Bioactive food and beverage compositions and methods

Abstract

The present invention provides bioactive foods, beverages, compositions and ingredients comprising a bioactive form of a beta-enorphin peptide (BND) peptide, as well as methods and compositions for producing a bioactive form of a beta-enorphin peptide (BND) peptide and such bioactive foods, beverages, compositions and ingredients. The invention also provides methods and compositions for the production of biologically active forms of beta-enorphin (BND) peptides in cells, tissues, and plants, as well as cells, tissues, and plants that produce biologically active forms of beta-enorphin (BND) peptides. The invention further provides methods and compositions for inducing, increasing, or maintaining relaxation comprising administering the bioactive food, beverage, composition, or ingredient described to a subject in need thereof.

Description

[0001] Cross - reference to related applications

[0002] The content of Australian Provisional Patent Application No. 2022904029, filed on December 24, 2022, is incorporated herein by reference in its entirety. Technical field

[0003] The present invention pertains to the fields of bioactive foods and beverages, as well as compositions and methods for their production. Background art

[0004] There are many reported benefits of relaxation, including: improved concentration, better digestion, increased blood flow, reduced anger and frustration, lower blood pressure, reduced stroke risk, promoted emotional well - being, reduced fatigue, reduced inflammation, and lower heart rate.

[0005] The ingestion of certain foods and beverages is associated with promoting relaxation. However, some such foods and beverages (e.g., alcoholic beverages), while promoting some of the above - mentioned relaxation benefits, have adverse effects on other health factors. In addition, beverages (e.g., alcoholic beverages) are also addictive, which can lead to more well - documented problems.

[0006] Therefore, there is a desire to provide compositions and methods for inducing or promoting relaxation that can overcome one or more drawbacks of existing foods and beverages ingested for the purpose of inducing relaxation.

[0007] Beta - Endorphin (β - Endorphin or BND) is an endogenous opioid neuropeptide and peptide hormone. The BND precursor is formed in the pituitary gland and is subsequently processed into BND. When BND binds to its homologous μ - opioid receptor, BND exerts its effects through various mechanisms in the central and peripheral nervous systems.

[0008] The functions of BND are known to be related to hunger, excitement, pain, maternal care, sexual behavior, and reward cognition. In the broadest sense, BND is mainly used in the body to relieve stress and maintain homeostasis. In behavioral studies, it has been shown that BND is released into the ventricular system by volume transmission in response to various stimuli.

[0009] Although BND may be regarded as a potential bioactive substance that can be used to manage relaxation, studies have shown that due to various reasons (including the harsh environment of the gastrointestinal tract), if it and many other bioactive peptides are orally ingested in an unprotected form, it is unlikely to exert any biological effects in the body, as further discussed below.

[0010] Therefore, despite limited effectiveness, efforts to utilize the beneficial effects of BND have focused on stimulants and bioactive substances that induce the endogenous production of BND.

[0011] In the emerging science of bioactive substances taken orally, the issue of absorption is of crucial importance. However, previous studies have found that, with the exception of dipeptides and tripeptides, there is little clear evidence that dietary bioactive peptides can cross the intestinal wall intact and enter the hepatic portal system at physiologically relevant concentrations (Miner-Williams et al., 2014, Nutr Res Rev, 27(2):308-29).

[0012] Miner-Williams et al. (2014) reported that there are two sources secreting proteolytic enzymes into the digestive lumen:

[0013] · The stomach secretes pepsinogen, which is converted into the active protease pepsin under the action of acid.

[0014] · The pancreas secretes a group of powerful proteases, mainly including trypsin, chymotrypsin and carboxypeptidase.

[0015] Other studies have also reached similar conclusions, so it is generally believed that, with very few exceptions, dietary proteins are not absorbed in their unchanged form. Instead, they must first be digested into amino acids or dipeptides and tripeptides.

[0016] Through the action of these pepsin and trypsin, dietary proteins are mainly hydrolyzed into medium and small peptides (oligopeptides) in the small intestinal lumen.

[0017] 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, not soluble enzymes. Their role is to further hydrolyze the luminal peptides, converting them into free amino acids and very small peptides. These end products of digestion are formed on the surface of intestinal epithelial cells and are available for absorption.

[0018] Previously, little absorption of peptides longer than four amino acids has been recorded. However, dipeptides and tripeptides are highly absorbed in the small intestine. These small peptides are co-transported with H+ ions by a transporter called PepT1 and are absorbed into small intestinal epithelial cells.

[0019] Once inside the intestinal epithelial cells, most of the absorbed dipeptides and tripeptides are digested into amino acids by cytoplasmic peptidases and exported from the cells into the blood. Only a very small number of these small peptides enter the blood intact.

[0020] As emphasized, absorption of intact proteins occurs only in a few cases. First, because very few proteins can withstand the test 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 penetrate the tight junctions.

[0021] An important exception to these general statements is that in the first few days after birth, neonates have the ability to absorb intact proteins. This rapidly lost ability is important because it enables neonatal animals to acquire passive immunity by absorbing immunoglobulins in colostrum.

[0022] Specifically, regarding opioid peptides, Asvadi et al., 2014, (Front Pharmacol. 2014; 5:18, and references therein) reported that it is well known that peptides (including opioid peptides) are susceptible to rapid enzymatic degradation. The main peptidases involved in opioid peptide degradation are aminopeptidases, angiotensin-converting enzyme (ACE), insulin-degrading enzyme, serine peptidases, dipeptidyl peptidase III and IV (DPP III, DPP IV).

[0023] Peptides are also reported to be prone to degradation by high temperatures. During temperature changes from 60 °C to 90 °C, heat causes protein denaturation and aggregation, which may lead to the formation of clusters of high-molecular-weight peptides, resulting in a decrease or loss of biological activity (Bloom et al. 2015).

[0024] In summary, these studies suggest that BND acts in the PNS and CNS and is unlikely (or at least unlikely without intestinal protection) to be used as a bioactive substance for managing relaxation by oral ingestion. This problem may be exacerbated if BND is subjected to high temperatures before ingestion.

[0025] The object of the present invention is to provide compositions and methods for inducing relaxation and / or to provide useful alternatives for the public. Summary of the Invention

[0026] The present applicant has unexpectedly shown for the first time that, contrary to the expectations of the prior art discussed in the background art section above, natural, unprotected BND peptides can exert biological effects by oral ingestion and can therefore be used for managing relaxation by oral ingestion.

[0027] The applicant has also unexpectedly shown that BND peptides can be used in food and beverage compositions, and that the observed biological effects are retained even after BND peptides have undergone the relatively extreme temperatures employed in the production of some such food and beverage compositions.

[0028] Accordingly, the present invention provides novel bioactive food and beverage compositions comprising BND, methods for their production, and uses in managing relaxation. The present invention further contemplates the recombinant expression of BND in an organism (such as a plant), and the use of biomaterials comprising or expressing the biomaterial to produce the bioactive food and beverage compositions according to the present invention.

[0029] Bioactive food or beverage composition or ingredient

[0030] In one aspect, the present invention provides a bioactive food or beverage composition or ingredient comprising a β-endorphin (BND) peptide in a bioactive form.

[0031] In one embodiment, the bioactivity of the food or beverage composition or ingredient is conferred by the BND peptide.

[0032] In one embodiment, the BND peptide comprises a sequence having at least 90% identity to the sequence of SEQ ID NO:1 or SEQ ID NO:2.

[0033] In a preferred embodiment, the BND peptide is not enteric-coated.

[0034] In one embodiment, the BND peptide is recombinantly produced.

[0035] In a preferred embodiment, recombinantly produced BND has higher bioactivity than synthetic BND.

[0036] In a further embodiment, recombinantly produced purified BND is purer than synthetic BND.

[0037] In a further embodiment, the production cost of recombinantly produced purified BND is lower than that of synthetic BND.

[0038] In one embodiment, the BND peptide is recombinantly produced in a cell, tissue or organism.

[0039] In one embodiment, recombinantly produced BND is purified from a cell, tissue or organism and added to a bioactive food or beverage composition or ingredient.

[0040] In a further embodiment, the bioactive food or beverage composition or ingredient comprises a cell, tissue, organism or a part thereof, wherein the BND is recombinantly produced.

[0041] In a further embodiment, the cell, tissue or organism is a plant cell, plant tissue or plant, respectively.

[0042] In a further embodiment, a cell, tissue, organism, plant cell, plant tissue, or plant is transgenic for a polynucleotide encoding a BND peptide.

[0043] In one embodiment, the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide or a chloroplast targeting signal peptide operably linked to the BND peptide.

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

[0045] In one embodiment, the polynucleotide is an expression cassette encoding a polypeptide cassette comprising:

[0046] · an ER targeting signal peptide, and

[0047] · a BND peptide.

[0048] In a further embodiment, the polypeptide cassette further comprises at least one of the following:

[0049] · a flexible linker,

[0050] · a first cleavage site,

[0051] · a second cleavage site,

[0052] · a detection / purification tag, and

[0053] · an ER retention sequence.

[0054] In a further embodiment, the polypeptide cassette further comprises, in the N- to C-direction:

[0055] · an ER targeting signal peptide,

[0056] · a flexible linker,

[0057] · a first cleavage site,

[0058] · a 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 cleavage site and the second cleavage site are different from each other.

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

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

[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] · a chloroplast targeting signal peptide, and

[0073] · the BND peptide.

[0074] In a further embodiment, the polypeptide cassette further comprises at least one of the following:

[0075] · a flexible linker,

[0076] · a first cleavage site,

[0077] · a second cleavage site, and

[0078] · a detection / purification tag.

[0079] In a further embodiment, the polypeptide cassette comprises, in the N-to-C direction:

[0080] · a chloroplast targeting signal peptide,

[0081] · a flexible linker,

[0082] · a first cleavage site,

[0083] · the BND peptide,

[0084] · a second cleavage site, and

[0085] · a detection / purification tag.

[0086] In one embodiment, the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0087] In a preferred embodiment, the first cleavage site is an enterokinase cleavage site.

[0088] In one embodiment, the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0089] In a preferred embodiment, the second cleavage site is a thrombin cleavage site.

[0090] In a preferred embodiment, the first cleavage site and the second cleavage site are different from each other.

[0091] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5His tag.

[0092] In one embodiment, the BND peptide is polymerized.

[0093] In one embodiment, the BND peptide is at least tandemly repeated.

[0094] In a further embodiment, the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to the BND peptide and at least one of the following:

[0095] · A first flexible linker,

[0096] · A detection / purification tag,

[0097] · A second flexible linker, and

[0098] · A cleavage site.

[0099] In a further embodiment, the polypeptide cassette comprises, in the N-to-C direction:

[0100] · A chloroplast targeting signal peptide,

[0101] · A first flexible linker,

[0102] · A detection / purification tag,

[0103] · A second flexible linker,

[0104] · A cleavage site, and

[0105] · The BND peptide.

