034] In one embodiment the BND peptide comprises a sequence with at least 90% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

[0035] In a preferred embodiment, the BND peptide is not enterically coated.

[0036] In one embodiment the BND peptide has been recombinantly produced.

[0037] In a preferred embodiment the recombinantly produced BND is more biologically active than synthesised BND.

[0038] In a further embodiment the recombinantly produced purified BND is more pure than synthesised BND. In a further embodiment the recombinantly produced purified BND is less expensive to produce than synthesised BND.

[0039] In one embodiment the BND peptide has been recombinantly produced in a cell, tissue, or organism.

[0040] In one embodiment the recombinantly produced BND has been purified from the cell, tissue or organism, and added to the bioactive food or beverage composition or ingredient.

[0041] In a further embodiment the bioactive food or beverage composition or ingredient comprises the cell, tissue, organism, or a part thereof in which the BND has been recombinantly produced.

[0042] In a further embodiment the cell, tissue, or organism is a plant cell, plant tissue or plant respectively.

[0043] In a further embodiment the cell, tissue, organism, plant cell, plant tissue or plant, is transgenic for a polynucleotide encoding the BND peptide.

[0044] In one embodiment the polynucleotide encodes an endoplasmic reticulum (ER) targeting, or chloroplast targeting signal peptide operably linked to the BND peptide.

[0045] In one embodiment the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide operably linked to the BND peptide.

[0046] In one embodiment the polynucleotide is an expression cassette encoding a polypeptide cassette comprising:

[0047] • ER targeting signal peptide, and

[0048] • the BND peptide.

[0049] In a further embodiment the polypeptide cassette additionally comprises at least one of:

[0050] • A flexible linker,

[0051] • A first cleavage site,

[0052] • A second cleavage site,

[0053] • A detection / purification tag, and

[0054] • An ER retention sequence. In a further embodiment the polypeptide cassette additionally comprises is an N- to C- direction:

[0055] • The ER targeting signal peptide,

[0056] • A flexible linker,

[0057] • A first cleavage site,

[0058] • The BND peptide,

[0059] • A second cleavage site,

[0060] • A detection / purification tag, and

[0061] • An ER retention sequence.

[0062] In one embodiment the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0063] In a preferred embodiment the first cleavage site is an enterokinase cleavage site.

[0064] In one embodiment the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0065] In a preferred embodiment the second cleavage site is a thrombin cleavage site.

[0066] In a preferred embodiment the first and second cleavage sites are different from one another.

[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 multimerised.

[0069] In one embodiment the BND peptide is at least tandemly repeated.

[0070] In one embodiment the polynucleotide encodes a chloroplast targeting signal peptide operably linked to the BND peptide.

[0071] In one embodiment the polynucleotide is an expression cassette encoding a polypeptide cassette comprising:

[0072] • chloroplast targeting signal peptide, and

[0073] • the BND peptide. In a further embodiment the polypeptide cassette additionally comprises at least one of:

[0074] • A flexible linker,

[0075] • A first cleavage site,

[0076] • A second cleavage site, and

[0077] • A detection / purification tag.

[0078] In a further embodiment the polypeptide cassette comprises is an N- to C- direction:

[0079] • The chloroplast targeting signal peptide,

[0080] • A flexible linker,

[0081] • A first cleavage site,

[0082] • The BND peptide,

[0083] • A second cleavage site, and

[0084] • A detection / purification tag.

[0085] In one embodiment the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0086] In a preferred embodiment the first cleavage site is an enterokinase cleavage site.

[0087] In one embodiment the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0088] In a preferred embodiment the second cleavage site is a thrombin cleavage site.

[0089] In a preferred embodiment the first and second cleavage sites are different from one another.

[0090] In one embodiment the detection / purification tag is a His tag. In a further embodiment the His tag is a V-5 His tag.

[0091] In one embodiment the BND peptide is multimerised.

[0092] In one embodiment the BND peptide is at least tandemly repeated.

[0093] In a further embodiment the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to the BND peptide and at least one of:

[0094] • A first flexible linker,

[0095] • A detection / purification tag,

[0096] • A second flexible linker, and A cleavage site.

[0097] In a further embodiment the polypeptide cassette comprises is an N- to C- direction:

[0098] • The chloroplast targeting signal peptide,

[0099] • A first flexible linker,

[0100] • A detection / purification tag,

[0101] • A second flexible linker,

[0102] • A cleavage site, and

[0103] • The BND peptide.

[0104] In one embodiment the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0105] In a preferred embodiment the cleavage site is an enterokinase cleavage site.

[0106] In one embodiment the detection / purification tag is a His tag. In a further embodiment the His tag is a V-5 His tag.

[0107] In one embodiment the BND peptide is multimerised.

[0108] In one embodiment the BND peptide is at least tandemly repeated.

[0109] In a further embodiment the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to the BND peptide and at least one of:

[0110] • A cleavage site,

[0111] • A flexible linker, and

[0112] • A detection / purification tag.

[0113] In a further embodiment the polypeptide cassette comprises is an N- to C- direction:

[0114] • The chloroplast targeting signal peptide,

[0115] • The BND peptide,

[0116] • A cleavage site,

[0117] • A first flexible linker, and

[0118] • A detection / purification tag.

[0119] In one embodiment the cleavage site is cleaved, by its corresponding protease, immediately prior to the N-terminus of the cleavage site. In one embodiment the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0120] In one embodiment the cleavage site is an enterokinase cleavage site.

[0121] In one embodiment the cleavage site is a thrombin cleavage site.

[0122] In one embodiment the detection / purification tag is a His tag. In a further embodiment the His tag is a V-5 His tag.

[0123] In one embodiment the BND peptide is multimerised.

[0124] In one embodiment the BND peptide is at least tandemly repeated.

[0125] In one embodiment the BND peptide accumulates in the ER or chloroplast of the cell, tissue, organism, plant cell, plant tissue or plant.

[0126] In one embodiment the BND peptide accumulates in the ER of the cell, tissue, organism, plant cell, plant tissue or plant.

[0127] In one embodiment the BND peptide accumulates in the chloroplast of the cell, tissue, organism, plant cell, plant tissue or plant.

[0128] In a preferred embodiment the BND peptide has the authentic N-terminus of naturally occurring BND.

[0129] In one embodiment the BND comprises at least one, preferably at least two, more preferably at least three, more preferably at least four, more preferably all of the first five N-terminal amino acids of SEQ ID NO: 1 or 2.

[0130] In one embodiment the cleaved BND has no more than five, preferably no more than four, more preferably no more than 3, more preferably no more than 2, more preferably no more than 1, more preferably no additional amino acids beyond the N-terminus of SEQ ID NO: 1 or 2.

[0131] In one embodiment the cleaved 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 cleave to produce the N-terminus as defined above.

[0132] In one embodiment the BND accumulates at a level of at least 0.1 % of total soluble protein in the cell, tissue, organism, plant cell, plant tissue or plant.

[0133] Preferably the BND accumulates at a level of 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%, 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% of total soluble protein in the cell, tissue, organism, plant cell, plant tissue or plant.

[0134] In one embodiment the plant cell, plant tissue or plant is that of plant suitable for human consumption.

[0135] In a further embodiment the plant cell, plant tissue or plant is that of plant suitable for animal consumption.

[0136] In a further embodiment the plant cell, plant tissue or plant is that of forage plant.

[0137] In a further embodiment the plant cell, plant tissue or plant is that 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.

[0138] In one embodiment the food or beverage composition is an alcoholic beverage. io In one embodiment the food or beverage composition is selected from: a) a hot beverage selected from a coffee, tea or a cocoa / hot chocolate beverage, b) an ingredient used to produce the hot beverage, and c) an ingredient used as an additive to the hot beverage.

[0139] In a further embodiment the plant cell, plant tissue or plant is that of a coffee plant, a tea plant, or a cocoa plant.

[0140] In a further embodiment the plant cell or plant tissue is from, or part of, a coffee bean, a tea leaf or a cocoa bean.

[0141] In a further embodiment the BND has been added to, or infused into the ingredient, plant part, plant tissue, coffee bean, tea leaf or cocoa bean.

[0142] In one embodiment the BND is recombinantly produced as described herein, and purified prior to infusion.

[0143] In one embodiment the coffee bean has been roasted, and the bioactivity of the BND peptide survives the roasting of coffee bean, or part thereof.

[0144] In one embodiment the coffee bean, or part thereof, has been roasted at a temperature of at least 150°C, for at least 10 minutes.

[0145] 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 bioactivity of the BND peptide survives the roasting of coffee bean, or part thereof.

[0146] Preferably the BND has been roasted 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, at the above temperature, and the bioactivity of the BND peptide survives the roasting of coffee bean, or part thereof. In a further embodiment the food or beverage composition is in liquid form, and bioactivity of the BND survives heating to at least 50°C.

[0147] Preferably the BND survives 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.

[0148] Expression cassette

[0149] In one aspect the invention provides an expression cassette encoding a polypeptide cassette comprising:

[0150] • ER targeting signal peptide, and

[0151] • a BND peptide.

[0152] In a further embodiment the polypeptide cassette additionally comprises at least one of:

[0153] • A flexible linker,

[0154] • A first cleavage site,

[0155] • A second cleavage site,

[0156] • A detection / purification tag, and

[0157] • ER retention sequence.

[0158] In a further embodiment the polypeptide cassette additionally comprises is an N- to C- direction:

[0159] • The ER targeting signal peptide,

[0160] • A flexible linker,

[0161] • A first cleavage site,

[0162] • The BND peptide,

[0163] • A second cleavage site,

[0164] • A detection / purification tag, and

[0165] • An ER retention sequence.

[0166] In one embodiment the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0167] 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.

[0168] In a preferred embodiment the second cleavage site is a thrombin cleavage site.

[0169] In a preferred embodiment the first and second cleavage sites are different from one another.

