3.1. Protein Characteristics
Canaryseeds have been compared extensively with wheat and other cereals in the same family, and one of their distinguishing factors is their higher protein content (Table 1
), which ranges between 20–23%, in comparison to 13% for wheat. Canaryseed proteins, along with other cereal proteins, can be separated into four fractions based on their solubility: prolamins, glutelins, globulins and albumins [11
]. The prolamin and glutelin fractions, which are principally storage proteins, are more abundant in canaryseeds than wheat, however, the globulin and albumin fractions represent the lowest amount of overall protein [3
], which is possibly indicative of a reduced amount of anti-nutritional factors, such as enzyme inhibitors [4
]. Regardless of the variations in protein fraction proportions, wheat remains unique because of its ability to make dough, due to the exceptional viscoelastic properties of its proteins [11
]. Nonetheless, to date, no published data is available on the breadmaking potential of 100% canary flour, although Abdel-Aal, et al. [12
] reported that replacement of up to 25% of wheat flour with canaryseed flour in bread had no significant effects on bread quality and loaf volume, except for crumb color.
A key trait of canaryseeds is their possible lack of gluten-like proteins, which elicit an allergic reaction known as coeliac disease in some sensitive individuals when they consume gluten-containing cereals, such as wheat, barley, and rye [18
]. Gluten is a complex mixture of proteins called prolamins, which play key roles in conveying dough viscosity/elasticity. Wheat prolamins are termed gliadins and glutenins, barley prolamins are hordeins, and those from rye secalin. A common characteristic of these proteins is the presence of multiple proline and glutamine residues, making them resistant to gastrointestinal digestion and more exposed to deamination by tissue transglutaminase [20
]. In a recent study conducted by Boye, et al. [21
] to establish the safety of canaryseeds for human consumption from a food allergy perspective, glabrous canaryseeds were analyzed using three separate techniques (enzyme-linked immunosorbent assay (ELISA), mass spectroscopy, and Western blotting) which all yielded negative results for gluten, indicating the cereal is an excellent alternative for individuals with coeliac disease. Although canaryseeds do not contain gluten and may be represented as gluten-free, canaryseeds do however contain a newly reported allergen named granule-bound starch synthase (GBSS), which is present in rice and maize [22
], and which cross-reacted with sera from wheat sensitive/allergic individuals [21
]. GBSS was simultaneously identified through mass spectroscopy analysis in several cereals (wheat, oat, sorghum, millet, teff, quinoa, buckwheat) [21
]. As such, Health Canada has deemed it inappropriate for canaryseed, or food containing canaryseed, to be labelled as “wheat-free”. Health Canada also requires canaryseed and foods containing canaryseed to be labelled with a statement to the effect that the product “may not be suitable for people with wheat allergy”, provided the food does not also contain wheat as an ingredient [10
The amino acid profile of canaryseeds (Table 2
) remains unique, due to its high content of tryptophan, an essential amino acid, which is usually lacking in most cereal grains. Abdel-Aal, et al. [4
] reported a higher tryptophan content in the Keet cultivar of hairy canaryseed proteins (2.8 g/100 g of protein) as compared to wheat (1.2 g/100 g) and casein (1.0 g/100 g) protein, as well as higher amounts of essential amino acids phenylalanine, leucine, and isoleucine as compared to wheat. Similarly to other cereals, canaryseeds are deficient in essential amino acids lysine, threonine, and methionine, but possess comparable levels to wheat [4
]. Glabrous canaryseeds would make an excellent addition to other cereal grain and legume products to ensure consumers meet the recommended dietary intake of essential amino acids. In addition, canaryseeds contain high amounts of glutamic acid. Glutamic acid is the most abundant amino acid in the brain, which plays significant roles in synaptic activity, memory, and learning, also, it was reported that changes in glutamic acid metabolism and regulation in the brain leads to the development of Alzheimer’s disease [23
]. Moreover, high content of glutamic acid in the seeds could indicate the presence of high gamma-aminobutyric acid (GABA), a functional compound produced in plants primarily by the decarboxylation of L-glutamic acid, which has several health promoting properties, including reducing blood pressure and blood cholesterol, anticancer, and anti-obesity activity [24
]. GABA concentration, however, has not been directly determined in canaryseeds.
3.2. Health Promoting Properties of Canaryseed Proteins
Chronic disease is of major global concern today and includes diseases such as cardiovascular disease, cancer, and diabetes, which are leading causes of death worldwide [27
]. A balance between an active lifestyle and good eating habits are critical in the long term to prevent and combat chronic diseases. Beyond their physiological and metabolic effects, dietary proteins are intrinsically associated with health improvement and prevention of nutrition related chronic diseases (ex. cardiovascular diseases, hypertension, cancer, oxidative damage, etc.), and which need to be also considered when assessing protein quality [28
]. This is particularly relevant as consumers are increasingly looking to natural food sources to help prevent specific diseases or illnesses. Some parts of world, such as Mexico, have utilized hairy canaryseeds as a traditional folk medicine for treatment of diabetes and hypertension for centuries [5
]. However, because of the presence of toxic hairs, the seeds were not consumed directly but soaked in water, drained, dried and then processed to make canaryseed “milk”, which can be safely consumed.