[0106] In one embodiment, the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0107] In a preferred embodiment, the cleavage site is an enterokinase cleavage site.

[0108] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5His tag.

[0109] In one embodiment, the BND peptide is multimerized.

[0110] In one embodiment, the BND peptide is at least tandemly repeated.

[0111] In a further embodiment, the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to the BND peptide and at least one of the following:

[0112] · A cleavage site,

[0113] · A flexible linker, and

[0114] · A detection / purification tag.

[0115] In a further embodiment, the polypeptide cassette comprises, in the N- to C-direction:

[0116] · A chloroplast targeting signal peptide,

[0117] · The BND peptide,

[0118] · A cleavage site,

[0119] · A first flexible linker, and

[0120] · A detection / purification tag.

[0121] In one embodiment, the cleavage site is cleaved by its corresponding protease immediately before the N-terminus adjacent to the cleavage site.

[0122] In one embodiment, the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0123] In one embodiment, the cleavage site is an enterokinase cleavage site.

[0124] In one embodiment, the cleavage site is a thrombin cleavage site.

[0125] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5His tag.

[0126] In one embodiment, the BND peptide is multimerized.

[0127] In one embodiment, the BND peptide is at least tandemly repeated.

[0128] In one embodiment, the BND peptide accumulates in the ER or chloroplasts of cells, tissues, organisms, plant cells, plant tissues or plants.

[0129] In one embodiment, the BND peptide accumulates in the ER of cells, tissues, organisms, plant cells, plant tissues, or plants.

[0130] In one embodiment, the BND peptide accumulates in the chloroplasts of cells, tissues, organisms, plant cells, plant tissues, or plants.

[0131] In a preferred embodiment, the BND peptide has the authentic N-terminus of a naturally occurring BND.

[0132] 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.

[0133] In one embodiment, the cleaved BND has no more than five, preferably no more than four, more preferably no more than three, more preferably no more than two, more preferably no more than one, more preferably no additional amino acids outside the N-terminus of SEQ ID NO: 1 or 2.

[0134] 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% correctly cleaved to produce the N-terminus as defined above.

[0135] In one embodiment, the BND accumulates at a level of at least 0.1% of the total soluble protein in cells, tissues, organisms, plant cells, plant tissues, or plants.

[0136] Preferably, 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 the total soluble protein in cells, tissues, organisms, plant cells, plant tissues or plants.

[0137] In one embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a plant suitable for human consumption.

[0138] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a plant suitable for animal consumption.

[0139] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a forage plant.

[0140] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of an alfalfa plant, rice plant, wheat plant, barley plant, corn plant, coffee plant, cocoa plant or tobacco plant.

[0141] In one embodiment, the food or beverage composition is an alcoholic beverage.

[0142] In one embodiment, the food or beverage composition is selected from:

[0143] a) hot drinks selected from coffee, tea or cocoa / hot chocolate drinks,

[0144] b) ingredients for making hot drinks, and

[0145] c) ingredients used as additives for hot drinks.

[0146] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a coffee plant, tea plant or cocoa plant.

[0147] In a further embodiment, the plant cell or plant tissue is from a coffee bean, tea leaf or cocoa bean, or is a part of a coffee bean, tea leaf or cocoa bean.

[0148] In a further embodiment, the BND is added or injected into the composition, plant part, plant tissue, coffee bean, tea leaf or cocoa bean.

[0149] In one embodiment, the BND is recombinantly produced as described herein and purified prior to injection.

[0150] In one embodiment, the coffee bean has been roasted and the biological activity of the BND peptide is retained after roasting of the coffee bean or a part thereof.

[0151] In one embodiment, the coffee bean or a part thereof has been roasted at a temperature of at least 150 °C for at least 10 minutes.

[0152] Preferably, the BND has been roasted at a temperature of at least 155 °C, more preferably at least 160 °C, more preferably at least 165 °C, more preferably at least 170 °C, more preferably at least 175 °C, more preferably at least 180 °C, more preferably at least 185 °C, more preferably at least 190 °C, more preferably at least 195 °C, more preferably at least 200 °C, more preferably at least 210 °C, more preferably at least 220 °C, more preferably at least 230 °C, more preferably at least 240 °C, more preferably at least 250 °C, and the biological activity of the BND peptide is retained after roasting of the coffee bean or a part thereof.

[0153] Preferably, the BND has been roasted at the above temperature for at least 15 minutes, more preferably at least 20 minutes, more preferably at least 25 minutes, more preferably at least 30 minutes, more preferably at least 35 minutes, more preferably at least 40 minutes, more preferably at least 45 minutes, more preferably at least 50 minutes, more preferably at least 55 minutes, more preferably at least 60 minutes, and the biological activity of the BND peptide is retained after roasting of the coffee bean or a part thereof.

[0154] In a further embodiment, the food or beverage composition is in liquid form and the biological activity of the BND is retained after heating to at least 50 °C.

[0155] Preferably, the BND is retained after 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.

[0156] Expression cassette

[0157] In one aspect, the present invention provides an expression cassette encoding a polypeptide cassette, comprising:

[0158] · An ER targeting signal peptide, and

[0159] · A BND peptide.

[0160] In a further embodiment, the polypeptide cassette further comprises at least one of the following:

[0161] · A flexible linker,

[0162] · A first cleavage site,

[0163] · A second cleavage site,

[0164] · A detection / purification tag, and

[0165] · An ER retention sequence.

[0166] In a further embodiment, the polypeptide cassette further comprises, in the N- to C-direction:

[0167] · An ER targeting signal peptide,

[0168] · A flexible linker,

[0169] · A first cleavage site,

[0170] · A BND peptide,

[0171] · A second cleavage site,

[0172] · A detection / purification tag, and

[0173] · An ER retention sequence.

[0174] In one embodiment, the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0175] In a preferred embodiment, the first cleavage site is an enterokinase cleavage site.

[0176] In one embodiment, the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0177] In a preferred embodiment, the second cleavage site is a thrombin cleavage site.

[0178] In a preferred embodiment, the first cleavage site and the second cleavage site are different from each other.

[0179] In one embodiment, the purification tag is a His tag. In a further embodiment, the His tag is a V-5His tag.

[0180] In one embodiment, the BND peptide is multimerized.

[0181] In one embodiment, the BND peptide is at least tandemly repeated.

[0182] In one embodiment, the polynucleotide encodes a chloroplast targeting signal peptide operably linked to the BND peptide.

[0183] In a further aspect, the present invention provides an expression cassette encoding a polypeptide cassette, comprising:

[0184] · a chloroplast targeting signal peptide, and

[0185] · a BND peptide.

[0186] In a further embodiment, the polypeptide cassette further comprises at least one of the following:

[0187] · a flexible linker,

[0188] · a first cleavage site,

[0189] · a second cleavage site, and

[0190] · a detection / purification tag

[0191] In a further embodiment, the polypeptide cassette further comprises, in the N- to C-direction:

[0192] · a chloroplast targeting signal peptide,

[0193] · a flexible linker,

[0194] · a first cleavage site,

[0195] · a BND peptide,

[0196] · a second cleavage site, and

[0197] · a detection / purification tag.

[0198] In one embodiment, the first cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0199] In a preferred embodiment, the first cleavage site is an enterokinase cleavage site.

[0200] In one embodiment, the second cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0201] In a preferred embodiment, the second cleavage site is a thrombin cleavage site.

[0202] In a preferred embodiment, the first cleavage site and the second cleavage site are different from each other.

[0203] In one embodiment, the purification tag is a His tag. In a further embodiment, the His tag is a V-5His tag.

[0204] In one embodiment, the BND peptide is multimerized.

[0205] In one embodiment, the BND peptide is at least tandemly repeated.

[0206] In a further embodiment, the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to the BND peptide and at least one of the following:

[0207] · A first flexible linker,

[0208] · A detection / purification tag,

[0209] · A second flexible linker, and

[0210] · A cleavage site.

[0211] In a further embodiment, the polypeptide cassette comprises, in the N-to-C direction:

[0212] · A chloroplast targeting signal peptide,

[0213] · A first flexible linker,

[0214] · A detection / purification tag,

[0215] · A second flexible linker,

[0216] · A cleavage site, and

[0217] · The BND peptide,

[0218] In one embodiment, the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0219] In a preferred embodiment, the cleavage site is an enterokinase cleavage site.

[0220] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5His tag.

[0221] In one embodiment, the BND peptide is multimerized.

[0222] In one embodiment, the BND peptide is at least tandemly repeated.

[0223] In a further embodiment, the polypeptide cassette comprises a chloroplast targeting signal peptide operably linked to the BND peptide and at least one of the following:

[0224] · A cleavage site,

[0225] · A flexible joint, and

[0226] · A detection / purification tag.

[0227] In a further embodiment, the polypeptide cassette comprises, in the N- to C-direction:

[0228] · A chloroplast targeting signal peptide,

[0229] · A BND peptide,

[0230] · A cleavage site,

[0231] · A first flexible joint, and

[0232] · A detection / purification tag.

[0233] In one embodiment, the cleavage site is cleaved by its corresponding protease immediately before the N-terminus adjacent to the cleavage site.

[0234] In one embodiment, the cleavage site is an enterokinase cleavage site or a thrombin cleavage site.

[0235] In one embodiment, the cleavage site is an enterokinase cleavage site.

[0236] In one embodiment, the cleavage site is a thrombin cleavage site.

[0237] In one embodiment, the detection / purification tag is a His tag. In a further embodiment, the His tag is a V-5His tag.

[0238] In one embodiment, the BND peptide is multimerized.

[0239] In one embodiment, the BND peptide is at least tandemly repeated.

[0240] A plant cell or a plant

[0241] In one aspect, the present invention provides a plant cell, a plant tissue, a plant or a part thereof that is genetically modified to express a biologically active form of a BND peptide.

[0242] In a further aspect, there is provided a plant cell, a plant tissue, a plant or a part thereof that comprises an expression cassette encoding the polypeptide cassette or the polypeptide cassette of the present invention.

[0243] In one embodiment, a plant cell, a plant tissue, a plant or a part thereof that is genetically modified to express a biologically active form of a BND peptide comprises an expression cassette encoding the polypeptide cassette or the polypeptide cassette of the present invention.

[0244] In one embodiment, the BND peptide has the authentic N-terminus of a naturally occurring BND.

[0245] Preferably, the BND comprises at least the first 5 nucleotides of SEQ ID NO:1.

[0246] In one embodiment, a plant cell, plant tissue or plant is transgenic for a polynucleotide encoding BND.

[0247] In one embodiment, the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide or a chloroplast targeting signal peptide operably linked to the BND peptide.

[0248] In one embodiment, the polynucleotide encodes an endoplasmic reticulum (ER) targeting signal peptide operably linked to the BND peptide.

[0249] In one embodiment, the polynucleotide encodes a chloroplast targeting signal peptide operably linked to the BND peptide.

[0250] In a further embodiment, the BND peptide accumulates in the ER or chloroplast of a cell, tissue, organism, plant cell, plant tissue or plant.