[0170] In one embodiment the purification tag is a His tag. In a further embodiment the His tag is a V-5 His tag.

[0171] In one embodiment the BND peptide is multimerised.

[0172] In one embodiment the BND peptide is at least tandemly repeated.

[0173] In one embodiment the polynucleotide encodes a chloroplast targeting signal peptide operably linked to the BND peptide.

[0174] In a further aspect the invention provides an expression cassette encoding a polypeptide cassette comprising:

[0175] • chloroplast targeting signal peptide, and

[0176] • the BND peptide.

[0177] In a further embodiment the polypeptide cassette additionally comprises at least one of:

[0178] • A flexible linker,

[0179] • A first cleavage site,

[0180] • A second cleavage site, and

[0181] • A detection / purification tag

[0182] In a further embodiment the polypeptide cassette additionally comprises is an N- to C- direction:

[0183] • The chloroplast targeting signal peptide,

[0184] • A flexible linker,

[0185] • A first cleavage site,

[0186] • The BND peptide,

[0187] • A second cleavage site, and

[0188] • A detection / purification tag. In one embodiment the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0189] In a preferred embodiment the first cleavage site is an enterokinase cleavage site.

[0190] In one embodiment the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0191] In a preferred embodiment the second cleavage site is a thrombin cleavage site.

[0192] In a preferred embodiment the first and second cleavage sites are different from one another.

[0193] In one embodiment the purification tag is a His tag. In a further embodiment the His tag is a V-5 His tag.

[0194] In one embodiment the BND peptide is multimerised.

[0195] In one embodiment the BND peptide is at least tandemly repeated.

[0196] In a further embodiment the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to the BND peptide and at least one of:

[0197] • A first flexible linker,

[0198] • A detection / purification tag,

[0199] • A second flexible linker, and

[0200] • A cleavage site.

[0201] In a further embodiment the polypeptide cassette comprises is an N- to C- direction:

[0202] • The chloroplast targeting signal peptide,

[0203] • A first flexible linker,

[0204] • A detection / purification tag,

[0205] • A second flexible linker,

[0206] • A cleavage site, and

[0207] • The BND peptide,

[0208] In one embodiment the cleavage site is an enterokinase cleavage site or a thrombin cleavage site. In a preferred embodiment the cleavage site is an enterokinase cleavage site.

[0209] In one embodiment the detection / purification tag is a His tag. In a further embodiment the His tag is a V-5 His tag.

[0210] In one embodiment the BND peptide is multimerised.

[0211] In one embodiment the BND peptide is at least tandemly repeated.

[0212] In a further embodiment the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to the BND peptide and at least one of:

[0213] • A cleavage site,

[0214] • A flexible linker, and

[0215] • A detection / purification tag.

[0216] In a further embodiment the polypeptide cassette comprises is an N- to C- direction:

[0217] • The chloroplast targeting signal peptide,

[0218] • The BND peptide,

[0219] • A cleavage site,

[0220] • A first flexible linker, and

[0221] • A detection / purification tag.

[0222] In one embodiment the cleavage site is cleaved, by its corresponding protease, immediately prior to the N-terminus of the cleavage site.

[0223] In one embodiment the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0224] In one embodiment the cleavage site is an enterokinase cleavage site.

[0225] In one embodiment the cleavage site is a thrombin cleavage site.

[0226] In one embodiment the detection / purification tag is a His tag. In a further embodiment the His tag is a V-5 His tag.

[0227] In one embodiment the BND peptide is multimerised.

[0228] In one embodiment the BND peptide is at least tandemly repeated. Plant cell or plant

[0229] In one aspect the invention provides a plant cell, plant tissue, plant or part thereof, genetically modified to express a BND peptide in a bioactive form.

[0230] In a further aspect the provides a plant cell, plant tissue, plant or part thereof, comprising an expression cassette encoding a polypeptide cassette, or a polypeptide cassette of the invention.

[0231] In one embodiment the plant cell, plant tissue, plant or part thereof, genetically modified to express a BND peptide in a bioactive form comprises an expression cassette encoding a polypeptide cassette, or a polypeptide cassette of the invention.

[0232] In one embodiment the BND peptide has the authentic N-terminus of naturally occurring BND.

[0233] Preferably the BND comprises at least the first 5 nucleotides of SEQ ID NO: 1.

[0234] In one embodiment the plant cell, plant tissue or plant is transgenic for a polynucleotide encoding the BND.

[0235] In one embodiment the polynucleotide encodes an endoplasmic reticulum (ER) targeting, or chloroplast targeting signal peptide operably linked to the BND peptide.

[0236] In one embodiment the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide operably linked to the BND peptide.

[0237] In one embodiment the polynucleotide encodes a chloroplast targeting signal peptide operably linked to the BND peptide.

[0238] In a further embodiment the BND peptide accumulates in the ER or chloroplast of the cell, tissue, organism, plant cell, plant tissue or plant.

[0239] In a one embodiment the BND peptide accumulates in the ER of the cell, tissue, organism, plant cell, plant tissue or plant. In a further embodiment the BND peptide accumulates in chloroplast of the cell, tissue, organism, plant cell, plant tissue or plant.

[0240] In one embodiment the plant cell, plant tissue or plant is that of plant suitable for human consumption.

[0241] In a further embodiment the plant cell, plant tissue or plant is that of plant suitable for animal consumption.

[0242] In a further embodiment the plant cell, plant tissue or plant is that of forage plant.

[0243] In a further embodiment the plant cell, plant tissue or plant is that 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.

[0244] In a further embodiment the plant cell, plant tissue or plant is that of a coffee plant, tea plant, cocoa plant.

[0245] In one embodiment the plant cell, plant tissue or plant or part thereof is selected from a coffee bean, tea leaf, and a cocoa bean.

[0246] Method for producing a bioactive food or beverage composition comprising BND.

[0247] In a further aspect the invention provides a method for producing bioactive food or beverage composition comprising a beta-endorphin (BND) peptide in a bioactive form.

[0248] In one embodiment the BND peptide comprises a sequence with at least 90% identity to SEQ ID NO: 1.

[0249] In a preferred embodiment the BND peptide is not enterically coated.

[0250] In one embodiment the method comprises providing a cell, tissue or organism comprising the BND peptide.

[0251] In a further embodiment the BND peptide is produced in the cell, tissue or organism or part thereof. In one embodiment the cell, tissue, or organism or part thereof is a plant cell, plant tissue or plant or part thereof respectively.

[0252] In one embodiment the food or beverage composition of a is selected from: a) a hot beverage selected from a coffee, tea or a cocoa / hot chocolate beverage. b) an ingredient used to produce the hot beverage c) an ingredient used as an additive to the hot beverage

[0253] In one embodiment the plant cell, plant tissue or plant or part thereof is that of a coffee plant, a tea plant, or a cocoa plant.

[0254] In one embodiment the plant cell or plant tissue is from or part of a coffee bean, a tea leaf or a cocoa bean.

[0255] In one embodiment the coffee bean has been roasted, and wherein the bioactivity of the BND peptide survives the roasting of coffee bean.

[0256] In one embodiment the coffee bean has been roasted at a temperature of at least 150°C, for at least 10 minutes.

[0257] 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 bioactivity of the BND peptide survives the roasting of coffee bean, or part thereof.

[0258] Preferably the BND has been roasted 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, at the above temperature, and the bioactivity of the BND peptide survives the roasting of coffee bean, or part thereof.

[0259] In a further embodiment the food or beverage composition is in liquid form, and bioactivity of the BND survives heating to at least 50°C. Preferably the BND survives 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 1OO°C.

[0260] Use of a BND peptide in the manufacture of a food or beverage composition for managing relaxation

[0261] In a further aspect the invention provides use of a BND peptide in the manufacture of a food or beverage or ingredient composition for managing relaxation.

[0262] 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.

[0263] In one embodiment the food or beverage composition is produced by processing a plant cell, plant tissue, plant or part thereof of the invention.

[0264] Method for managing relaxation

[0265] In a further aspect the invention provides a method of inducing, increasing or maintaining relaxation, the method comprising administering to a subject in need thereof, a food beverage or ingredient composition of the invention, or produced by the method of the invention.

[0266] In one embodiment the subject is a mammal.

[0267] In one embodiment the mammal is selected from the group consisting of human, dog, cat, horse, pig, cow and sheep.

[0268] In a further embodiment, the subject is a human.

[0269] In one embodiment the subject is a bird.

[0270] In one embodiment the bird is selected from the group consisting of chicken and turkey. DETAILED DESCRIPTION OF THE INVENTION

[0271] In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

[0272] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

[0273] BND

[0274] Human beta endorphin (BND) is a 31 amino acid straight chain peptide.

[0275] Amino acid residues 1-25 sharing 100% sequence identity with BND of other species, including ovine, equine, bovine, rat and camel. In an embodiment, the BND is human BND. In an embodiment, the human BND comprises an 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.

[0276] Previous studies have shown that the amino acids 28-31 impart the antigenic characteristics of BND, and that a chain of amino acids 1-27 has equivalent activity to the full-length BND when tested in vitro, in a guinea pig ileum assay (Yeung et al., 1978, Int J Pept Protein Res, 12(1), 42-6.)

[0277] In a further embodiment, the human BND comprises an amino acid sequence of SEQ ID NO: 1 (below) or a functional variant thereof having at least 70% sequence identity thereto.

[0278] In a further embodiment, the human BND comprises an amino acid sequence of SEQ ID NO:2 (below) or a functional variant thereof having at least 70% sequence identity thereto. Polypeptide variants

[0279] Polypeptide sequence identity can be determined in the following manner. The 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, which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ).

[0280] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http: / www. ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[0281] A preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)

[0282] Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0283] Methods for producing constructs and vectors

[0284] The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and / or polynucleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms.

[0285] Methods for producing and using genetic constructs and vectors are well known in the art and are described generally 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). Methods for producing host cells comprising polynucleotides, constructs or vectors

[0286] The invention provides a host cell which comprises a genetic construct or vector of the invention.