The health benefits associated with drinking canaryseed “milk” were found to be largely related to the bioactive peptides produced during digestion. Bioactive peptides are small, specific and active protein fragments released from food proteins by proteolytic enzymes during protein digestion, which positively affect an individual’s overall health [29
]. Bioactive peptides have been reported from many food sources, such as fish and crustaceans, dairy products (milk, cheese, yoghurt), eggs, meat, and vegetal sources (grains, legumes, seeds) [31
]. Depending on the amino acid composition and sequence, bioactive peptides possess different types of activity, including antioxidant, antimicrobial, antihypertensive, radical scavenging, anti-inflammatory, opioid, immunomodulatory, anticancer, chelation activity, and antidiabetic activity among others [31
]. In recent years, a lot of research has been focused on the ability of plant proteins from cereals, nuts, and pulses to generate bioactive peptides with measurable health benefits. Thus far, very little research has been conducted on the bioactivity of glabrous canaryseeds. Research on canaryseed proteins and peptide bioactivity has been tested exclusively in vitro to date, with no animal or human subjects, and predominantly using the hairy varieties. Although the nutrient profile between hairless and hairy canaryseeds are very similar, further investigation into hairless canaryseed bioactivity is required and ongoing.
3.2.1. Antidiabetic Activity
Dipeptidyl peptidase IV (DPP-IV) enzyme plays a major role in the development of hyperglycemia in individuals with type II diabetes, because it inactivates incretin hormones, thereby increasing blood glucose levels [29
]. Incretin-based therapy is a common treatment for type II diabetes, but it remains less effective, because the half-life of the hormone is very short, due to inactivation by DPP-IV enzymes [30
]. DPP-IV inhibitors improve the efficiency of incretin-based therapy by inactivating the enzyme and increasing the activity of the incretin hormones. Estrada-Salas, et al. [5
] found that peptides produced by in vitro gastrointestinal digestion of canaryseed milk using pepsin, trypsin, and pancreatin, displayed inhibitory activity in a dose dependent manner against DPP-IV enzyme from porcine kidney. In addition, an in vivo and in vitro study have demonstrated an anti-obesity effect of a lipid extract (produced by hexane extraction) of hairless canaryseed [33
]. The anti-obesity effect of canaryseeds in addition to the inhibitory action of DPP-IV by canaryseed peptides would make this grain an excellent nutritional approach to improve the efficiency of synthetic drugs, since food derived DPP-IV inhibitors lack the potency of synthetic drugs inhibitors [35
]. Further characterization of the DPP-IV inhibitor peptides in canaryseeds remains necessary to establish their antidiabetic effects and capacity.
3.2.2. Antihypertensive Activity
The angiotensin-I converting enzyme (ACE) increases blood pressure and causes hypertension in inclined individuals. ACE converts the inactive angiotensin-I into angiotensin-II (a very powerful vasoconstrictor) and inactivates bradykinin (a vasodilator), which both lead to the direct increase in blood pressure [5
]. Synthetic ACE inhibitors are produced as a treatment for hypertension, and although effective, the synthetic inhibitors cause side effects, including coughing, food taste alterations, rashes and reduced efficiency when used in the long term [36
]. Food sources of ACE inhibitors are of great interest, since individuals with hypertension can consume them as part of a healthy diet to reduce their high blood pressure [37
Recent research studies revealed that canaryseed bioactive peptides have great potential to lower blood pressure through the inhibition of the ACE enzyme. Estrada-Salas, et al. [5
] showed that canaryseed flour proteins digested in vitro using pepsin, trypsin, and pancreatin, exhibited a maximum percent inhibition against the ACE enzyme of 73.5% and an IC50
value of 322 μg/mL, which was similar to the IC50
value of other peptides from chickpea, pea, soybean, wheat gliadin, and sardine muscle. Undigested canaryseed proteins had significantly lower inhibition activity, meaning the antihypertensive bioactive peptides are produced upon protein digestion [5
]. Similarly, Valverde, et al. [6
] found that canaryseed flour proteins from the prolamin fraction had the highest inhibition activity against the ACE enzyme, with an IC50
value of 217.4 μg/mL, after in vitro digestion with pepsin and pancreatin. They further identified five peptides by mass spectroscopy (LSLGT, TDQPAG, QQLQT, FEPLQLA, and KPQLYQPF) in the digested prolamin fraction that had both ACE and DPP-IV inhibition activity. Additionally, Passos, et al. [38
] administered to rats an aqueous extract of canaryseeds (obtained by soaking the seeds in water), which successfully reduced systolic blood pressure in the animals while having no renal or toxicological effects. All these studies demonstrated the potential positive effect of canaryseeds on cardiovascular disease control.