[0251] In one embodiment, the BND peptide accumulates in the ER of a cell, tissue, organism, plant cell, plant tissue or plant.

[0252] In a further embodiment, the BND peptide accumulates in the chloroplast of a cell, tissue, organism, plant cell, plant tissue or plant.

[0253] In one embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a plant suitable for human consumption.

[0254] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a plant suitable for animal consumption.

[0255] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a forage plant.

[0256] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of an alfalfa plant, rice plant, wheat plant, barley plant, corn plant, coffee plant, cocoa plant or tobacco plant.

[0257] In a further embodiment, the plant cell, plant tissue or plant is a plant cell, plant tissue or plant of a coffee plant, tea plant or cocoa plant.

[0258] In one embodiment, the plant cell, plant tissue or plant or a part thereof is selected from coffee beans, tea leaves and cocoa beans.

[0259] A method for producing a bioactive food or beverage composition comprising BND.

[0260] In a further aspect, the present invention provides a method for producing a bioactive food or beverage composition comprising a bioactive form of the β-endorphin (BND) peptide.

[0261] In one embodiment, the BND peptide comprises a sequence having at least 90% identity to SEQ ID NO:1.

[0262] In a preferred embodiment, the BND peptide is not enteric-coated.

[0263] In one embodiment, the method comprises providing a cell, tissue or organism comprising the BND peptide.

[0264] In a further embodiment, the BND peptide is produced in a cell, tissue or organism or a part thereof.

[0265] In one embodiment, the cell, tissue or organism or a part thereof is a plant cell, plant tissue or plant or a part thereof, respectively.

[0266] In one embodiment, the food or beverage composition of a is selected from:

[0267] a) a hot drink selected from coffee, tea or cocoa / hot chocolate drinks,

[0268] b) an ingredient for making a hot drink,

[0269] c) an ingredient used as an additive to a hot drink.

[0270] In one embodiment, the plant cell, plant tissue or plant or a part thereof is a plant cell, plant tissue or plant or a part thereof of a coffee plant, tea plant or cocoa plant.

[0271] In one embodiment, the plant cell or plant tissue is from coffee beans, tea leaves or cocoa beans or is a part of coffee beans, tea leaves or cocoa beans.

[0272] In one embodiment, the coffee beans have been roasted and the bioactivity of the BND peptide is retained after roasting of the coffee beans.

[0273] In one embodiment, the coffee beans have been roasted at a temperature of at least 150 °C for at least 10 minutes.

[0274] Preferably, the BND has been baked at a temperature of at least 155 °C, more preferably at least 160 °C, more preferably at least 165 °C, more preferably at least 170 °C, more preferably at least 175 °C, more preferably at least 180 °C, more preferably at least 185 °C, more preferably at least 190 °C, more preferably at least 195 °C, more preferably at least 200 °C, and the biological activity of the BND peptide is retained after baking of the coffee beans or parts thereof.

[0275] Preferably, the BND has been baked at the above temperature for at least 15 minutes, more preferably at least 20 minutes, more preferably at least 25 minutes, more preferably at least 30 minutes, more preferably at least 35 minutes, more preferably at least 40 minutes, more preferably at least 45 minutes, more preferably at least 50 minutes, more preferably at least 55 minutes, more preferably at least 60 minutes, and the biological activity of the BND peptide is retained after baking of the coffee beans or parts thereof.

[0276] In a further embodiment, the food or beverage composition is in liquid form, and the biological activity of the BND is retained after heating to at least 50 °C.

[0277] Preferably, the BND is retained after 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.

[0278] Use of the BND peptide in the production of a food or beverage composition for managing relaxation

[0279] In a further aspect, the present invention provides the use of the BND peptide in the production of a food or beverage or ingredient composition for managing relaxation.

[0280] In one embodiment, the BND peptide is produced in the plant cells, plant tissues, plants or parts thereof of the present invention, or is produced by the method of the present invention.

[0281] In one embodiment, the food or beverage composition is produced by processing the plant cells, plant tissues, plants or parts thereof of the present invention.

[0282] Method for managing relaxation

[0283] In a further aspect, the present invention provides a method for inducing, increasing or maintaining relaxation, the method comprising administering to a subject in need thereof the food beverage or ingredient composition of the present invention, or a food beverage or ingredient composition produced by the method of the present invention.

[0284] In one embodiment, the subject is a mammal.

[0285] In one embodiment, the mammal is selected from the group consisting of a human, dog, cat, horse, pig, cow, and sheep.

[0286] In a further embodiment, the subject is a human.

[0287] In one embodiment, the subject is a bird.

[0288] In one embodiment, the bird is selected from the group consisting of a chicken and a turkey. Detailed Description

[0289] In this specification, patent specifications, other external documents, or other information sources are cited, which is generally for providing the background for discussing the features of the present invention. Unless otherwise clearly stated, the citation of such external documents should not be construed as an admission that such documents or such information sources are prior art in any jurisdiction or form part of the common general knowledge in the art.

[0290] As used in this specification, the term "comprising" means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", there may also be features other than that term or features before that term. Related terms such as "including" and "containing" should be interpreted in the same way.

[0291] BND

[0292] Human beta-endorphin (BND) is a 31-amino acid linear peptide.

[0293] Amino acid residues 1-25 have 100% sequence identity with BNDs of other species including sheep, horse, cow, rat, and camel. In one embodiment, the BND is human BND. In one embodiment, the human BND comprises the amino acid sequence of SEQ ID NO:2 (below) or a functional variant thereof that has 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 therewith.

[0294] Previous studies have shown that amino acids 28-31 confer antigenic properties to BND, and in the guinea pig ileum assay, the chain of amino acids 1-27 has activity equivalent to that of full-length BND when tested in vitro (Yeung et al., 1978, Int J Pept Protein Res, 12(1), 42-6).

[0295] In a further embodiment, the human BND comprises the amino acid sequence of SEQ ID NO:1 (below) or a functional variant thereof that has at least 70% sequence identity therewith.

[0296] In a further embodiment, the human BND comprises the amino acid sequence of SEQ ID NO:2 (below) or a functional variant thereof having at least 70% sequence identity thereto.

[0297] Polypeptide variant

[0298] Polypeptide sequence identity can be determined as follows. Using BLASTP in bl2seq (from the BLAST program suite, version 2.2.5 [November 2002]), which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ), compare the target polypeptide sequence with the candidate polypeptide sequence.

[0299] A global sequence alignment program can also be used to calculate polypeptide sequence identity over the entire length of overlap between the candidate polynucleotide sequence and the target polynucleotide sequence. EMBOSS-needle (available from 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 global sequence alignment programs suitable for calculating polypeptide sequence identity.

[0300] The preferred method for calculating the % polypeptide sequence identity is based on using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.) to align the sequences to be compared

[0301] The invention also includes making one or several conservative substitutions of amino acids in the polypeptide sequence without significantly altering its biological activity. Those skilled in the art will know methods for making phenotypically silent amino acid substitutions (see, for example, Bowie et al., 1990, Science 247, 1306).

[0302] Methods for generating constructs and vectors

[0303] The genetic constructs of the invention comprise one or more polynucleotide sequences of the invention and / or polynucleotides encoding the polypeptides of the invention and can be used to transform, for example, bacterial, fungal, insect, mammalian or plant organisms.

[0304] Methods for generating and using genetic constructs and vectors are well known in the art and are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).

[0305] Methods for generating host cells containing polynucleotides, constructs or vectors

[0306] The present invention provides host cells comprising the genetic constructs or vectors of the present invention.

[0307] Host cells comprising the genetic constructs (e.g., expression constructs) of the present invention can be used 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 the recombinant production of the polypeptides of the present invention. Such methods can involve culturing the host cells in a suitable medium under conditions suitable or conducive to the expression of the polypeptides of the present invention. The expressed recombinant polypeptides can optionally be secreted into the culture and can then be isolated from the medium, host cells or medium by methods well known in the art (e.g., Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).

[0308] Methods for generating plant cells and plants containing constructs and vectors

[0309] The present invention further provides plant cells comprising genetic constructs for expressing BND according to the present invention, and plant cells modified to alter the expression of the polynucleotides or polypeptides of the present invention or the polynucleotides or polypeptides used in the methods of the present invention. Plants containing such cells also constitute an aspect of the present invention.

[0310] Methods for transforming plant cells, plants, and parts thereof with polypeptides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual. Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.

[0311] Methods for plant genetic manipulation

[0312] There are many plant transformation strategies available (e.g., Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297, Hellens RP, et al (2000) Plant Mol Biol 42:819-32, Hellens R et al Plant Meth 1:13). For example, strategies can be designed to increase the expression of polynucleotides / polypeptides in plant cells, organs, and / or at specific developmental stages when they are normally expressed, or to ectopically express polynucleotides / polypeptides in cells, tissues, organs, and / or at specific developmental stages when they are not normally expressed.

[0313] Signal peptide

[0314] Signal peptides are well known to those skilled in the art and can be used to direct the accumulation of recombinant proteins and peptides to subcellular locations in plants.

[0315] ER targeting signal peptide

[0316] Endoplasmic reticulum (ER) targeting signal peptides are known to those skilled in the art and are described, for example, in Kim and Hwang, 2013, Traffic; 14:613-621.

[0317] In one embodiment, the ER targeting signal peptide for use in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to the sequence of SEQ ID NO:7.

[0318] Chloroplast targeting signal peptide

[0319] Chloroplast targeting signal peptides are known to those skilled in the art and are described, for example, in Bruce et al., 2000, Trends Cell Biol; 10(10):440-7.

[0320] In one embodiment, the chloroplast targeting signal peptide for use in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to a sequence selected from SEQ ID NO:31 and SEQ ID NO:39.

[0321] Expression cassette

[0322] As used herein, an expression cassette refers to a polynucleotide sequence having elements encoding a polypeptide to be expressed.

[0323] An expression cassette typically includes a promoter operably linked to a sequence encoding a polypeptide to be expressed. An expression cassette typically also includes a terminator, which is operably linked to the polypeptide to be expressed.

[0324] Polypeptide cassette

[0325] As used herein, a polypeptide cassette refers to the polypeptide sequence encoded by an expression cassette. In addition to the target peptide (in this case BND, or a multimer of BND), the polypeptide cassette may also include a plurality of additional peptide elements, such as those described below.

[0326] Purification / detection tag

[0327] A purification or detection tag is preferably included in the polypeptide cassette to facilitate detection or purification of the polypeptide cassette. Such tags are known to those skilled in the art and include, for example: FLAG-tag, His-tag, V%-HIS tag, Myc-tag, Strep-tag, TC tag, and HA-tag. Examples of such sequences are shown in Table 1 below.

[0328] Table 1 - Examples of purification / detection tags

[0329]

[0330] In one embodiment, the purification / detection tag for use in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to the purification / detection tag sequences disclosed herein.

[0331] The purification / detection tag can facilitate the detection of the polypeptide cassette or the tag itself, for example, by Western blotting.