[0287] Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (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) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide 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).

[0288] Methods for producing plant cells and plants comprising constructs and vectors

[0289] The invention further provides plant cells which comprise a genetic construct for expressing BND in accordance with the, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention or used in the methods of the invention. Plants comprising such cells also form an aspect of the invention.

[0290] Methods for transforming plant cells, plants and portions 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 transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.

[0291] Methods for genetic manipulation of plants

[0292] A number of 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 may 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 which / when it is not normally expressed. Signal peptides

[0293] Signal peptides are well known to those skilled in the art and can be used to direct accumulation of recombinant proteins and peptides to subcellular locations in plants.

[0294] ER targeting signal peptides

[0295] Endoplasmic reticulum (ER) targeting signal peptides are known to those skilled in the art and are described for example Kim and Hwang, 2013, Traffic; 14:613-621

[0296] In one embodiment the ER targeting signal peptides for use in the invention comprises a sequence with 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.

[0297] Chloroplast targeting signal peptides

[0298] Chloroplast targeting signal peptides are known to those skilled in the art and are described for example, Bruce et al., 2000, Trends Cell Biol;10(10):440-7.

[0299] In one embodiment the chloroplast targeting signal peptides for use in the invention comprises a sequence with 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 NO: 31 and 39.

[0300] Expression cassette

[0301] An expression cassette as used herein refers to a polynucleotide sequence with elements encoding a polypeptide to be expressed.

[0302] The expression cassette will typically include a promoter operably linked to the sequence encoding the polypeptide to be expressed. The expression cassette will also typically include a terminator, operably linked to the polypeptide to be expressed.

[0303] Polypeptide cassette

[0304] A polypeptide cassette as used herein refers to a polypeptide sequence encoded by the expression cassette. In addition to the target peptide, in this case BND or, a multimer of BND, the polypeptide cassette may include multiple additional peptide elements, as those described below. Purification / detection tags

[0305] A purification or detection tag is preferably included in the polypeptide cassette to facilitate detection or purification of the polypeptide cassette. Such tags are know 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.

[0306] Table 1 - Examples of purification / detection tags

[0307] In one embodiment the purification / detection tag for use in the invention comprises a sequence with 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.

[0308] Purification / detection tags can facilitate detection of the polypeptide cassette, or the tag itself, by for example Western blotting. Purification / detection tags can facilitate purification of the polypeptide cassette via use of affinity columns known to those skilled in the art.

[0309] Cleavage site A cleavage site may be included to facilitate cleavage and separation of elements on either side of the cleavage site. Such cleavage sites are known to those skilled in the art and include for example: enterokinase cleavage sites, thrombin cleavage sites. Examples of such sequences are shown in Table 2 below. Table 2 - Examples of cleavage sites

[0310] In one embodiment the cleavage site for use in the invention comprises a sequence with 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 those disclosed herein.

[0311] ER retention sequence

[0312] An ER retention sequence can be used for retaining a target peptide, in this case BND in the endoplasmic reticulum in a plant cell. Examples of such ER retention sequences include KDEL and HDEL sequences. Examples of such sequences are shown in Table 3 below.

[0313] Table 3 - Examples of ER retention sequences

[0314] In one embodiment the ER retention sequence for use in the invention comprises a sequence with 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 ER retention sequences sequence disclosed herein.

[0315] Flexible linker

[0316] In a preferred embodiment the flexible peptide linker is soluble. In one embodiment the flexible peptide linker comprises the sequence (GGGS)n or (Gly- Gly-Gly-Ser)n. In one embodiment n is a number between 1 and 5. Examples of such sequences are shown in Table 4 below.

[0317] Table 4 - Examples of flexible linker sequence In one embodiment the ER retention sequence for use in the invention comprises a sequence with 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 ER retention sequences sequence disclosed herein.

[0318] Plant transformation protocols

[0319] The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: 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, pages243-251); 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); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (US Patent Serial 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 (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); grasses (US Patent Nos. 5, 187, 073 and 6. 020, 539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci.104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US Patent Nos. 5, 416, 011 ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830); pineapple (US Patent Serial No. 5, 952, 543); poplar (US Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871); cereals (US Patent No. 6, 074, 877); pear (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(l):38-45); strawberry (Oosumi et al., 2006 Planta. 223(6): 1219-30; Folta et al., 2006 Planta Apr 14; PMID: 16614818), rose (Li et al., 2003), Rubus (Graham et al., 1995 Methods Mol Biol. 1995;44: 129-33), tomato (Dan et al., 2006, Plant Cell Reports V25:432-441), apple (Yao et al., 1995, Plant Cell Rep. 14, 407-412), Canola (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), ryegrass (Altpeter et al, 2004 Developments in Plant Breeding ll(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 Actinidia eriantha (Wang et al., 2006, Plant Cell Rep. 25,5: 425-31). Transformation of other species is also contemplated by the invention. Suitable methods and protocols are available in the scientific literature.

[0320] Plants

[0321] The plant cells, plant tissues, plants and parts thereof in which the BND is produced in accordance with the invention, or which comprise constructs of the invention, can be from any plant species.

[0322] In one embodiment the cells, plant tissues, plants and parts thereof, is / are derived from a gymnosperm plant species.

[0323] In a further embodiment they are derived from an angiosperm plant species.

[0324] In a further embodiment they are derived from a dicotyledonous plant species.

[0325] In a further embodiment they are derived from a monocotyledonous plant species.

[0326] In one embodiment they are derived from a commercial crop species.

[0327] Preferred dicotyledonous genera include: Amygdalus, Anacardium, Arachis, Brassica, Cajanus, Cannabis, Carthamus, Carya, Ceiba, Cicer, Cocos, Coriandrum, Coronilla, Cossypium, Crotalaria, Dolichos, Elaeis, lycine, Gossypium, Helianthus, Lathyrus, Lens, Lespedeza, Linum, Lotus, Lupinus, Macadamia, Medicago, Melilotus, Mucuna, Olea, Onobrychis, Ornithopus, Papaver, Phaseolus, Phoenix, Pistacia, Pisum, Prunus, Pueraria, Ribes, Ricinus, Sesamum, Theobroma, Tr]folium, Trigonella, Vicia and Vigna.

[0328] Preferred dicotyledonous species include: Amygdalus communis, Anacardium occidental, Arachis hypogaea, Arachis hypogea, Brassica napus Rape, Brassica nigra. Brassica campestris, Cajanus cajan, Cajanus indicus, Cannabis sativa, Carthamus tinctorius, Carya illinoinensis, Ceiba pentandra, Cicer arietinum, Cocos nucifera, Coriandrum sativum, Coronilla varia, Cossypium hirsutum, Crotalaria juncea, Dolichos lablab, Elaeis guineensis, Gossypium arbor eum, Gossypium nanking, Gossypium barbadense, Gossypium herbaceum, Gossypium hirsutum, Glycine max, Glycine ussuriensis, Glycine gracilis, Helianthus annus, Lathyrus angustifolius, Lathyrus luteus, Lathyrus mutabilis, Lathyrus sericea, Lathyrus striata, Lathyrus uliginosus, Lathyrus sativus, Lens culinaris, Lespedeza stipulacea, Linum usitatissimum, Lotus corniculatus, Lupinus albus, Medicago arabica, Medicago arborea, Medicago falcate, Medicago hispida, Medicago officinalis, Medicago sativa, Medicago tribuloides, Macadamia integrifolia, Melilotus albus, Mucuna pruriens, Olea europaea, Onobrychis viciifolia, Ornithopus sativus, Phaseolus aureus, Phaseolus aureus cerasifera, Phaseolus aureus cerasus, Phaseolus aureus coccineus, Phaseolus aureus domestica, Phaseolus aureus lunatus, Phaseolus aureus maheleb, Phaseolus aureus mungo, Phaseolus aureus persica, Phaseolus aureus pseudocerasus, Phaseolus aureus vulgaris, Papaver somniferum, Phaseolus acutifolius, Phoenix dactylifera, Pistacia vera, Pisum sativum, Prunus amygdalus, Prunus armeniaca, Pueraria thunbergiana, Ribes nigrum, Ribes rubrum, Ribes grossularia, Ricinus communis, Sesamum indicum, Trifolium augustifolium, Trifolium diffusum, Trifolium hybridum, Trifolium incarnatum, Trifolium ingrescens, Tri folium pratense, Tri folium repens, Trifolium '' resupinatum, Trifolium subterraneum, Theobroma cacao, Trifolium alexandrinum, Trigonella foenumgraecum, Vigna angustifolia, Vigna atropurpurea, Vigna calcarata, Vigna dasycarpa, Vigna ervilia, Vigna oxycoccos, Vigna pannonica, Vigna sesquipedalis, Vigna sinensis, Vigna villosa, Viciafaba, Vida sative and Vigna angularis.

[0329] Preferred monocotyledonous genera include: Agropyron, Allium, Alopecurus, Andropogon, Arrhenatherum, Asparagus, Avena, Bambusa, Bothrichloa, Bouteloua, Bromus, Cenchrus, Chloris, Cymbopogon, Cynodon, Dactylis, Dichanthium, Digitaria, Eleusine, Elymus, Eragrostis, Fagopyrum, Festuca, Hordeum, Lolium, Oryza, Panicum, Paspalum, Pennisetum, Phalaris, Phleum, Poa, Saccharum, Secale, Setaria, Sorghastrum, Sorghum, Triticum, Vanilla, x Triticosecale and Zea.