3.2.3. Antioxidant Activity
The antioxidant potential of plants has received a great deal of attention, because increased oxidative stress has been identified as a major causative factor in the development and progression of several life threatening diseases, including neurodegenerative and cardiovascular diseases. Free radical species that are generated in the body by various endogenous systems cause extensive damage to body tissues by destroying cell membrane structure, modifying enzyme activity, and changing DNA leading to cancer development [39
]. In this regard, bioactive peptides of canaryseeds demonstrated antioxidant activity by reacting with free radical species, thereby preventing tissue damage and decay. Valverde, et al. [6
] used two in vitro radical scavenging assays on digested canaryseed protein fractions and found that the prolamins had the overall highest antioxidant activity. Mass spectroscopy analysis of the digested prolamin fraction identified five peptides, of which only one had antioxidant activity (KPQLYQPF). Protein fractions from digested canaryseeds had higher antioxidant activity in general as compared to raw flour, because the seed proteins undergo hydrolysis, increasing their antioxidant activity [6
3.2.4. Other Bioactivities
Only very limited studies have been conducted on other bioactive properties of hairy canaryseed proteins. As an example, acetylcholinesterase inhibitors are currently employed as a form of treatment for individuals with Alzheimer’s disease, because they help maintain levels of acetylcholine in the brain, which is essential for nerve impulses and transmission [40
]. Kchaou, et al. [41
] found that a methanol extract of a hairy Tunisian canaryseed variety had a percent inhibition against acetylcholinesterase enzyme of 65% at a concentration of 1 mg/mL, which was attributed predominantly to polyphenols and flavonoids in the extract. An antibacterial activity of hairy Tunisian canaryseed extracts, especially against gram-positive bacteria, was also reported by Kchaou, et al. [41
]. These bioactivities could possibly be the result of canaryseed peptides, as it was previously demonstrated for hemp seed protein hydrolysates, which exhibited acetylcholinesterase inhibition [40
], or for other cereal proteins, such as wheat and barley, for which antibacterial activity was reported [42
]. Proteins and peptides from cereal grains and legumes (wheat, barley, amaranth, oat, rye, soybean etc.) are known to have antithrombotic, immunomodulatory, and anticancer activity [42
]. Bioactivities of Canadian glabrous canaryseed peptides remain largely unknown, but because of the diverse bioactivity reported in similar cereal grains from the same family, it remains highly likely that canaryseed peptides possess additional health promoting properties, which still need to be confirmed.
3.3. Protein Digestibility
Protein digestibility is an important parameter to consider when assessing protein quality [53
]. The health advantages of glabrous canaryseeds depends on their digestibility and bioavailability. Several in vivo studies indicated excellent protein digestibility of canaryseed in animals. Broiler chickens fed hairless canaryseed groats and hulled seeds exhibited similar ileal protein digestibility as other feed components, including corn, wheat, sorghum, and peas [54
]. The same study showed high apparent ileal digestibility of amino acids cysteine (86%), phenylalanine (88%), and tryptophan (93%). Furthermore, weight gain between broiler chickens fed with wheat and chickens fed with canaryseeds were similar.
Later, Classen et al. [55
] fed broiler chickens yellow glabrous canaryseeds and glabrous brown seeds and determined the seeds were equivalent in terms of feeding value. Magnuson, et al. [56
] found no evidence of toxicity in rats when fed glabrous canaryseeds for a 90 day study and, furthermore, rat diets supplemented with 50% hulled and dehulled glabrous canaryseeds were comparable in terms of growth, hematology, and clinical parameters as rats with diets supplemented with 50% wheat. Thacker [57
] showed that crude protein digestibility in pigs increased linearly with increasing proportions of canaryseeds in their diets. Moreover, he found that a pig’s diet containing 25% canaryseeds promoted the highest growth rates in the pigs with a crude protein digestibility of approximately 78%. All these studies indicate that hairless canaryseeds make an excellent addition or supplement to conventional animal feed, as it promotes growth, but also enhances protein digestibility.
For human digestibility of canaryseed proteins, no in vivo study has been reported in the literature despite several in vitro studies that have been carried out to mimic human protein digestibility of canaryseeds under gastrointestinal conditions. Abdel-Aal, et al. [4
] used a multienzyme approach with trypsin, chymotrypsin, and peptidase and established an in vitro protein digestibility of 84% in hairy canaryseeds. Interestingly, Rajamohamed, et al. [58
] compared the effects of thermal treatment on canaryseed protein digestibility. The in vitro protein digestibility of raw, roasted, and boiled glabrous canaryseed flours was determined by gastric, duodenal, and sequential gastric-duodenal methods. The sequential gastric-duodenal method was most effective at digesting the proteins and, overall, thermal processing enhanced protein digestion. As a cereal, canaryseeds can be used in various forms, such as a whole groat, whole meal, or whole grain flour in several applications, such as a cereal, in pasta, and in baking to make products, such as bread, muffins, and cereal grain bars [10
]. Since thermal processing increased protein digestibility, the heating and thermal processing of canaryseeds in the development and production of baked goods will contribute to its improved nutritive value.