[0332] The purification / detection tag can facilitate the purification of the polypeptide cassette by using affinity columns known to those skilled in the art.

[0333] Cleavage site

[0334] A cleavage site may be included to facilitate the cleavage and separation of the elements located on either side of the cleavage site. Such cleavage sites are known to those skilled in the art and include, for example: enterokinase cleavage site, thrombin cleavage site. Examples of such sequences are shown in Table 2 below.

[0335] Table 2 - Examples of cleavage sites

[0336] Cleavage site Exemplary sequence SEQ ID NO Thrombin cleavage site LVPRGS 16 Enterokinase cleavage site DDDDK 13

[0337] In one embodiment, the cleavage site for use in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to the cleavage site sequences selected from those disclosed herein.

[0338] ER retention sequence

[0339] The ER retention sequence can be used to retain the target peptide (BND herein) in the endoplasmic reticulum of plant cells. Examples of such ER retention sequences include the KDEL sequence and the HDEL sequence. Examples of such sequences are shown in Table 3 below.

[0340] Table 3 - Examples of ER retention sequences

[0341] ER retention sequence Exemplary sequence SEQ ID NO KDEL KDEL 49 KDEL AAAKDEL 22 HDEL HDEL 50

[0342] In one embodiment, the ER retention sequence for use in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to the ER retention sequences disclosed herein.

[0343] Flexible linker

[0344] In a preferred embodiment, the flexible peptide linker is soluble.

[0345] 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 from 1 to 5. Examples of such sequences are shown in Table 4 below.

[0346] Table 4 - Examples of flexible linker sequences

[0347]

[0348]

[0349] In one embodiment, the ER retention sequence for use in the present invention comprises a sequence having at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to the ER retention sequences disclosed herein.

[0350] Plant transformation protocol

[0351] The following are representative publications that disclose gene transformation protocols that can be used for gene transformation of 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, pages 243-251); rice (Alam et al., 1999, Plant Cell Rep. 18, 572); alfalfa / Medicago (Wand et al., 2016, Protein Pept Lett., 23(5):495-502); apple (Yao et al., 1995, Plant Cell Reports 14, 407-412); maize (U.S. Patent Serial Numbers 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Patent Serial Number 5,159,135); potato (Kumar et al., 1996 Plant J. 9, :821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Patent Serial Numbers 5,846,797 and 5,004,863); grasses (U.S. Patent Serial Numbers 5,187,073 and 6.020,539); mint (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 (U.S. Patent Serial No. 5,792,935); soybean (U.S. Patent Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Patent Serial No. 5,952,543); poplar (U.S. Patent No. 4,795,855); general monocotyledonous plants (U.S. Patent Nos. 5,591,616 and 6,037,522); Brassica (U.S. Patent Nos. 5,188,958; 5,463,174 and 5,750,871); cereal (U.S. Patent No. 6,074,877); pear (Matsuda et al., 2005, Plant Cell Rep. 24(1):45 - 51); Prunus (Ramesh et al., 2006 PlantCell Rep. 25(8):821 - 8; Song and Sink 2005 Plant Cell Rep. 2006; 25(2):117 - 23; Gonzalez Padilla et al., 2003 Plant Cell Rep. 22(1):38 - 45); 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), rapeseed (Brassica napus L.). (Cardozaand Stewart, 2006 Methods Mol Biol. 343:257 - 66), safflower (Orlikowska et al, 1995, PlantCell Tissue and Organ Culture 40:85 - 91), ryegrass (Altpeter et al, 2004 Developments in Plant Breeding 11(7):255 - 250), rice (Christou et al, 1991 Nature Biotech.9:957 - 962), maize (Wang et al., 2009 In: Handbook of Maize pp. 609 - 639) and Actinidia eriantha (Wang et al., 2006, Plant Cell Rep. 25, 5: 425 - 31). The present invention also contemplates the transformation of other species. Suitable methods and protocols are available in the scientific literature.

[0352] plant

[0353] Plant cells, plant tissues, plants and parts thereof that produce BND or contain the constructs of the present invention according to the present invention can be from any plant species.

[0354] In one embodiment, the cells, plant tissues, plants and parts thereof are derived from gymnosperm species.

[0355] In a further embodiment, they are derived from angiosperm species.

[0356] In a further embodiment, they are derived from dicotyledonous species.

[0357] In a further embodiment, they are derived from monocotyledonous species.

[0358] In one embodiment, they are derived from economically important crop species.

[0359] Preferred genera of dicotyledonous plants include: Amygdalus, Anacardium, Arachis, Brassica, Cajanus, Carthamus, Carya, Ceiba, Cicer, Cocos, Coriandrum, Coronilla, Cossypium, Crotalaria, Dolichos, Elaeis, Glycine, Gossypium, Helianthus, Lathyrus, Lens, Lespedeza, Linum, Lotus, Lupinus, Macadamia, Medicago, Melilotus, Mucuna, Olea, Onobrychis, Ornithopus, Phaseolus, Phoenix, Pistacia, Pisum, Prunus, Pueraria, Ribes, Ricinus, Sesamum, Theobroma, Trifolium Trigonella, Vicia and Vigna.

[0360] Preferred species of dicotyledonous plants include: Amygdalus communis, Anacardium occidentale, Arachis hypogaea, Arachis hypogea, Brassica napus Rape, Brassica nigra, Brassica campestris, Cajanus cajan, Cajanus indicus, Carthamus tinctorius, Carya illinoinensis, Ceiba pentandra, Cicer arietinum, Cocos nucifera, Coriandrum sativum, Coronilla varia, Gossypium hirsutum, Crotalaria juncea, Dolichos lablab, Elaeis guineensis, Gossypium arboreum, Gossypium nanking, Gossypium barbadense, Gossypium herbaceum, Gossypium hirsutum, Glycine max, Glycine ussuriensis, Glycine gracilis, 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, Medicagoarborea), 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, 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, TrifoliumTrifolium diffusum, Trifolium hybridum, Trifolium incarnatum, Trifolium ingrescens, Trifolium pratense, Trifolium 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, Vicia faba, Vicia sative, and Vigna angularis.

[0361] Preferred genera of monocotyledonous plants 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.

[0362] Preferred monocotyledonous plant species include: Agropyron desertorum, Agropyron elongatum, Agropyron spicatum, Agropyrum 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, Paspalumdilatatum), 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, Dactylis aristatum, Dactylis sericeum, Digitaria smutsii, Elymus junceus, Festuca ovina, Festuca pratensis, Festuca rubra, Panicum purpurascens, Panicum virgatum, Paspalum notatum, Pennisetum purpureum, Pennisetum spicatum, PhleumLolium pratense, Poa pratensis, Saccharum officinarum, Saccharum spontaneum, Sorghum sudanense, Triticum durum, Triticum monococcum, Vanilla fragrans, and Zea mays.

[0363] Preferred plants are from the genera Lolium and Trifolium. Particularly preferred are Lolium perenne and Trifolium repens.

[0364] Particularly preferred monocotyledonous plant species are: Lolium perenne and Oryza sativa.

[0365] The preferred genus is Coffea. The preferred coffee species is Coffea arabica.

[0366] A further preferred genus is Oryza. The preferred species of the genus Oryza is Oryza sativa.

[0367] Another preferred genus is Medicago. Preferred species of the genus Medicago include Medicago sativa and Medicago truncatula. The particularly preferred Medicago sativa species is Medicago sativa, commonly known as alfalfa.

[0368] Another preferred genus is Glycine. Preferred species of the genus Glycine include Glycine max and Glycine wightii (also known as Neonotonia wightii). The particularly preferred Glycine max species is Glycine max, commonly known as soybean. The particularly preferred Glycine wightii species is Glycine wightii, commonly known as perennial soybean.

[0369] Plant parts, propagules, and progeny

[0370] The term "plant" is intended to include the whole plant, any part of the plant, seeds, fruits, propagules, and progeny of the plant.

[0371] The term "propagule" refers to any plant part that can be used for sexual or asexual regeneration or propagation, including seeds and cuttings.

[0372] The plants of the present invention can grow and self-pollinate or cross with different plant lines, and the resulting progeny containing the polynucleotide or construct of the present invention and / or expressing the BND sequence / construct also form part of the present invention.

[0373] Preferably, the plant, plant part, propagule and progeny contain the polynucleotide or construct of the invention, and / or express the BND sequence according to the invention.

[0374] Method for producing food and beverage compositions

[0375] Methods for producing food and beverage compositions, including compositions incorporating bioactive ingredients, are known in the art and are described, for example, in: WO2017037263, WO2019045576, WO2011146140 and WO2017124075. BRIEF DESCRIPTION OF THE DRAWINGS

[0376] The present invention will be described with reference to the following non-limiting drawings.

[0377] Figure 1 A graph showing the relaxing effect on subjects of oral ingestion of BND dissolved in saline compared to a saline control is shown.

[0378] Figure 2 A graph showing the relaxing effect on subjects of oral ingestion of coffee infused with BND compared to a coffee control is shown.

[0379] Figure 3 A graph showing the relaxing effect on subjects of oral ingestion of BND samples that have undergone different temperature regimes prior to ingestion is shown.

[0380] Figure 4 A graph showing the relaxing effect on subjects of oral ingestion of coffee samples produced from coffee beans infused with BND and roasted at different temperatures prior to production and ingestion of the coffee samples is shown.

[0381] Figure 5 A graph showing the effect on subjects of oral ingestion of beer infused with BND compared to a beer control is shown.

[0382] Figure 6 The TargetP analysis output of the full peptide sequence of the ER targeting cassette is shown.

[0383] Figure 7 The output of NetGene2 is shown. The sequence of SEQ ID NO:32 was analyzed by NetGene2 to predict splicing.

[0384] Figure 8 The TargetP analysis output of the full peptide sequence of the chloroplast targeting cassette is shown.

[0385] Figure 9Shows the output of NetGene2. The sequence of SEQ ID NO:35 was analyzed by NetGene2 to predict its splicing.

[0386] Figure 10 Shows the output of TargetP analysis of full-length ER-targeted, tandem repeat BND.

[0387] Figure 11 Shows the output of TargetP analysis of full-length chloroplast-targeted, tandem repeat BND.

[0388] Figure 12 Shows the immunoblot analysis of soluble protein extracts from leaves of Nicotiana benthamiana transiently expressing BND fusion constructs. Replicate leaf extracts were run on SDS-PAGE, then immunoblotted and probed with anti-V5 antibody. The upper and lower insets represent different exposure times (10 seconds and 30 seconds respectively) of the immunoblot. Inset A shows plant extracts expressing a single BND construct targeted to the ER and chloroplast. Inset B shows plant extracts expressing a tandem BND construct targeted to the ER and chloroplast. VC = vector control.

[0389] Figure 13 Shows the ELISA results of leaves expressing a single β-endorphin construct.