[0330] Preferred monocotyledonous species include: Agropyron desertorum, Agropyron elongatum, Agropyron spicatum, Agropyron trachycaulum, Agropyron trichophorum, Allium fistulosum, Allium, sativum, Alopecurus pratensis, Andropogon gerardi, Arrhenatherum elatius, Asparagus officinalis, Avena sativa, Bambusa vulgaris, Bothrichloa barbinodis, Bothrichloa ischaemum, Bouteloua curipendula, Bouteloua gracilis, Bromus erectus, Cenchrus ciliaris, Chloris gayana, Cymbopogon nardus, Cynodon dactylon, Dactylis glomerata, Dichanthium annulatum, Digitaria decumbens, Eleusine coracan, Elymus angustus, Eragrostis curvula, Eragrostis tef, Fagopyrum esculentum, Fagopyrum tataricum, Festuca arundinacea, Hordeum distichum, Hordeum vulgare, Lolium. perenne, Lolium multiflorum, Oryza sativa, Panicum italicium, Panicum maximum, Panicum miliaceum, Paspalum dilatatum, Pennisetum clandestinum, Pennisetum glaucum, Phalaris arundinacea, Phleum bertolinii, Poa fendleriana, Poa nemoralis, Saccharum robustum, Saccharum sinense, Secale cereale, Setaria sphacelata, Sorghastrum nutans, Sorghum dochna, Sorghum halepense, Sorghum bicolor, Triticum aestivum, Triticum dicoccum, X Triticosecale, Zea mays, Agropyron cristatum, Agropyron intermedium, Agropyron smithii, Allium ascalonicum, Allium cepa, Allium chinense, Allium porrum, Allium schoenoprasum, Avena nuda, Bambusa vulgaris, Bothrichloa saccharoides, Bouteloua eriopoda, Bromus inermis, Bromus riparius, Dactyli's aristatum, Dactyli's sericeum, Digitaria smutsii, Elymus junceus, Festuca ovina, Festuca pratensis, Festuca rubra, Panicum purpurascens, Panicum virgatum, Paspalum notatum, Pennisetum purpureum, Pennisetum spicatum, Phleum pratense, Poa pratensis, Saccharum officinarum, Saccharum spontaneum, Sorghum sudanense, Triticum durum, Triticum monococcum, Vanilla fragrans and Zea mays.

[0331] Preferred plants are from the genera Lolium and Trifolium. Particularly preferred are the species Lolium perenne and Trifolium repens.

[0332] Particularly preferred monocotyledonous plant species are: Lolium perenne and Oryza sativa.

[0333] A preferred genus is Coffea. A preferred Coffea species is Coffea arabica.

[0334] A further preferred genus is Oryza. A preferred Oryza species is Oryza sativa.

[0335] 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.

[0336] 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.

[0337] Plant parts, propagules and progeny

[0338] The term "plant" is intended to include a whole plant, any part of a plant, a seed, a fruit, propagules and progeny of a plant.

[0339] The term 'propagule' means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.

[0340] The plants of the invention may be grown and either selfed or crossed with a different plant strain and the resulting progeny, comprising the polynucleotides or constructs of the invention, and / or expressing the BND sequences / constructs, also form a part of the present invention. Preferably the plants, plant parts, propagules and progeny comprise a polynucleotide or construct of the invention, and / or express a BND sequence in accordance with the invention.

[0341] Methods for production of food and beverage compositions

[0342] Methods for producing food and beverage compositions, including those incorporation bioactive ingredients, are known in the art and described for example in: WO2017037263, WO2019045576; WO2011146140 and WO2017124075.

[0343] BRIEF DESCRIPTION OF THE FIGURES

[0344] This invention is illustrated with reference to the following non-limiting figures.

[0345] Figure 1 shows a graph demonstrating the relaxation effect on a subject, of orally ingested BND dissolved in normal saline, compared to that of a normal saline control.

[0346] Figure 2 shows a graph demonstrating the relaxation effect on a subject, of orally ingested coffee infused with BND, compared to that of a coffee control.

[0347] Figure 3 shows a graph demonstrating the relaxation effect on a subject, of orally ingested BND samples that were subjected to various temperature regimes prior to ingestion.

[0348] Figure 4 shows a graph demonstrating the relaxation effect on a subject, of orally ingested coffee samples produced from beans infused with BND and roasted at various temperature prior to production and ingestion of the coffee samples.

[0349] Figure 5 shows a graph demonstrating the effect on a subject, of orally ingested beer infused with BND, compared to that of a beer control.

[0350] Figure 6 shows shows the output from TargetP analysis of ER targeting cassette full peptide sequence.

[0351] Figure 7 shows the output from NetGene2. The sequence from SEQ ID NO: 32 was analysed by NetGene2 to predict splicing. Figure 8 shows the output from TargetP analysis of Chloroplast targeting cassette full peptide sequence.

[0352] Figure 9 shows the output from NetGene2. The sequence from SEQ ID NO: 35 was analysed by NetGene2 to predict splicing.

[0353] Figure 10 shows the output from TargetP analysis of the full-length ER targeting, tandem repeat BND.

[0354] Figure 11 shows output from TargetP analysis of the full-length chloroplast targeting, tandem repeat BND.

[0355] Figure 12 shows an immunoblot analysis of soluble protein extracts from N. benthamiana leaves transiently expressing BND fusion constructs. Replicate leaf extracts were run on SDS-PAGE before immunoblotting and probing with the anti V5 antibody. Upper and lower panels represent different time exposures of the immunoblots (10 and 30 seconds respectively). Panel A shows the extracts from plants expressing single BND constructs targeted to the ER and chloroplast. Panel B shows the extracts from plants expressing tandem BND constructs targeted to the ER and chloroplast. VC = vector control.

[0356] Figure 13 shows ELISA results for leaves expressing single p-endorphin constructs.

[0357] Figure 14 shows Immunoblot analysis of soluble proteins extracted in buffer containing IM urea and 1% Triton X-100 from N. benthamiana leaves transiently expressing BND fusion constructs. White arrow shows the chloroplast signal cleaved tandem endorphin cassette of 12.6 kDa. Black arrow shows the ER-signal cleaved tandem repeat endorphin cassette of 8.7 kDa.

[0358] Figure 15 shows quantification of non-purified recombinant BND accumulating in N. benthamiana leaves. A) dilution series from leaf extracts alongside a dilution series of 2, 5, 10, 20, and 40 ng of a V5 fusion protein standard. White arrows show the signal cleaved single and tandem repeat endorphin cassettes of 8.7 kDa and 12.9, respectively. B) Standard curve generated by the Image Lab 5.2.1 software after scanning the dilution series results in Panel A. C) Average production of BND was calculated as pg / g FW of transient expressing leaves. Figure 16 shows immunoblotting analysis of endorphin purification using Ni-affinity chromatography. Top and bottom panel images showed the stain-free gel and immunoblotted membrane, respectively.

[0359] Figure 17 shows immunoblotting analysis of endorphin purification using Ni-affinity binding and peptide concentration by ultrafiltration using a Amicon® 3 kDa cut-off filter. Top and bottom panel images showed the stain-free gel and immunoblotted membrane, respectively. Arrows indicate the ER targeting BND dimer and oligomers.

[0360] EXAMPLES

[0361] This invention is illustrated with reference to the following non-limiting examples.

[0362] Example 1 - Bioactive effect of orally ingested BND peptide

[0363] Materials and methods

[0364] The BND peptide used in the present study comprised amino acids 1-27, that is a 27-mer of the human B-endorphin peptide sequence. The BND 27-mer was synthesized by Leon Biological Technology Co Ltd, Nanjing, China. lOmg of the BND peptide was dissolved in 200ml of normal saline, and this BND test sample was consumed by Subject A (an adult male), and relaxation index monitored over a period of 4 to 6 hours. A control test was carried out using a control test sample of 200ml of normal saline (without the BND) on the same subject, under the same conditions.

[0365] The relaxation index was self-assessed by the subject as set out below, immediately after consumption of the test and control samples. The degree of relaxation (as indicated in Table 5 below), was recorded at short intervals, during the test periods. Physical data including pulse, blood pressure and other physical parameters were noted, and closely aligned with the self-assessed relaxation index.

[0366] Table 5 - Relaxation index

[0367] Results

[0368] The results are shown in Figure 1.

[0369] This data surprisingly shows, for the first time as far as the applicant is aware, that BND can produce a bioactive effect when orally ingested. Existing literature strongly indicates that BND like many other bioactive peptides, is unlikely to exert any biological effect inside the body if orally ingested in an unprotected form, for various reasons including the harsh environment of the gastro-intestinal tract, as discussed in the Background section of this specification.

[0370] Figure 1 shows that the orally ingested BND produces a net relaxing effect, relative to the control, lasting up to four hours after ingestion.

[0371] Example 2 - Bioactive effect of BND infused into pre-made hot coffee

[0372] Materials and methods

[0373] The BND peptide used, and the relaxation index assessment was as described in Example 1. lOmg of BND peptide was infused into 200ml of brewed coffee at 60oC, and after 5 minutes at room temperature, the BND infused coffee was consumed by the test subject over a period of 5 minutes. A control test was carried out using a control test sample of 200ml of coffee (without the BND) on the same subject, under the same conditions on another day. Stress index assessment began immediately after consumption of each coffee sample. Results

[0374] The results are shown in Figure 2, which shows that the BND infused coffee produced a much stronger relaxing effect than did the control coffee, and that the effect lasts for up to four hours after ingestion.

[0375] These results show that the surprising bioactive effect of BND as demonstrated in Example 1, is surprisingly maintained even when the BND has been added to coffee at 60oC, indicating that neither the presence of coffee nor the heating to 60°C have a deleterious effect on the efficacy of the BND in inducing a relaxed state in the test subject.

[0376] Example 3 - Bioactive effect of BND subjected to various temperatures including conditions mimicking coffee roasting

[0377] The aim of these tests was to assess the effect of higher temperatures on the bioactive efficacy of BND demonstrated in the Examples above, and to see if the efficacy would be maintained after exposure to conditions comparable to the roasting of coffee beans.

[0378] Materials and methods

[0379] The BND peptide used, and the relaxation index assessment was as described in Example 1.