[0390] Figure 14 Shows the immunoblot analysis of soluble proteins extracted from leaves of Nicotiana benthamiana transiently expressing BND fusion constructs in a buffer containing 1 M urea and 1% Triton X-100. The white arrow shows the 12.6 kDa tandem endorphin cassette of chloroplast signal cleavage. The black arrow shows the 8.7 kDa tandem repeat endorphin cassette of ER-signal cleavage.

[0391] Figure 15 Shows the quantification of unpurified recombinant BND accumulated in leaves of Nicotiana benthamiana. A) Dilution series of leaf extracts and 2 ng, 5 ng, 10 ng, 20 ng, and 40 ng dilution series of V5 fusion protein standards. The white arrows show the singly endorphin cassette and tandem repeat endorphin cassette (8.7 kDa and 12.9 kDa) of signal cleavage respectively. B) Standard curve generated by Image Lab 5.2.1 software after scanning the results of the dilution series in inset A. C) The average yield of BND was calculated as μg / g FW of transiently expressing leaves.

[0392] Figure 16 Shows the immunoblot analysis of endorphin purification using Ni-affinity chromatography. The images of the upper and lower insets show the unstained gel and immunoblot membrane respectively.

[0393] Figure 17 shows immunoblot analysis of endorphin purification using Ni-affinity binding, and peptide concentration by ultrafiltration using a 3 kDa cut-off filter. The images of the upper and lower insets show the unstained gel and the immunoblot membrane, respectively. Arrows indicate ER-targeted BND dimers and oligomers.

[0394] Example

[0395] The present invention will be illustrated with reference to the following non-limiting examples.

[0396] Example 1 - Biological Activity of Orally Administered BND Peptide

[0397] Materials and Methods

[0398] The BND peptide used in this study comprised amino acids 1 - 27, i.e., a 27-mer of the human β-endorphin sequence. The BND 27-mer was synthesized by Leon Biological Technology Co Ltd, Nanjing, China.

[0399] 10 mg of the BND peptide was dissolved in 200 ml of physiological saline, and the BND test sample was taken by Subject A (an adult male), and the relaxation index was monitored within 4 to 6 hours. Under the same conditions, 200 ml of physiological saline (without BND) was used as a control test sample for the same subject for a control test.

[0400] The subject self-assessed the relaxation index immediately after taking the test sample and the control sample as follows. During the test, the degree of relaxation was recorded at short time intervals (as shown in Table 5 below). Physical data (including pulse, blood pressure, and other physical parameters) were recorded and closely compared with the self-assessed relaxation index.

[0401] Table 5 - Relaxation Index

[0402] Self-description Relaxation index Extremely calm, relaxed 100 Very calm and relaxed 80 Moderately calm and relaxed 60 Moderately calm 40 Slightly calm and relaxed 20 Neither relaxed nor unrelaxed 0 Slightly unrelaxed -20

[0403] Results

[0404] The results are as Figure 1 shown.

[0405] These data unexpectedly showed (to the applicant's knowledge, for the first time) that BND can exert a biological activity when orally administered. The existing literature fully indicates that, as discussed in the background section of this specification, due to various reasons, including the harsh environment of the gastrointestinal tract, BND, like many other bioactive peptides, is unlikely to exert any biological effect in the body if orally administered in an unprotected form.

[0406] Figure 1 Oral ingestion of BND produced a net relaxing effect relative to the control, lasting up to four hours after ingestion.

[0407] Example 2 - Biological Activity of BND Injected into Prepared Hot Coffee

[0408] Materials and Methods

[0409] The BND peptide used and the relaxation index assessment were as described in Example 1.

[0410] At 60 °C, 10 mg of the BND peptide was injected into 200 ml of brewed coffee. Five minutes later at room temperature, the subject drank the BND-injected coffee over a 5-minute period. On another day, under the same conditions, the same subject was given a control test sample of 200 ml of coffee (without BND). Stress index assessment began immediately after each coffee sample was consumed.

[0411] Results

[0412] The results were as Figure 2 shown, showing that the BND-injected coffee produced a stronger relaxing effect than the control coffee, and the effect lasted up to four hours after ingestion.

[0413] These results show that even when BND was added to coffee at 60 °C, the unexpected biological activity of BND demonstrated in Example 1 was unexpectedly maintained, indicating that the presence of coffee and heating to 60 °C had no adverse effect on the efficacy of BND in inducing a relaxed state in the subjects.

[0414] Example 3 - Biological Activity of BND Exposed to Different Temperatures (Including Conditions Simulating Coffee Roasting)

[0415] The purpose of these tests was to evaluate the effect of higher temperatures on the BND biological activity efficacy demonstrated in the above examples and to observe whether its efficacy was maintained after exposure to conditions comparable to coffee bean roasting.

[0416] Materials and Methods

[0417] The BND peptide used and the relaxation index assessment were as described in Example 1.

[0418] A 10 mg BND sample was placed on filter paper and individually subjected to the following temperature treatments:

[0419] · Room temperature (20 °C) for 14 minutes

[0420] · 160 °C in a fan forced oven for 14 minutes

[0421] · In a forced convection oven at 230 °C for 14 minutes

[0422] The non - ambient temperatures (160 °C and 230 °C) and heating times were chosen because these parameters are commonly used in the coffee roasting process.

[0423] Then, each test sample was dissolved in 200 ml of normal saline (at 20 °C) for 5 minutes, ingested by the subjects, and then the relaxation index was evaluated.

[0424] Results

[0425] The results are as Figure 3 shown, and indicate that the BND effects produced according to each temperature treatment are roughly equivalent, and unexpectedly show that even though the BND peptide has experienced relatively harsh (160 °C and 230 °C) temperature environments (equivalent to the conditions used for coffee roasting), the BND effect is still retained.

[0426] Example 4 - Biological activity of BND in coffee beans containing BND after roasting the beans

[0427] The purpose of this example was to evaluate the effect of BND on roasted beans containing BND to test the hypothesis that plant materials expressing BND can be processed into beverages providing the same biological activity. To this end, BND was injected into raw coffee beans and then exposed to conditions equivalent to coffee roasting.

[0428] Materials and methods

[0429] The BND peptide used and the relaxation index evaluation were as described in Example 1.

[0430] The BND peptide was injected into raw (unroasted) coffee beans by soaking the beans in a 1 mg / ml BND solution dissolved in normal saline for 12 hours.

[0431] Then, in separate trials, the raw bean samples injected with BND were roasted in a forced convection oven at 160 °C and 230 °C for 14 minutes. The pre - roasted and post - injected beans served as controls.

[0432] Then, the roasted (BND - injected) test beans and the control beans were ground and used separately to make coffee.

[0433] The subjects drank 200 ml of each test coffee (made at each roasting temperature) and the control bean coffee after 5 minutes, and then the relaxation index was evaluated.

[0434] Results

[0435] The results are as Figure 4 shown.

[0436] As shown, coffee made from beans infused with BND that had undergone two roasting temperatures unexpectedly demonstrated the effect of BND on relaxing the subjects. This effect was similar to that shown by coffee made from pre-roasted beans infused with BND.

[0437] This indicates that if BND can be recombinantly expressed in a biomaterial (such as a plant material) in the correct form, then the biomaterial can be processed under relatively harsh processing conditions to produce a food or beverage composition in which the shown BND bioactivity is retained.

[0438] Example 5 - BND in an alcoholic beverage

[0439] Materials and methods

[0440] The BND used and the relaxation index assessment were as described in Example 1.

[0441] At 20 °C, 10 mg of BND was infused into 200 ml of Stella Artois beer for 5 minutes and then consumed by the subjects, followed by relaxation index assessment.

[0442] On different days, a control beer (without BND) was tested under the same conditions.

[0443] Results

[0444] The results were as Figure 5 shown.

[0445] The data showed that the relaxation effect when BND was combined with beer was greater than that shown by beer alone, lasting up to 4 hours after ingestion.

[0446] These data indicate that the alcohol content in the beer and / or the beer did not have an adverse effect on the efficacy of BND to induce a relaxed state.

[0447] Furthermore, in the subjects, the relaxation effect of beer and BND together was more pronounced than that of beer alone.

[0448] Example 6 - Expression of BND in plants

[0449] Background

[0450] To the applicant's knowledge, BND has never been expressed in plants, let alone expressed in an active form or at a commercially meaningful level.

[0451] It is alleged that the N-terminus of BND is important for activity. Therefore, in order to produce active BND in plants, it may be important or essential to express a peptide with the correct N-terminal sequence. The sequence of the mature peptide fragment (31 residues) is highly conserved as YGGFMTSEKSQTPLVTLFKNAIIKNAYKKGE (SEQ ID NO:2).

[0452] Although proteins, polypeptides and even peptides have been expressed in plants, the production levels of any peptide are unpredictable. Signal peptides have been used to target proteins, polypeptides and peptides to subcellular organelles in order to increase accumulation, but this is also unpredictable.

[0453] The cleavage of the targeting signal sequence is also unpredictable and may be affected by factors such as sequence differences (hydrophobicity, charge, size, etc.) between the downstream peptide and the signal sequence. There are various software applications for predicting where the cleavage will occur, but these are far from guaranteeing a correct prediction.

[0454] Therefore, some unpredictable challenges need to be overcome before producing a reasonable level of BND in plants in an active form / correctly cleaved manner.

[0455] Materials and Methods

[0456] Construct design

[0457] In the model plant Nicotiana benthamiana, constructs were generated for the expression and targeting of BND to the endoplasmic reticulum (ER) and chloroplasts, with signal peptide sequences (discussed further below). A C-terminal V5-His tag was also included for detection / quantification and / or purification.

[0458] Expression Cassette Features

[0459] Both cassettes were designed to use the same GATEWAY TM recombinant directional cloning steps to place them in a binary vector, in which the BND fusion peptide is controlled by the CaMV35s promoter (constitutive) and the NOS terminator. Both cassettes were optimized for expression in Nicotiana benthamiana; this includes introns with appropriate predicted splicing sites and efficiencies; Kozak sequences, removal of the polyadenylation signal sequence, removal of mRNA instability sequences, double stop codons and tetranucleotides (Scott et al, 2010, Plant Biotechnology Journal. 8:912-927). Those skilled in the art should understand that these optimizations are not essential but may be beneficial. The sequences used in the constructs are shown in the sequence listing discussed below.

[0460] Endoplasmic reticulum (ER) targeting of a single BND

[0461] The Arabidopsis thaliana purple acid phosphatase signal peptide sequence (SEQ ID NO:7) is used to target the BND to the endoplasmic reticulum. The expression cassette is based on Winichayakul et al (2009).

[0462] The total peptide sequence to be expressed (SEQ ID NO:25) includes:

[0463] · ER targeting signal peptide (SEQ ID NO:7)

[0464] · Flexible linker (SEQ ID NO:18)

[0465] · Enterokinase cleavage site (SEQ ID NO:13)

[0466] · BND peptide (SEQ ID NO:2)

[0467] · Thrombin cleavage site (SEQ ID NO:16)

[0468] · V-5His tag (SEQ ID NO:20)

[0469] · ER retention sequence (SEQ ID NO:22)

[0470] 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 as Figure 6 shown and indicate that the signal peptide will be cleaved to leave the authentic N-terminal sequence of the BND peptide.

[0471] The peptide coding sequence targeting the ER was optimized for tobacco, created and custom synthesized by GenScript. The total GATEWAY TM flanked BND-V5-His (with intron) nucleic acid sequence targeting the ER is shown in SEQ ID NO:26.

[0472] After subcloning using Geneious Prime GATEWAY, the sequence (SEQ ID NO:32) determined from the TATATAA box of the CaMV35S promoter to the double stop codon (TAATGA) was subjected to splicing prediction by NetGene2( Figure 7 ). These results show that the intron is predicted to be correctly spliced.

[0473] Chloroplast targeting of a single BND

[0474] The truncated Nicotiana tabacum NtRBCs rubisco signal peptide (SEQ ID NO:31) is used to target BND to the chloroplast (Eseverrie et al., 2020). The chloroplast targeting cassette is shown in SEQ ID NO:27.

[0475] The total peptide sequence to be expressed (SEQ ID NO:27) includes:

[0476] · Chloroplast targeting signal peptide (SEQ ID NO:31)

[0477] · Flexible linker (SEQ ID NO:18)

[0478] · Enterokinase cleavage site (SEQ ID NO:13)

[0479] · BND peptide (SEQ ID NO:2)

[0480] · Thrombin cleavage site (SEQ ID NO:16)

[0481] · V-5His tag (SEQ ID NO:20)

[0482] The total peptide sequence encoded by the chloroplast targeting cassette (theoretically 13.47 kDa) was run through the signal prediction software TargetP. The results are as Figure 8 shown, indicating that the signal peptide will be cleaved to leave the authentic N-terminal sequence of the BND peptide.

[0483] The peptide coding sequence targeting the chloroplast was optimized for expression in Nicotiana benthamiana to create the sequence shown in SEQ ID NO:33.

[0484] The attL1, 5’UTR, AtDGAT1 intron 3, double stop codons, and attL2 sequence (NB changed from A to T to eliminate the mRNA instability sequence, which changes the CCA codon to CCT, both encoding proline) were added to the optimized chloroplast targeting sequence, as shown in SEQ ID NO:34.

[0485] After subcloning using Geneious Prime GATEWAY, the sequence from the TATATAA box of the CaMV35S promoter to the (TAATGA) double stop codons is shown in SEQ ID NO:35. The sequence was analyzed by NetGene2 for splicing prediction ( Figure 9 ).

[0486] Tandem repeat BND construct

[0487] Constructs were also prepared to express the BND as a tandem array of peptide sequences separated by a flexible linker, as shown in SEQ ID NO 36. The sequence having an enterokinase cleavage site, tandem repeats of BND, a thrombin cleavage site, and engineered cloning restriction sites is shown in SEQ ID NO 37.

[0488] The nucleotide sequences were optimized for expression in Nicotiana benthamiana; this included introns with appropriate predicted splice sites and efficiencies; Kozak sequences, removal of polyadenylation signal sequences, and removal of mRNA instability sequences. The nucleic acid and peptide sequences of the tandem repeat fragments are shown in the sequence listing.

[0489] Subsequently, the tandem repeat BND cassette was subcloned into the above ER-targeting cassette and chloroplast-targeting cassette.

[0490] The peptide sequence of the ER-targeting tandem repeat BND (SEQ ID NO:29) (predicted to be 16.08 kDa) was run through the signal sequence recognition software TargetP ( Figure 10 )

[0491] For the chloroplast-targeting tandem repeat BND expression cassette, a modified version of the chloroplast transit peptide (SEQ ID NO:39) was used.

[0492] The peptide sequence of the chloroplast-targeting tandem repeat BND expression cassette (predicted to be 22.35 kDa) (SEQ ID NO:38) was also run through the TargetP software ( Figure 11 )

[0493] Cloning and transformation

[0494] Cloning of plant organelle-targeted BND

[0495] The designed ER-targeting BND cassette and chloroplast-targeting BND cassette were cloned into the binary vector pRSh1 (Scott et al., 2010) by Gateway TM LR Clonase TM reactions, respectively. The conformation of the plasmid constructs was confirmed by restriction enzyme mapping and sequencing.

[0496] Cloning of plant organelle-targeted tandem repeat BND

[0497] The tandem 2× repeat BND fragment was cloned into the 056488pPCR Script-ER targeting cassette and 056486pPCR Script-CHL targeting cassette. The plasmid constructs 056488-ER-2xEdph and 056486-CHL-2xEdph were screened and confirmed by restriction enzyme mapping. By Gateway TMLR Clonase TM The reaction cloned ER-targeted 2xEdph and chloroplast-targeted 2xEdph into the binary vector pRSh1 (Scott et al., 2010) respectively. The conformation of the plasmid construct was confirmed by restriction enzyme mapping and sequencing.

[0498] The binary vector containing the BND expression cassette was transformed into Agrobacterium cells.

[0499] The plasmid DNAs of pRSh1-ER-Edph (single BND targeting ER), pRSh1-CHL-Edph (single BND targeting chloroplast), pRSh1-ER-2xEdph (tandem BND targeting ER), and pRSh1-CHL-2xEdph (tandem BND targeting chloroplast) were transformed into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method and selected on a medium containing the appropriate antibiotics. Agrobacterium tumefaciens cells containing the plasmid DNA were selected and confirmed by PCR using pRSh1 forward and reverse primers.

[0500] Transient expression of the BND gene cassette in tobacco by Agrobacterium-mediated

[0501] Agrobacterium cells containing the BND expression cassette were infiltrated into the leaves of tobacco (Nicotiana benthamiana). Leaf samples were harvested, soluble proteins were extracted, and BND expression was analyzed by SDS-PAGE immunoblotting ( Figure 12 ) and ELISA ( Figure 13 ).

[0502] Results

[0503] Immunoblotting

[0504] The immunoblotting results of ER-targeted BND (ER-Edph) and tandem repeat BND (ER-2xEdph) showed that they both accumulated to detectable levels ( Figure 12 ). However, no peptides were observed for chloroplast-targeted BND (CHL-Edph), and very low amounts of peptides were observed for chloroplast-targeted tandem repeat BND (CHL-2xEdph), which could not be predicted by those skilled in the art. No immunoblotting signal was observed when transiently expressing Nicotiana benthamiana using Agrobacterium cells containing the vector control (VC).

[0505] ELISA

[0506] Enzyme-linked immunosorbent assay (ELISA) is a technique used for quantification and generally has a broader dynamic range than gel scanning. Optionally, BND with a V5::6xHis tag contained in the total soluble protein extract matrix of leaves was absorbed onto nickel-coated plates (Pierce TM ), and then immunologically detected with an anti-V5 antibody ( Figure 13 ). ELISA results also confirmed the accumulation of ER-targeted BND (ER-Edph). No color development was observed for the empty (no protein coating), vector control (VC), and chloroplast-targeted BND (CHL-Edph) soluble protein extracts. The reaction was incubated dynamically at 37 °C for 10 min, and the change in optical density at 405 nm was recorded.

[0507] Transient expression of the chloroplast-targeted BND cassette was detected in Nicotiana benthamiana leaves only after using an alternative extraction buffer

[0508] The lack of recombinant protein in the chloroplast-targeted cassette was unexpected; to see if this might be due to low extraction efficiency (chloroplasts are small discrete organelles with multiple membranes), total protein extraction was repeated using different buffers. It contained 1 M urea and 1% triton x-100 (solubilized bilayer membrane proteins).

[0509] Immunoblotting showed relatively strong accumulation of the chloroplast-targeted tandem repeat BND (CHL-2xEdph), but not of the chloroplast-targeted single BND (CHL-Edph) peptide ( Figure 14 ). The reason for the lack of accumulation observed in chloroplasts may be due to the tandem repeat arrangement (compared to a single peptide). However, unpublished results suggest that this is more likely due to the use of different chloroplast-targeting sequences in the two constructs. The tandem construct used a previously tested targeting sequence (Winichayakul et al 2009), while the monomeric cassette used a recently published shorter version (Eseverri et al 2020).

[0510] The immunoblot band pattern indicates that the peptides targeted to the ER and chloroplasts were correctly processed in terms of signal cleavage

[0511] The predicted size of the uncleaved ER-Edph peptide is 11.9 kDa, while the predicted size of the signal-cleaved peptide is 8.7 kDa. Thus, it seems that most of the detected ER-Edph peptides were appropriately cleaved ( Figure 12 , small panel A and Figure 14 ). The relatively darker higher band may be a miscleaved peptide or dimerization of the cleaved peptide; considering the size difference suggests it is more likely to be a dimer.

[0512] The predicted size of the uncleaved ER-2xEdph peptide is 16.1 kDa, and the signal-cleaved version is 12.9 kDa; however, immunoblotting ( Figure 12 panel B) shows a band pattern very similar to that of the single ER-Edph peptide ( Figure 12 panel A). Cleavage of the ER signal sequence and the first BND could explain the band pattern, but this is unlikely given the absence of specific peptidase sites between the BND repeats. Given the relatively small size of the peptides, they are likely to be cleaved and subsequently dimerized, and in this particular 4%-15% gradient acrylamide gel, they migrate faster than expected. This can be confirmed by running ER-Edph and ER-2xEdph extracts side by side on the same gel and / or using a higher%-polyacrylamide gel with a different running buffer (such as Tris-tricine) to resolve smaller protein sizes (<10 kDa).

[0513] The predicted size of the uncleaved CHL-2XEdph is 22 kDa, and the cleaved version is 12.6 kDa (or 18.5 kDa if cleaved at the repeat transit peptide cleavage site). Figure 14 The immunoblot in [reference] indicates the presence of the 12.6 kDa version, indicating that the first transit peptide cleavage site is recognized. The larger immunoreactive band may be caused by miscleaved peptides or dimerization of the cleaved peptides.

[0514] Quantification of ER-targeted BNDs from crude extracts

[0515] The amount of BND expressed in the transient leaf expression system was determined as follows. Leaf extracts of ER-targeted BND (ER-Edph) and tandem repeat BND (ER-2xEdph) were immunoblotted in the same gel with 2 ng, 5 ng, 10 ng, 20 ng, and 40 ng of PEAPOD-V5 fusion protein standards (prepared in another project by expression in bacterial cells and purification by affinity gel binding, Figure 15 panel A). The band intensities of the standards were scanned by Image Lab 5.2.1 software and plotted as a standard curve ( Figure 15 panel B). From the curve, we could calculate the average accumulation amount (μg / g FW) of BND in Nicotiana benthamiana leaves ( Figure 15 panel C).

[0516] The total recombinant protein level and the level of accumulated recombinant BND were converted to % total soluble protein, as shown in Table 6.

[0517] Table 6. Quantification of accumulated unpurified recombinant proteins.