[0380] 10 mg BND samples were placed on filter paper and separately subjected to the following temperature regimes:

[0381] • room temperature (20°C) for 14 minutes

[0382] • 160°C in a fan forced oven for 14 minutes

[0383] • 230°C in a fan forced oven for 14 minutes

[0384] The non-ambient temperatures (160°C and 230°C) as well as the heating times, were chosen as these parameters are those that are often used in the coffee roasting process.

[0385] Each of the test samples was then dissolved in 200ml of normal saline (at 20°C) for 5 minutes the ingested by the test subject, followed by relaxation index assessment. Results

[0386] The results are shown in Figure 3, and demonstrate that the BND effect produced following each temperature regime, is roughly equivalent, and shows surprisingly, that the BND effect is retained even after the BND peptide has been subjected to the relatively harsh (160°C and 230°C) temperature environments, that are equivalent to conditions used for coffee roasting.

[0387] Example 4 - Bioactive effect of BND in coffee beans comprising the BND, after roasting of the beans

[0388] The aim of this Example was to assess the effect on BND of roasting beans comprising the BND, to test the hypothesis that plant material expressing BND could be processed into a beverage that would provide the same bioactive effect. To this end BND was infused into raw coffee beans, which were then exposed to conditions equivalent to coffee roasting.

[0389] Materials and methods

[0390] The BND peptide used, and the relaxation index assessment was as described in Example 1.

[0391] The BND peptide was infused into raw (unroasted) coffee beans, by soaking the beans in a lmg / ml solution of BND dissolved in normal saline for 12 hours.

[0392] Samples of the raw BND infused beans were then roasted in separate trials, at 160°C and 230°C in a fan-forced oven for 14 minutes. The pre-roasted and later infused beans were used as controls.

[0393] The roasted (BND infused) test beans and control beans were then ground and separately used to make coffee.

[0394] 200 ml of each of the test coffee (produced via each roasting temperature), and the control beans coffee were then consumed by the test subject after 5 minutes, followed by relaxation index assessment. Results

[0395] The results are shown in Figure 4.

[0396] As shown, the BND effect on relaxing the subject is surprisingly demonstrated in coffee produced from BND-infused beans subjected to both roasting temperatures. The effect is similar to that shown by coffee produced from pre-roasted BND-infused beans.

[0397] This indicates that if BND could be recombinantly expressed in biological material (e.g. plant material) in the right form, that the biological material could be processed under relatively harsh processing conditions to produce a food or beverage composition that retains the BND bioactivity demonstrated.

[0398] Example 5 - BND in an alcoholic beverage

[0399] Materials and methods

[0400] The BND used, and the relaxation index assessment was as described in Example 1. lOmg of BND was infused into 200ml of Stella Artois beer for 5 minutes at 20°C, and then consumed by the test subject, followed by relaxation index assessment.

[0401] A control beer test was carried out using control beer (without BND) under the same conditions on a different day.

[0402] Results

[0403] The results are shown in Figure 5.

[0404] This data shows that the BND has a relaxing effect when combined with beer greater than the relaxing effect demonstrated by beer alone lasting up to 4 hours after ingestion.

[0405] This data demonstrates that the beer, and / or the alcoholic content of the beer, did not have a deleterious effect on the efficacy of the BND in inducing a relaxed state.

[0406] Furthermore, the relaxing effect of the beer and the BND together was more pronounced than the relaxing effect of the beer alone in the test subject. Example 6 - Expression of BND in plants

[0407] Background

[0408] To the best of the applicant's knowledge BND has never been expressed in plants, let alone expressed in an active form, or at commercially significant levels.

[0409] The N-terminus of the BND is reputed to be important in activity. Therefore, for production of active BND in plants it is likely important or essential to express the peptide with the correct N-terminal sequence. The sequence of the mature peptide fragment (31 residues) is highly conserved YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE (SEQ ID NO:2).

[0410] While proteins, polypeptides and even peptides have been expressed in plants, the level of production of any peptide is unpredictable. Signal peptides have been used to target proteins, polypeptides and peptides to subcellular organelles with a view to increasing accumulation, but again this is unpredictable.

[0411] Cleavage of the targeting signal sequences is also unpredictable and may be influenced by factors such as the difference in sequence (hydrophobicity, charge, size, etc.) between the downstream peptide and the signal sequence. There are various software applications for predicting where cleavage will occur, but these are far from guaranteed to predict correctly.

[0412] Thus, there are several unpredictable, challenges to overcome before BND can be produced in plants, in an active form / correctly cleaved, at a reasonable level.

[0413] Materials and methods

[0414] Construct design

[0415] Constructs were produced for expressing and targeting BND to the Endoplasmic Reticulum (ER) and the chloroplast, with the signal peptide sequences (as discussed further below) in the model plant Nicotiana benthamiana. A C-terminal V5-His tag was also included for detection / quantification and / or purification. Expression cassette features

[0416] Both cassettes were designed to use the same GATEWAY™ recombinant directional cloning step to place them into a binary vector where the BND fusion peptide is under the control of a CaMV35s promoter (constitutive) and NOS terminator. Both cassettes were optimized for expression in Nicotiana benthamiana; this includes an intron with the appropriate predicted splicing sites and efficiency; Kozak sequence, poly adenylation signal sequences removed, mRNA instability sequences removed, double stop codon and tetranucleotide (Scott et al, 2010, Plant Biotechnology Journal. 8:912-927). Those skilled in the art will understand that these optimizations, are not essential but may be beneficial. Sequences used in the constructs are shown in the Sequence Listing as discussed below.

[0417] Endoplasmic Reticulum (ER) targeting of single BND

[0418] The Arabidopsis thaliana Purple Acid Phosphatase signal peptide sequence (SEQ ID NO:7) was used for targeting BND to the ER. The expression cassette was based on Winichayakul et al (2009).

[0419] The total peptide sequence to be expressed (SEQ ID NO: 25) included:

[0420] • The ER targeting signal peptide (SEQ ID NO: 7)

[0421] • A flexible linker (SEQ ID NO: 18)

[0422] • An enterokinase cleavage site (SEQ ID NO: 13)

[0423] . The BND peptide (SEQ ID NO: 2)

[0424] • A thrombin cleavage site (SEQ ID NO: 16)

[0425] • V-5 His tag (SEQ ID NO: 20)

[0426] • ER retention sequence (SEQ ID NO: 22)

[0427] The total peptide sequence (theoretically 11.92 kDa) encoded by the ER targeting cassette (SEQ ID NO: 26) was run through the signal prediction software TargetP. The results are shown in Figure 6 and indicate that the signal peptide will be cleaved to leave the authentic N-terminal sequence of the BND peptide.

[0428] The ER targeted peptide coding sequence was optimized for tobacco, created and custom synthesized by GenScript. The total GATEWAY™ flanked ER targeted BND-V5-His with intron nucleic acid sequence is shown in SEQ ID NO:26.

[0429] After using Geneious Prime GATEWAY subcloning, the sequence (SEQ ID NO:32) determined from TATATAA box of the CaMV35S promoter to the double stop codon (TAATGA), was analysed by NetGene2 to predict splicing (Figure 7). These results show that the intron is predicted to be correctly spliced.

[0430] Chloroplast targeting of single BND

[0431] A truncated Nicotiana tabacum NtRBCs rubisco signal peptide (SEQ ID NO:31) was used for targeting BND to the chloroplast (Eseverrie et al., 2020). The chloroplast targeting cassette is shown in SEQ ID NO: 27.

[0432] The total peptide sequence (SEQ ID NO: 27) to be expressed included:

[0433] • The chloroplast targeting signal peptide (SEQ ID NO: 31)

[0434] • A flexible linker (SEQ ID NO: 18)

[0435] • An enterokinase cleavage site (SEQ ID NO: 13)

[0436] . The BND peptide (SEQ ID NO: 2)

[0437] • A thrombin cleavage site (SEQ ID NO: 16)

[0438] • V-5 His tag (SEQ ID NO: 20)

[0439] The total peptide sequence (theoretically 13.47 kDa) encoded by the chloroplast targeting cassette was run through the signal prediction software TargetP. The results, shown in Figure 8, indicate that the signal peptide will be cleaved to leave the authentic N-terminal sequence of the BND peptide.

[0440] Chloroplast targeted PEPTIDE coding Sequence was optimized for expression in Nicotiana benthamiana, creating the sequence shown in SEQ ID NO:33.

[0441] To the optimized chloroplast targeting sequence has attLl, 5'UTR, AtDGATl intron3, double stop codon, attL2, sequences added (NB T was changed from A to eliminate an mRNA instability sequence, this changed the CCA codon to CCT, both of which code for proline) as shown in SEQ ID NO:34.

[0442] After using Geneious Prime GATEWAY subcloning, the sequence from the TATATAA box of the CaMV35S promoter to (TAATGA) double stop codon is shown in SEQ ID NO: 35. The sequence was analysed by NetGene2 to predict splicing (Figure 9). Tandem repeat BND constructs

[0443] Constructs were also prepared for expressing BND as a tandemly arranged peptide sequence separated by a flexible linker as shown in SEQ ID NO 36. The sequence with an Enterokinase Cleavage Site, the tandemly repeated BND, a Thrombin cleavage site and engineered cloning restriction sites is shown in SEQ ID NO 37.

[0444] The nucleotide sequence was optimized for expression in Nicotiana benthamiana; this includes an intron with the appropriate predicted splicing sites and efficiency; Kozak sequence, poly adenylation signal sequences removed, and mRNA instability sequences removed. The nucleic acid and peptide sequences of the tandem repeat fragment are shown in the Sequence Listing.

[0445] The tandem repeat BND cassette was subsequently subcloned into the ER- and chloroplasttargeting cassettes described above.

[0446] The peptide sequence (predicted 16.08 kDa) of ER-targeting tandem repeat BND (SEQ ID NO:29) was run through the signal sequence recognition software TargetP (Figure 10).