[0518] Extract ER-Edph ER-2xEdph Total soluble protein (TSP), (mg) 24 1.33 Average recombinant protein produced (μg) 24.32 2.6 Average recombinant protein produced (% TSP) 0.1 0.2 Average BND produced (% TSP) 0.03 0.08

[0519] Purification and Re - quantification of ER - targeted BND

[0520] Although the recombinant protein appears to accumulate to relatively low levels in the transient expression system, it can be purified and concentrated to produce sufficient amounts for further studies, such as cleavage mass spectrometry conformation and appropriate sequence, bioassays, etc. In this proof - of - concept study, we added a C - terminal V5::His tag to the BND fragment to assist in purification and concentration.

[0521] Leaves (15.87 g FW) transiently expressing ER - targeted BND were harvested 72 h after Agrobacterium infiltration and homogenized in 10 mL of ice - cold 2.5X extraction buffer containing 50 mM sodium phosphate buffer pH 7.4, 2.5 M NaCl, and 2.5% Triton X - 100. The crude extract was centrifuged at 10000×g for 5 min at 4 °C to remove leaf debris, protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was added to a final concentration of 1 mM, and the final volume of the soluble extract was adjusted to 25 mL with cold sterile milliQ H2O.

[0522] Initially, after adding imidazole (5 - 15 mM), the soluble extract precipitated, and most of the histidine - tagged material did not bind to the Ni2+ column. This may be due to unknown compounds in the Nicotiana benthamiana leaf extract. Subsequently, the pre - diluted crude extract was passed through a 3 kDa cut - off filter (to retain the recombinant peptide), then imidazole was added and the extract was loaded onto the column ( Figure 16 ).

[0523] In Figure 16 , immunoblotting showed that the C - terminal tagged ER - targeted BND was present in the soluble extract (lane 2) and the filtered extract (lane 4). After loading the filtered extract onto the column, the peptide was eluted with a series of imidazole concentrations (80 - 400 mM). The disappearance of the monomer (smaller band ∼8.7 kDa) and the appearance of a larger band (previously hypothesized to be a dimer or uncleaved signal BND) were observed. This may be due to the concentration of the recombinant protein using a 3 kDa cut - off filter and an increase in the degree of oligomerization.

[0524] In eluate 1, eluate 2, and eluate 3, we could not detect the protein on the unstained gel; therefore, these eluates were combined and concentrated (Supplementary Protocol 2). Subsequently, the protein samples were analyzed by immunoblotting, as Figure 17 shown (lower panel, lane 14 below).

[0525] Immunoblotting showed that the concentrated soluble eluate contained ER-targeted BND, although it is currently present as dimers and larger oligomers (Figure 27, lane 14). From this, we were able to recalculate the recombinant protein levels in the total soluble leaf protein extract, which showed that the recombinant BND levels (as a percentage of total soluble leaf protein) were significantly higher than the levels determined initially (Table 7 compared to Table 6).

[0526] Table 7. Quantification after purification of recombinant proteins targeted to the ER.

[0527] Transient expression leaf (g fresh weight) 15.87 Soluble protein concentration in filtered extract (mg / mL) 9.97 Total soluble protein (g) 0.25 Purified ER-endorphin-V5::6xHis (mg) 5.13 Yield (total soluble protein %) 2.06

[0528] Conclusions and further experiments

[0529] The present applicant has demonstrated for the first time that mature BND peptides can be recombinantly synthesized and accumulated in plants. This was achieved by targeting the peptide (via a cleavable signal sequence) to the endoplasmic reticulum (ER) or chloroplasts. In the latter case, a truncated version of the chloroplast transit peptide sequence did not result in detectable BND accumulation. Immunoblot analysis showed that the ER-targeting signal sequence was efficiently cleaved, while the non-truncated chloroplast transit peptide appeared to be removed in approximately 50% of the cases.

[0530] Purification of the recombinant protein (by size exclusion and affinity chromatography) enabled more accurate quantification of the accumulated BND levels; when targeted to the ER, this reached approximately 2% of the total soluble protein. The ability to purify and concentrate BND should allow for further characterization of the peptide.

[0531] The first step could involve further determining the efficiency and accuracy of signal cleavage. This could be achieved by excising gel slices from appropriate migration points and subjecting them to trypsin digestion followed by mass spectrometry. Similarly, the efficiency and accuracy of β-enterokinase cleavage immediately upstream of the mature BND peptide could be achieved by initially treating the gel slices with enterokinase prior to trypsin digestion; followed by mass spectrometry. These analyses would also allow examination of the C-terminus to confirm the exact sequence.

[0532] The N-terminal targeting sequence of the fragment targeted to chloroplasts could be efficiently cleaved, leaving the correct N-terminal residue of the mature BND peptide. The use of a β-enterokinase site could facilitate this. The advantage of chloroplast targeting is that it may produce peptides with the correct C-terminus, as no additional sequences are retained in chloroplasts (unlike the ER).

[0533] Example 7 - Further confirmation of the N-terminus and activity of recombinantly expressed BDN

[0534] The current recombinant peptides could be subjected to mass spectrometry, and in the future, commercially available antibodies against the mature peptide could facilitate the handling of the mature peptide alone (without tags).

[0535] For example, mass spectrometry can be used to confirm the sequence of recombinant peptides (with or without enterokinase treatment) from tandem endorphin repeat cassettes targeted to chloroplasts. This can show between which residues the chloroplast targeting peptide is cleaved and the percentage of the cleaved peptide. Similarly, this can demonstrate that enterokinase cleaves at the appropriate position, the percentage of the cleaved peptide, and that the cleavage leaves the expected N-terminal residues of β-endorphin. The synthetic BND described in Example 1 can be used as a standard for mass spectrometry analysis.

[0536] Optionally, without using anti-BND antibodies, it may also be possible to purify and quantify the recombinant mature peptide. Optionally, a V5::His tag can be placed between the chloroplast signal sequence and the β-enterokinase site upstream of the BND peptide. This will allow purification and concentration from the chloroplast, as well as the ability to remove the N-terminal signal and tag.

[0537] If mass spectrometry analysis shows that the sequence and enterokinase cleavage are correct, an expression cassette containing the following can be generated:

[0538] Chloroplast transit peptide (from Winichayakul et al 2009)::Internal His Tag::Enterokinase cleavage site::Single BND peptide.

[0539] For example, the amino acid sequence of such an expression cassette is shown in SEQ ID NO:40.

[0540] The total peptide sequence to be expressed (SEQ ID NO:40) includes:

[0541] · Chloroplast targeting signal peptide (SEQ ID NO:31)

[0542] · Flexible linker (SEQ ID NO:18)

[0543] · V-5His tag (SEQ ID NO:20)

[0544] · Flexible linker (SEQ ID NO:18)

[0545] · Enterokinase cleavage site (SEQ ID NO:13)

[0546] · BND peptide (SEQ ID NO:1)

[0547] For example, the polynucleotide sequence encoding such a cassette is shown in SEQ ID NO:41.

[0548] By standard procedures, this cassette can be ligated into an expression cassette with a CaMV35S promoter and terminator to generate the sequence shown in SEQ ID NO 42.

[0549] The entire expression cassette can be used to transform Agrobacterium by standard procedures and / or as described above, and then introduced into plants by transient or stable expression as described herein.

[0550] Further alternative expression cassettes can be generated that contain the following:

[0551] Chloroplast transit peptide (from Winichayakul et al 2009)::Single BND peptide::Enterokinase cleavage site::His tag.

[0552] For example, the amino acid sequence of such an expression cassette is shown in SEQ ID NO:51.

[0553] The total peptide sequence to be expressed (SEQ ID NO:51) includes:

[0554] · Chloroplast targeting signal peptide (SEQ ID NO:31)

[0555] · BND peptide (SEQ ID NO:1)

[0556] · Enterokinase cleavage site (SEQ ID NO:13)

[0557] · Flexible linker (SEQ ID NO:18)

[0558] · V-5His tag (SEQ ID NO:20)

[0559] For example, the polynucleotide sequence encoding such a cassette is shown in SEQ ID NO:52.

[0560] By the standard procedures discussed above, this cassette can be ligated into an expression cassette with a CaMV35S promoter and terminator.

[0561] The entire expression cassette can be used to transform Agrobacterium by standard procedures and / or as described above, and then introduced into plants by transient or stable expression as described herein.

[0562] The polynucleotide coding sequence can be codon optimized to suit the species to be transformed.

[0563] After generating and purifying the recombinantly expressed BND peptide, a bioassay can be performed to determine the activity of the recombinant BND compared to the chemically synthesized peptide (see, for example, Examples 1-5 herein).

[0564] Example 8 - Stable Transformation of Plants to Recombinantly Express Active BND

[0565] The expression cassettes described in Examples 6 and 7 can be cloned into a suitable vector for stable (and transient) transformation of plants.

[0566] For example, the expression cassettes described in Examples 6 and 7 can be cloned into pRSh1 (Scott et al 2010), replacing the constitutive promoter cauliflower mosaic virus 35S (CaMV35Sp)-driven adaptive expression cassette to create a binary vector, or cloned into pBR2 from pDONR 221 by TM LR cloning (Thermo Fisher Scientific).

[0567] Alfalfa

[0568] Alfalfa can be stably transformed, for example, as described in Wand et al., 2016, Protein Pept Lett., 23(5):495-502

[0569] Rice

[0570] Rice can be stably transformed, for example, as described in Alam et al., 1999, Plant Cell Rep. 18, 572

[0571] Wheat

[0572] Wheat can be stably transformed, for example, as described in Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877

[0573] Barley

[0574] Barley can be stably transformed, for example, as described in Lazzeri, P. 1995, Methods Mol Biol:49:95-106

[0575] Maize

[0576] Maize can be stably transformed, for example, as described in U.S. Patent Serial No. 5,177,010 and U.S. Patent Serial No. 5,981,840

[0577] Tobacco

[0578] Tobacco can be stably transformed, for example, as described in Horsch et al., 1985, Science 227, 1229

[0579] Coffee

[0580] It can stably transform coffee, for example, as described in Ribas et al., BMC Plant Biol. 2011; 11:92.

[0581] Tea

[0582] It can stably transform tea, for example, as described in Chen et al., 2022 Front. Plant Sci., Sec. Plant Systematics and Evolution Volume 13.

[0583] Cocoa

[0584] It can stably transform cocoa, for example, as described in Sain SL et al., 1994, Plant Cell, Tissue and Organ Culture volume 37, pages 243 - 251 and Maximova et al., 2003, Plant Cell Rep 21, 872 - 883.

[0585] Other plant species can be stably transformed as described herein and by other protocols known in the art.

[0586] Example 9 - Analysis of BND Expressed by Stable Transformation

[0587] The recombinantly expressed BND produced by stable transformation (and by transient expression) can be purified and analyzed as described in Examples 6 and 7 above.