[0447] For the chloroplast targeting tandem repeat BND expressing cassette, a modified version of the chloroplast transit peptide (SEQ ID NO: 39) was used.

[0448] The peptide sequence (SEQ ID NO:38) of the chloroplast targeting tandem repeat BND expressing cassette (predicted 22.35 kDa) was also run through the TargetP software (Figure 11).

[0449] Cloning and transformation

[0450] Cloning of plant organelle-targeting BND

[0451] Both designed ER- and chloroplast-targeting BND cassettes were individually cloned into a binary vector pRShl (Scott et al., 2010) by Gateway™ LR Clonase™ reaction. Conformation of the plasmid constructs was confirmed by restriction enzyme mapping and sequencing.

[0452] Cloning of plant organelle-targeting tandem repeat BND The tandem 2x repeat BND fragment was cloned into 056488 pPCR Script-ER targeting cassette and 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 ER- and chloroplast-targeting 2xEdph were individually cloned into a binary vector pRShl (Scott et al., 2010) by Gateway™ LR Clonase™ reaction. Conformation of the plasmid constructs was confirmed by restriction enzyme mapping and sequencing.

[0453] Transformation of binary vectors containing BND expressing cassette into Agrobacterium cells

[0454] Plasmid DNA of pRShl-ER-Edph (ER-targeted single BND), pRShl-CHL-Edph (Chloroplast- targeted single BND), pRShl-ER-2xEdph (ER-targeted tandem BND) and pRShl-CHL- 2xEdph (Chloroplast-targeted tandem BND) were transformed into Agrobacterium tumefaciens GV3101 strain by freeze-thaw method and selected on appropriate antibiotics containing medium. A. tumefaciens cells containing the plasmid DNA were selected and confirmed by PCR using pRShl-forward and reverse primers.

[0455] Transient expression of BND containing cassette in tobacco by Agrobacterium mediation

[0456] Agrobacterium cells containing the BND expressing cassettes were infiltrated into leaves of tobacco Nicotina benthamiana). Leaf samples were harvested, extracted for soluble proteins and analyzed for BND expression by SDS-PAGE Immunoblot (Figure 12) and ELISA (Figure 13).

[0457] RESULTS

[0458] Immunoblotting

[0459] Immunoblotting results of the ER targeting BND (ER-Edph) and tandem repeat BND (ER- 2xEdph) showed they both accumulated to detectable levels (Figure 12). However, no peptide was observed for the chloroplast targeting BND (CHL-Edph) and very low quantities of the peptide was observed for the chloroplast targeting tandem repeat BND (CHL-2xEdph), which could not have been predicted by those experienced in the art. No immunoblot signal was observed from transient expression of N. benthamiana with Agrobacterium cells containing vector control (VC). ELISA

[0460] Enzyme-Linked ImmunoSorbent Assay (ELISA) is a technique used for quantification which typically has a broader dynamic range than gel scanning. Selectively, BND tagged with V5: :6xHis containing in the matrix of leaf total soluble protein extract was absorbed onto the Nickle coated plate (Pierce™) and subsequently detected immunologically by anti-V5 antibody (Figure 13). The ELISA results also confirmed the accumulation of the ER targeting BND (ER-Edph). No colour developing was observed for the none (no protein coating), the vector control (VC) and the chloroplast targeting BND (CHL-Edph) soluble protein extracts. The reactions were kinetically incubated at 37°C for 10 min and the change in optical density at 405 nm was recorded.

[0461] Transient expression of chloroplast targeted BND cassettes was detected in N. benthamiana leaves only after using an alternative extraction buffer

[0462] The lack of recombinant protein from the chloroplast targeting cassette was surprising; to see if this could be due to poor extraction efficiency (chloroplasts are small, discreet organelles with multiple membranes) extraction of total proteins was repeated using a different buffer. This contained 1 M urea and 1% triton x-100 (solubilized bilayer membrane proteins).

[0463] The immunobloting showed there was relatively strong accumulation of the chloroplast targeting tandem repeat BND (CHL-2xEdph) but not for the chloroplast targeting single BND (CHL-Edph) peptide (Figure 14). It is possible that the reason for the lack of accumulation seen in the chloroplast was because of the tandem repeat arrangement compared to the single peptide. However, unpublished results show that it is more likely due to the use of different chloroplast targeting sequences used in the two constructs. Where the tandem construct used a targeting sequence previously tested (Winichayakul et al 2009), the monomer cassette used a shortened version that was recently published (Eseverri et al 2020).

[0464] Immunoblot banding patterns suggest the ER and chloroplast targeted peptides are correctly processed in terms of signal cleavage

[0465] The predicted size of the uncleaved ER-Edph peptide is 11.9kDa while the predicted size of the signal cleaved peptide is 8.7kDa. Thus, it appears that the majority of the detected ER-Edph peptide is cleaved appropriately (Figure 12, panel A and Figure 14). The relatively faint higher band is likely to be either be incorrectly cleaved peptide or dimerization of the cleaved peptide; given the size difference suggests it is more likely to be a dimer.

[0466] The predicted size of the uncleaved ER-2xEdph peptide is 16.1kDa and the signal cleaved version is 12.9kDa; however, the immunoblot (Figure 12 panel B) showed a very similar banding pattern to the single ER-Edph peptide (Figure 12 panel A). Cleavage of the ER signal sequence and first BND would explain the banding pattern but this is unlikely to have occurred given there is no specific peptidase site located between the BND repeats. Given the relatively small size of the peptides it is more probable that they are the cleaved and subsequently dimerized and in this particular 4-15% gradient acrylamide gel they have migrated faster than would be expected. This could be confirmed by running ER- Edph and ER-2xEdph extracts side by side on the same gel and / or using higher % polyacrylamide gel with different running buffer like Tris-tricine to resolve smaller protein sizes (<10 kDa).

[0467] The predicted size of the uncleaved CHL-2XEdph is 22kDa and the cleaved version is 12.6kDa (or 18.5kDa if cleaved at the repeat transit peptide cleavage site). The immunoblot in Figure 14 suggests that the 12.6kDa version exists which means the first transit peptide cleavage site was recognised. The larger immunoreactive band is likely to be either by incorrectly cleaved peptide or dimerization of the cleaved peptide.

[0468] Quantification of ER targeted BND from crude extracts

[0469] The quantities of BND expressed in the transient leaf expression systems were determined as follows. Leaf extracts from the ER targeting BND (ER-Edph) and tandem repeat BND (ER-2xEdph) were immunoblotted in the same gel as 2, 5, 10, 20, and 40 ng of a PEAPOD- V5 fusion protein standard (prepared in another project by expressing in bacterial cells and purifying by affinity gel binding, Figure 15 panel A). The band intensities of the standards were scanned and plotted as a standard curve by Image Lab 5.2.1 software (Figure 15 panel B). From the curve we were able to calculate the average accumulation of BND (pg / g FW) in the N. benthamiana leaves (Figure 15 panel C).

[0470] The level of total recombinant protein as well as level of recombinant BND accumulated is converted to a % of total soluble protein, shown in Table 6. Table 6. Quantification of non-purified recombinant proteins accumulated.

[0471] Purification and re-quantification of ER targeting BND

[0472] Although the recombinant protein appears to have accumulated to a relatively low level in the transient expression system, it can be purified and concentrated to generate sufficient quantities for further studies, e.g., mass spectroscopy conformation of cleavage and appropriate sequence, bioassay, etc. In this proof of concept study we added a C-terminal V5: : His tag to the BND fragment to assist with purification and concentration.

[0473] Leaves transiently expressing the ER-targeting BND were harvested (15.87 g FW) 72 h after Agrobacterium infiltration and homogenised in 10 mL of ice-cold 2.5X extraction buffer containing 50 mM sodium phosphate buffer pH 7.4, 2.5 M NaCI, and 2.5% triton X- 100. The crude extract was centrifuged at 10,000 x g for 5 min at 4 °C to remove the leaf debris, the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1 mM and the final volume of soluble extract was adjusted to 25 mL with cold-sterile milliQ H2O.

[0474] Initially the soluble extract precipitated after the addition of imidazole (5-15 mM) and the majority of histidine tagged material did not bind to the Ni2+ column. This was likely due to unknown compounds in the extract from N. benthaniama leaves. Subsequently prediluted crude extract was passed this through a 3kDa cut-off filter membrane (which retained the recombinant peptides) prior to the addition of imidazole and loading extract onto the column (Figure 16).

[0475] In Figure 16, the immunoblot showed the C-terminally tagged ER targeting BND was present in the soluble extract (Lane 2) and 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). Disappearance of the monomer (smaller band ~ 8.7 kDa) and appearance of a larger band (previously assumed to be dimer or uncleaved signal BND) was observed. This may have resulted from using of the 3 kDa cut-off filter served to concentrate the recombinant protein and led to an increase in the degree of oligomerization.

[0476] In elutions 1, 2 and 3 we were unable to detect protein on the stain-free gel from; consequently, these were pooled and concentrated (Supplementary Protocol 2). Protein samples were subsequently analysed by immunoblotting as shown in Figure 17 (lower panel, lane 14).

[0477] The immunoblot showed the concentrated soluble eluent contained the ER targeting BND although now present both as a dimer and larger oligomer (Figure 27, Lane 14). From this we were able to re-calculate the level of recombinant protein in the total soluble leaf protein extract which showed the level of recombinant BND (as a percentage of the total soluble leaf protein) was considerably higher than first determined (Table 7 vs Table 6).

[0478] Table 7. Quantification after purification of recombinant proteins targeted to the ER.

[0479] Conclusion and further experiments

[0480] The applicants have demonstrated for the first time that the mature BND peptide can be recombinantly synthesised and accumulate in planta. This was achieved by targeting the peptide (by way of cleavable signal sequences) to the endoplasmic reticulum (ER) or the chloroplast. In the case of the latter, a truncated version of the chloroplast transit peptide sequence did not result in accumulation of detectable BND. Immunoblot analysis suggested the ER targeting signal sequence was efficiently cleaved while the nontruncated chloroplast transit peptide appeared to have been removed approximately 50% of the time.

[0481] Purification of the recombinant protein (by both size exclusion and affinity chromatography) enabled more accurate quantification of the level of BND that accumulated; this reached approximately 2% of the total soluble protein when targeted to the ER. The ability to purify and concentrate the BND should allow further characterization of the peptide. The first steps can include further determination of the efficiency and accuracy of signal cleavage. This can be achievable by taking gel slices from the appropriate migration points and subjecting these to trypsin digestion mass spectrometry analysis. Similarly, the efficacy and accuracy of p-enterokinase cleavage just upstream of the mature BND peptide can be achieved by initially subjecting the gel slices to enterokinase prior to trypsin digestion; followed by mass spectrometry analysis. These analyses will also allow examination of the C-terminus to confirm the exact sequence.

[0482] A chloroplast targeted fragment can have its N terminal targeting sequence efficiently cleaved leaving the correct N-terminal residue of the mature BND peptide. Use of a p- enterokinase site could facilitate this. The advantage of chloroplast targeting is that it should be possible to produce the peptide with the correct C-terminus since retention in the chloroplast (unlike the ER) does not require an additional sequence.

[0483] Example 7 - Further confirmation of N-terminus and activity of recombinantly expressed BDN

[0484] Mass spectroscopy analysis can be performed on the current recombinant peptides, working with the mature peptide alone (no tags) in the future can be facilitated using commercial antibodies against the mature peptide.

[0485] For example, mass spectroscopy can be used to confirm the sequences of the recombinant peptides (with and without enterokinase treatment) from the chloroplast targeted tandem endorphin repeat cassette. This can show between which residues the chloroplast targeting peptide is being cleaved as well as the percentage of peptides that are being cleaved. Similarly, this can demonstrate that the enterokinase cleaves at the appropriate site, the percentage of peptides that are cleaved and that cleavage is leaving the expected N- terminal residue of p-endorphin. The synthesised BND described in Example 1 can be used as a standard for mass spec analysis.

[0486] Alternatively, without use of an anti-BND antibody, purification and quantification of the recombinant mature peptide would also be possible. As an alternative the V5: : H is tag can be placed between the chloroplast signal sequence and a p-enterokinase site upstream of the BND peptide. This would allow purification and concentration from the chloroplast as well as enabling removal of the N-terminal signals and tags.

[0487] Provided the mass spectrometry shows the sequence and the cleavage by enterokinase are correct, an expression cassette can be produced containing the following: Chloroplast transit peptide (from Winichayakul et al 2009) " internal His

[0488] Tag: :enterokinase cleavage site: :single BND peptide.

[0489] By way of example, the amino acid sequence of such an expression cassette is shown in SEQ ID NO: 40.

[0490] The total peptide sequence (SEQ ID NO: 40) to be expressed includes:

[0491] • chloroplast targeting signal peptide (SEQ ID NO:31)

[0492] • flexible linker (SEQ ID NO: 18)

[0493] • V-5 His tag (SEQ ID NO: 20)

[0494] • flexible linker (SEQ ID NO: 18)

[0495] • Enterokinase cleavage site (SEQ ID NO: 13)

[0496] • BND peptide (SEQ ID NO: 1)

[0497] By way of example, a polynucleotide sequence encoding such a cassette is shown in SEQ ID NO: 41.

[0498] This cassette can be ligated into expression cassette with a CaMV35S promoter and terminator by standard procedures to produce the sequence shown in SEQ ID NO 42.

[0499] The whole expression cassette can be used to transform Agrobacterium by standard procedures, and / or as described above, before introduction into plants by transient or stable expression as described herein.

[0500] A further alternative expression cassette can be produced containing the following:

[0501] Chloroplast transit peptide (from Winichayakul et al 2009): :single BND peptide: :enterokinase cleavage site: : His Tag.

[0502] By way of example, the amino acid sequence of such an expression cassette is shown in SEQ ID NO: 51.

[0503] The total peptide sequence (SEQ ID NO: 51) to be expressed includes:

[0504] • Chloroplast targeting signal peptide (SEQ ID NO:31)

[0505] • BND peptide (SEQ ID NO: 1)

[0506] • Enterokinase cleavage site (SEQ ID NO: 13)

[0507] • flexible linker (SEQ ID NO: 18)

[0508] • V-5 His tag (SEQ ID NO: 20)

[0509] By way of example, a polynucleotide sequence encoding such a cassette is shown in SEQ ID NO: 52. This cassette can be ligated into expression cassette with a CaMV35S promoter and terminator by standard procedures as discussed above.

[0510] The whole expression cassette can be used to transform Agrobacterium by standard procedures, and / or as described above, before introduction into plants by transient or stable expression as described herein.

[0511] The polynucleotide coding sequences can be codon optimised to suit the species to be transformed.

[0512] Bioassays to determine the activity of the recombinant BND compared to the chemically synthesised peptide can be performed (see for example Examples 1-5 herein) following generation and purification of the recombinantly expressed BND peptide.

[0513] Example 8 - Stable transformation of plants to recombinantly express active BND

[0514] The expression cassettes described in Example 6 and 7 can be cloned into suitable vectors for stable (as well as transient) transformation of plants.

[0515] For example, the expression cassettes described in Example 6 and 7 can be cloned into into pRShl (Scott et al 2010) replacing the constitutive promoter cauliflower mosaic virus 35S (CaMV35Sp) driven GATEWAY® adapted expression cassette, to create binary vectors, or into pBR2 from pDONR™221 by GATEWAY® LR cloning (Thermo Fisher Scientific).

[0516] Alfalfa

[0517] Alfalfa can be stably transformed for example, as described in Wand et al., 2016, Protein Pept Lett., 23(5):495-502

[0518] Rice

[0519] Rice can be stably transformed for example, as described in Alam et al., 1999, Plant Cell Rep. 18, 572. Wheat

[0520] Wheat can be stably transformed for example, as described in Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877.

[0521] Barley

[0522] Barley can be stably transformed for example, as described in Lazzeri, P. 1995, Methods Mol Biol:49: 95-106.

[0523] Com

[0524] Corn can be stably transformed for example, as described in US Patent Serial Nos. 5, 177, 010 and 5, 981, 840

[0525] Tobacco

[0526] Tobacco can be stably transformed for example, as described in Horsch et al., 1985, Science 227, 1229.

[0527] Coffee

[0528] Coffee can be stably transformed for example, as described in Ribas et al., BMC Plant Biol. 2011; 11: 92.

[0529] Tea

[0530] Tea can be stably transformed for example, as described in Chen et al., 2022 Front. Plant Sci., Sec. Plant Systematics and Evolution Volume 13.

[0531] Cocoa

[0532] Cocoa can be stably transformed for example, as described in Sain SL et al., 1994, Plant Cell, Tissue and Organ Culture volume 37, pages243-251, and in Maximova et al., 2003, Plant Cell Rep 21, 872-883.

[0533] Other plant species can be stably transformed as described herein, and with other protocols known in the art. Example 9 - Analysis of BND expressed via stable transformation

[0534] The recombinantly expressed BND produced via stable transformation (as well as via transient expression), can be purified and analysed as described in Examples 6 and 7 above.

[0535] Example 10 - Further confirmation of the activity of synthetic and recombinantly expressed BND via animal trial.

[0536] Examples 1-5 above, the applicants have surprisingly shown for the first time that, contrary to what is expected from the prior art, as discussed in the Background section above, that native, unprotected, BND peptide can exert a biological effect via oral ingestion, and can thus be used to manage relaxation via oral ingestion.

[0537] To further confirm and explore biological activity of BND (which may be synthetically or recombinantly produced, a larger scale trial can be performed in which the BND is ingested by rats. Such a trial can be performed for example at The Howard Florey Institute of Neuroscience & Mental Health (Floor 2, 161 Barry Street, Carlton, Victoria 3053, Australia) with the protocol described below.

[0538] AIM

[0539] The aim of the trial is to test the relaxation effect of human beta endorphin (BND) administered orally to groups of Sprague Dawley rats in a double blinded controlled trial over a period of six weeks. This study can be used to test the effect of synthetic and recombinantly produced BND as described herein, and to confirm the biological activity of the recombinantly produced BND.

[0540] The aim of the study is to test that the orally administered BND groups show demonstrable signs of enhanced relaxion in reference to the control groups.

[0541] Group Composition

[0542] Each of the four treatments will consist of 15 randomly blindly assigned male (or female) Sprague Dawley rats caged in groups of 4 in separate areas. TRIAL

[0543] The Groups are as follows:

[0544] 1. Sprague Dawley Rats n = 15 Treated Oral Vehicle, No Restraint (Control for Impact of Stress)

[0545] 2. Sprague Dawley Rats n = 15 Treated Oral Test recombinant Compound, No Restraint (Control for Impact of Compound)

[0546] 3. Sprague Dawley Rats n = 15 Treated Oral Vehicle, Restraint (Impact of Stress)

[0547] 4. Sprague Dawley Rats n = 15 Treated Oral synthesized Test Compound, Restraint (Impact of Compound on Stress)

[0548] In an alternative trial, the rats for each treatment can be split into 3 groups for replication. Optionally either all male, or all female rats can be used.

[0549] END OF TRIAL

[0550] At 6 weeks all groups will be sacrificed.

[0551] Post-mortem studies of organs will be carried out and cardiac puncture performed. Specimens for Haematological and Biochemical studies will be taken.

[0552] Cortisol tests

[0553] Serum cortisol testing will be carried out on all test subjects at time Zero, 2 weeks, 4 weeks and at sacrifice at 6 weeks.