[0588] Example 10 - Further Confirmation of the Activity of Synthetic and Recombinantly Expressed BND by Animal Experiments.

[0589] In Examples 1 - 5 above, the Applicant unexpectedly showed for the first time that, contrary to the expectations of the prior art discussed in the background art section above, the native, unprotected BND peptide can exert a biological effect by oral ingestion and can thus be used to manage relaxation by oral ingestion.

[0590] To further confirm and explore the biological activity of BND (which can be produced synthetically or recombinantly), larger - scale experiments can be conducted in which BND is ingested by rats. Such experiments can be carried out, for example, at The Howard Florey Institute of Neuroscience & Mental Health (Floor 2, 161 Barry Street, Carlton, Victoria 3053, Australia) according to the following protocol.

[0591] AIM

[0592] The purpose of this experiment is to test the relaxation effect of orally administering human beta-endorphin (BND) to a group of Sprague Dawley rats in a six-week double-blind controlled trial. This study can be used to test the effects of the synthetic and recombinantly produced BND described herein and to confirm the biological activity of the recombinantly produced BND.

[0593] The purpose of this study is to test whether the orally administered BND group shows significant signs of enhanced relaxation compared to the control group.

[0594] Group compositions

[0595] Each of the four treatment methods will consist of 15 male (or female) Sprague Dawley rats randomly and blindly assigned, 4 rats per group, housed in different areas of the cage.

[0596] Experiment

[0597] The groups are as follows:

[0598] 1. Sprague Dawley rats, n = 15, orally administered carrier treatment, no restraint (to control the stress effect)

[0599] 2. Sprague Dawley rats, n = 15, orally administered test recombinant compound treatment, no restraint (to control the effect of the compound)

[0600] 3. Sprague Dawley rats, n = 15, orally administered carrier treatment, restraint (stress effect)

[0601] 4. Sprague Dawley rats, n = 15, orally administered synthetic test compound treatment, restraint (the effect of the compound on stress)

[0602] In an alternative experiment, the rats in each treatment can be divided into 3 groups for repeated experiments. Optionally, all male or all female rats can be used.

[0603] End of experiment

[0604] At six weeks, all groups will be sacrificed.

[0605] Organ autopsy studies and cardiac punctures will be performed, and specimens for hematological and biochemical studies will be collected for research.

[0606] Cortisol test

[0607] Serum cortisol levels will be measured in all subjects at zero time, 2 weeks, 4 weeks, and at the time of sacrifice at 6 weeks.

[0608] Collect blood (0.5 ml) by tail vein puncture. Rodents will be carefully helped to familiarize with this procedure, and a local anesthetic will be applied at the tail vein puncture site to minimize trauma.

[0609] BND dosage regimen

[0610] Daily dose equivalent (by body weight ratio), equivalent to a 20 mg dose for a 70 kg human.

[0611] The dose of BND produced synthetically and recombinantly as described in the above examples will be used.

[0612] Optionally: Dose equivalent (by weight ratio), equivalent to a 20 mg dose for a 70 kg human. The dose of BND produced synthetically and recombinantly as described in the above examples can be used during weeks 1 - 2 and then during week 5 to test stress reduction at multiple stages.

[0613] Stressor mechanism

[0614] Restraint stress for 2 hours per day can be used for 3 weeks. The restraint time can be rotated so that the animals have different restraint times on consecutive days.

[0615] After completion of the 3 - week restraint or non - restraint period, all rats will be tested by the following 4 tests (these tests will be run for a further 3 weeks):

[0616] Elevated plus - maze (anxiety test)

[0617] The elevated plus - maze is custom - made of light - colored Perspex and consists of two open arms (10 cm × 44 cm for rats) and two closed arms (10 cm × 44 cm × 10 cm for rats), extending from a central platform (12 × 12 cm for rats). It is mounted on a base 40 cm above the ground. This is a test of rodent anxiety as they prefer the safe closed arms and are cautious when entering the open arms. Rodents face a conflict between their preference for new spaces, the opportunities presented by new spaces, and the potential danger of unprotected areas.

[0618] At the start of the experiment, the rodents will be placed in the center of the maze, facing the open arms, and the following variables will be tracked using the Cleversys Topscan tracking system: the time and number of entries into the open and closed arms. The time spent in the open and closed arms is expressed as a percentage of the total time in the maze. The number of entries into the open and closed arms is defined as the number of times all four paws of the rodent enter one arm of the maze. Indoor lighting is approximately 10 - 20 lux. The 10 - minute trial is only tested once.

[0619] Locomotor Test (Motor Function Test)

[0620] The rodents are removed from their home cages and placed in the middle of the Med Associates locomotor chamber. The system monitors a series of predefined parameters, including but not limited to distance traveled, movement time, number of movements, and rearing time. The system can also be used to evaluate general locomotor activity typically tested within 60 - 90 minutes. General locomotor activity measured using the aforementioned parameters can provide insights into general phenotypes caused by gene manipulation or drug administration.

[0621] Light / Dark Test (Anxiety Test)

[0622] This test can also be automated using the Med Associates locomotor system, where a black Perspex box is inserted into half of the chamber. This provides a light and a dark area. The black Perspex box has small openings to allow the rats to move from the dark area to the open light area (450 lux). At the start of the test, the rats are placed in the dark area and allowed to explore both areas during a 10 - minute trial. The software program will record the number of transitions from light to dark, the time spent in each area, and the latency time from the dark area to the light area. Rats that spend more time in the dark area than in the light area compared to control rats may exhibit higher levels of basal anxiety - like behavior.[[ID=IO]]

[0623] Large Open Field Test (Anxiety Test)

[0624] The rodents are removed from their home cages and then placed in the center of a square arena (110 cm × 110 cm). The rodents are exposed to a brightly lit open field. Their movement trajectories are tracked using automated tracking software (Cleversys Topscan). The trial lasts for 10 minutes, and then the rodents are placed back into the transport box and returned to their home cages. The large, well - lit area (450 lux) poses a threat to rodents that prefer darker environments and smaller spaces. For rats, this again presents a conflict between the potential danger of the large area (i.e., aerial predators) and the opportunity to find new food sources / mates in a new space. As a result, this elicits various responses, which may include high levels of exploration, crossing the central part of the arena, or remaining immobile and occupying the sides and corners of the test arena. Our CleverSys rodent tracking software captures and analyzes rats that spend more time in the peripheral and corner areas of the arena and considers these rats to have higher levels of anxiety - like behavior.

[0625] [[ID=IS]]The results of these tests will contribute to a better understanding of the behavioral manifestations of restraint and the ability of the test compounds to act as viable anxiolytics.

[0626] References

[0627] Baulcombe D. (2004) RNA silencing in plants. Nature 431: 356 - 363.

[0628] 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.

[0629] 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.

[0630] Mazur BJ, Chui CF(1985) Sequence of a genomic DNA clone for the small subunit of ribulose bis - phosphate carboxylase - oxygenase from tobacco. Nucleic Acids Research, 13:2373 - 2386. Scott RW, Winichayakul S, Roldan M, Cookson R, Willingham M, Castle M, Pueschel R, Peng C, Tzen JTC, Roberts NJ. (2010) Elevation of oil body integrity and emulsion stability by polyoleosins, multiple oleosin units joined in tandem head - to - tail fusions. Plant Biotechnology Journal, 8(8):912 - 927.

[0631] Skala W, Goettig P, Brandstetter H. (2013) Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox. Journal of Biotechnology, 168(4): 421-425. Terpe K. (2003) Overview of tag-protein fusions: from molecular and biochemical fundamentals to commercial systems. Applied Microbiology and Biotechnology, 60: 523-533. Winichayakul S, Pernthaner A, Scott R, Vlaming R, Roberts N. (2009) Head-to-tail fusions of camelid antibodies can be expressed in planta and bind in rumen fluid. Biotechnology and Applied Biochemistry, 53(2): 111-122.

[0632] Sequence summary

[0633]

[0634]

[0635]

[0636]

Claims

1. A bioactive food or beverage composition or ingredient comprising a bioactive form of a β-endorphin (BND) peptide.

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

2.

4. The bioactive food or beverage composition or ingredient according to any one of the preceding claims, wherein the BND peptide is not enteric-coated.

5. The bioactive food or beverage composition or ingredient according to any one of the preceding claims, wherein the BND peptide is recombinantly produced.

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

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

8. The bioactive food or beverage composition or ingredient according to claim 6, comprising the cell, tissue, organism or part thereof, wherein the BND is recombinantly produced.

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

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

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

16. The food or beverage composition according to any one of the preceding claims, which is: a) A hot drink selected from coffee, tea or cocoa / hot chocolate beverages, b) Ingredients for making said hot drink, c) Ingredients used as additives for said hot drink.

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

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

19. The food or beverage composition according to claim 18, wherein the coffee beans have been roasted and wherein the biological activity of the BND peptide is retained after roasting of the coffee beans.

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

21. The food or beverage composition according to any one of the preceding claims, which is in liquid form and wherein the biological activity of the BND is retained after heating to at least 50 °C.

22. An expression cassette encoding a polypeptide cassette, comprising: a. At least one of the following: i. An ER targeting signal peptide, and ii. A chloroplast targeting signal peptide, and b. A BND peptide 23. A plant cell, plant tissue or plant that has been genetically modified to express a biologically active form of the BND peptide.

24. The plant cell, plant tissue or plant according to claim 23, which comprises the expression cassette of claim 22, or a polypeptide cassette encoded by said expression cassette.

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

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

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

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

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

31. The plant part according to any one of the preceding claims, which is selected from coffee beans, tea leaves, and cocoa beans.

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

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

2.

34. The method according to any one of the preceding claims, wherein the BND peptide is not enteric-coated.

35. The method according to any one of the preceding claims, wherein the BND peptide has the authentic N-terminus of a naturally occurring BND.

36. The method according to any one of the preceding claims, which comprises providing a cell, tissue, organism, or a part thereof comprising the BND peptide.

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

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

39. The method according to any one of the preceding claims, wherein the food or beverage composition of a) is selected from: a) a hot drink selected from coffee, tea, or cocoa / hot chocolate drink, b) an ingredient for making the hot drink, and c) an ingredient used as an additive to the hot drink 40. The method according to any one of the preceding claims, wherein the plant cell, plant tissue, plant, or a part thereof in the food or beverage composition is a plant cell, plant tissue, plant, or a part thereof of a coffee plant, tea plant, or cocoa plant.

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

42. The method according to any one of the preceding claims, wherein the coffee beans have been roasted, and wherein the bioactivity of the BND peptide is retained after roasting of the coffee beans.

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

44. The method according to any one of the preceding claims, wherein the food or beverage composition of any one of the preceding claims is in liquid form, and wherein the bioactivity of the BND is retained after heating to at least 50 °C.

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

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

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

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 of the preceding claims, a food or beverage composition produced by the method of any one of the preceding claims, or a food or beverage composition extracted from the cells, plant cells, tissues, plant tissues, organisms or plants of any one of the preceding claims.

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

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

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