[0554] Blood(0.5ml) will be obtained by venepuncture from the tail. Care will be taken to familiarize the rodents with this procedure and topical anaesthetic will be applied to the tail venepuncture site to minimize trauma.

[0555] BND dosage regime

[0556] Daily dosage equivalent (Pro rata by weight), equivalent to 20mg dose in 70 kg human.

[0557] Doses of synthetic and recombinantly produced BND as described in the Examples above will be used. Alternatively: Dosage equivalent (Pro rata by weight), equivalent to 20mg dose in 70 kg human. Doses of synthetic and recombinantly produced BND as described in the Examples above can be used during weeks 1-2, then week 5 to test for multiple phases of stress reduction.

[0558] Stressor Regime

[0559] Restraint stress 2 hrs per day for a period of 3 weeks, can be used. The restraint time can be rotated around, so that animals would have a different restraint time, each consecutive day.

[0560] At the completion of the 3-week period of either Restraint or No Restraint all rats will be tested via the following 4 tests (these will run for a further 3 weeks):

[0561] Elevated Plus Maze (Anxiety Test)

[0562] The elevated plus-maze is custom-made of light-coloured Perspex consisting of two open arms (10cm x 44cm for rats) and two enclosed arms (10cm x 44cm x 10cm for rats) extending from a central platform (12 xl2 cm for rats). It is mounted on a base raised 40 cm above the floor. This is a test for anxiety in rodents as they prefer the safer closed arms and are cautious when entering the open arms. Rodents are presented with a conflict between their affinity for novel spaces and the opportunities it presents and the potential danger of an unprotected area.

[0563] At the beginning of the experiment, the rodent is placed in the centre of the maze facing an open arm and the following variables are tracked with the Cleversys Topscan tracking system: time and entries in open and closed arms. The time spent in the open and closed arms is expressed as a percentage of total time in this maze. The number of entries into open and closed arms is defined as the entry of all four feet of the rodent into one arm of the maze. The room lighting is approximately 10-20 lux. The test is only performed once for a 10-minute trial.

[0564] Locomotor Test (Motor Function Test)

[0565] The rodent is removed from its home cage and placed into the middle of the Med Associates locomotor chamber. The system monitors a range of prescribed parameters including, but not restricted to- distance travelled, time spent moving, number of moves, time spent rearing. This system can also be used to assess general locomotor activity usually tested over a 60-90 minute period. General locomotor activity measured using the aforementioned parameters can provide insights into the general phenotype elicited by gene manipulation or drug administration.

[0566] The Light / Dark Test (Anxiety Test)

[0567] This test can also be done automatically using the Med Associates locomotor system with black Perspex box inserts positioned into one half of the locomotor chamber. This provides a light and dark area. The black Perspex box has a small opening to allow the rat to move from the dark zone to the open light zone (450 lux). The rat is placed inside the dark zone at the beginning of the test and is allowed to explore both zones for a 10 minute trial. The software program will record the number of transitions from light to dark, the amount of time spent in each zone, and the latency to emerge from the dark zone to light. Rats that spend more time in the dark zone versus the light when compared with control rats may indicate greater levels of basal anxiety-like behaviour

[0568] Large Open Field Test (Anxiety Test)

[0569] The rodent is removed from its home cage and then placed inside the centre of the square arena (110cm x 110cm), exposing the rodent to the brightly light open field area. Its movements are tracked with by automated tracking software (Cleversys Topscan). The duration of the trial is 10 minutes and the rodent is then placed back in its transport box and returned to its home cage. The well lit (450 lux), large area poses a threat to the rodent that prefers darker conditions and smaller spaces. This once again presents a conflict for the rat between the possible danger of large areas (i.e. aerial predators) and the opportunies for new food sources / mating in novel spaces. Consequently, this will elicit a variety of responses that may include high levels of exploration, crossing through the central portion of the arena or remaining motionless and occupying the sides and corners of the test arena. Rats spending more time in the perimeter and corner zones of the arena, as captured and analysed by our CleverSys rodent tracking software are considered to have greater levels of anxiety-like behaviour.

[0570] The results of these tests will provide insight into the behavioural manifestations of restraint and the capacity of the test compound as a viable anxiolytic.

[0571] REFERENCES

[0572] 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.

[0573] 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.

[0574] 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.

[0575] 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.

[0576] 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.

[0577] Terpe K. (2003) Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 60: 523- 533.

[0578] 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.

[0579] SUMMARY OF SEQUENCES

[0580]

[0581]

Claims

Claims1. A bioactive food or beverage composition or ingredient comprising a betaendorphin (BND) peptide in a bioactive form.

2. The bioactive food or beverage composition or ingredient of claiml wherein the bioactivity of the food or beverage composition or ingredient is conferred by the BND peptide.

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

24. The bioactive food or beverage composition or ingredient of any preceding claim wherein the BND peptide is not enterically coated.

5. The bioactive food or beverage composition or ingredient of any preceding claim wherein the BND peptide has been recombinantly produced.

6. The bioactive food or beverage composition or ingredient of any preceding claim wherein the BND peptide has been recombinantly produced in a cell, tissue, or organism or part thereof.

7. The bioactive food or beverage composition or ingredient of claim 6 wherein the recombinantly produced BND has been purified from the cell, tissue or organism or part thereof, and added to the bioactive food or beverage composition or ingredient.

8. The bioactive food or beverage composition or ingredient of claim 6 that comprises the cell, tissue, organism or part thereof, in which the BND has been recombinantly produced.

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

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

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. The food or beverage composition of any one of claims 6 to 12 wherein the BND peptide has the authentic N-terminus of naturally occurring BND.

14. The food or beverage composition of any preceding claim wherein the BND accumulates at a level of at least 0.1 % of total soluble protein.

15. The food or beverage composition of any preceding claim wherein the plant cell, plant tissue or plant or part thereof is that of an alfalfa plant, or a rice plant.

16. The food or beverage composition of any preceding claim that is: a) a hot beverage selected from a coffee, tea or a cocoa / hot chocolate beverage. b) an ingredient used to produce the hot beverage c) an ingredient used as an additive to the hot beverage.

17. The food or beverage composition of any one preceding wherein the plant cell, plant tissue or plant or part thereof is that of a coffee plant, a tea plant, or a cocoa plant.

18. The food or beverage composition of any one of claims 6 to 12 wherein the plant cell or plant tissue is from or part of a coffee bean, a tea leaf or a cocoa bean material.

19. The food or beverage composition of claim 18 wherein the coffee bean has been roasted, and wherein the bioactivity of the BND peptide survives the roasting of coffee bean.

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

21. The food or beverage composition of any preceding claim that is in liquid form, and wherein bioactivity of the BND survives heating to at least 50°C.

22. An expression cassette encoding a polypeptide cassette comprising: a. at least one of: i. an ER targeting signal peptide, and ii. a chloroplast targeting signal peptide, and b. a BND peptide23. A plant cell, plant tissue or plant genetically modified to express a BND peptide in a bioactive form.

24. The plant cell, plant tissue or plant of claim 23 comprising an expression cassette of claim 22, or a polypeptide cassette encoded by the expression cassette.

25. The plant cell, plant tissue or plant of any one of claims 23 to 24 in which the BND peptide has the authentic N-terminus of naturally occurring BND.

26. A plant cell, plant tissue or plant of any one of claims 23 to 25 that is transgenic for a polynucleotide encoding the BND.

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

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

29. The plant cell, plant tissue or plant of any preceding claim wherein the plant cell, plant tissue, plant or part thereof, is that 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. The plant cell, plant tissue or plant of any preceding claim wherein the plant cell, plant tissue, plant or part thereof, is that of a coffee plant, tea plant, cocoa plant.

31. The plant part of any preceding claim that is selected from a coffee bean, tea leaf, and a cocoa bean.

32. A method for producing bioactive food or beverage composition comprising a BND peptide in a bioactive form.

33. The method of claim 32 wherein the BND peptide comprises the sequence with at least 90% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

34. The method of any preceding claim wherein the BND peptide is not enterically coated.

35. The method of any preceding claim wherein the BND peptide has the authentic N- terminus of naturally occurring BND.

36. The method of any preceding claim that comprises providing a cell, tissue, organism or part thereof, comprising the BND peptide.

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

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

39. The method of any preceding claim wherein the food or beverage composition of a is selected from: a) a hot beverage selected from a coffee, tea or a cocoa / hot chocolate beverage, b) an ingredient used to produce the hot beverage, and c) an ingredient used as an additive to the hot beverage40. The method of any preceding claim wherein the food or beverage composition wherein the plant cell, plant tissue, plant or part thereof, is that of a coffee plant, a tea plant, or a cocoa plant.

41. The method of any preceding claim wherein the wherein the plant cell, plant tissue is from or part of a coffee bean, a tea leaf or a cocoa bean material.

42. The method of any preceding claim wherein the wherein the coffee bean has been roasted, and wherein the bioactivity of the BND peptide survives the roasting of coffee bean.

43. The method of any preceding claim wherein the coffee bean material that has been roasted at a temperature of at least 150°C, for at least 10 minutes.

44. The method of any preceding claim wherein the food or beverage composition of any preceding claim that is in liquid form, and wherein bioactivity of the BND survives heating to at least 50°C.

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

46. The use of claim 45, wherein the BND peptide is produced in a plant or plant tissue, of any preceding claim, or by a method of any preceding claim.

47. The use of claim 46, wherein the food or beverage composition is produced by processing in a plant, plant tissue, or plant material, of any preceding claim.

48. A method of inducing, increasing or maintaining relaxation, the method comprising administering to a subject in need thereof a food or beverage composition of any one preceding claim, produced by the method of any preceding claim, or extracted from a cell, plant cell, tissue, plant tissue, organism or plant of any preceding claim.

49. The method of claim 48, wherein the subject is selected from the group consisting of a mammal, a human, an animal, a dog, a cat, a horse, a pig, a cow, a sheep and a bird.

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

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