Connoisseurs can find a wide range of products containing cannabis in the Netherlands, where it has long been practically legal: Cannabis popsicles, lollipops, chocolate and soap are but a few of the products available for purchase in the Dutch capital.
But don’t expect to have an easy time of it if you’re looking for something to hold your lunchtime turkey slices. For that, you will need to take a trip to neighboring Belgium, where a Jewish baker is about to launch Europe’s first commercial line of cannabis bread.
Cannabread will be available for purchase in Carrefour supermarkets in Brussels and two other Belgian cities later in November, according to a report last month in Vice Belgium. The bread is already on sale in at least one of five Lowy’s bakery shops in Brussels.
Lowy’s owner Charly Lowy said about 15 percent of the dough in Cannabread is made from cannabis seeds, but eating the bread will not get you high.
The level of THC, the psychoactive chemical in cannabis, is low, which is also why it can be sold without restrictions in Belgium, where marijuana laws are more restrictive.
Cannabread is also certified organic and, according to Lowy, full of minerals, vitamin E, Omega 3 and 6, fibers, carotene and magnesium.
“The bread is intended first and foremost for people who just love bread, and different kinds of it,” Lowy told the Jewish Telegraphic Agency.
“But it’s true that cannabis products are in right now.”
Boutique bakers in the Netherlands and beyond have occasionally offered cannabis bread in the past, but Lowy is the first to mass produce it, according to media reports.
While not intoxicating, the bread does taste and smell like cannabis, the Vice report said. Which may be why Belgium’s Federal Agency for the Safety of the Food Chain raided the bakery in 2018 and destroyed Lowy’s entire stock of Cannabread, citing the absence of certificates proving it does not get people high.
Lowy is tall and handsome. The Vice writer found him to resemble Don Draper, the lead character portrayed by Jon Hamm in the hit television drama “Mad Men.”
And he has a history of baking innovative breads, including one with beer and a purple bread containing wild rice.
Cannabread contains 15% marijuana. (Courtesy of Charly Lowy/via JTA)
His family story is also a common European Jewish tale of success amid adversity.
His late father, Otto, fled to Belgium from his native Austria, when it was annexed by Nazi Germany in 1938. After the Nazis invaded Belgium in 1940, Otto went underground. It was then, during the most perilous period of his life, that he met his wife, Hania, a Jewish immigrant from Poland. They wed in 1942 and had three children. Charly is the youngest.
When Otto died in 1980, Charly, who was then studying political science, took over the bakery and massively expanded the family business that his father had established in 1947.
Back then, the bakery’s motto was: “Bread, that’s all.”
No longer.
Hemp (Cannabis sativa L.), an annual herbaceous plant that belongs to the Cannabinaceae family, has been an important source of food, fiber, medicine, and a psychoactive/religious drug (Cromack, 1998). Historically, the cultivation of hemp has been limited due to the presence of the psychoactive compound tetrahydrocannabinol (THC). Since 1990, dozens of countries have authorized the licensed growth and processing of the hemp cultivars with substantially reduced levels (˂0.3%) of THC (Cherney & Small, 2016).
Canada, Australia, Austria, China, Great Britain, France, and Spain are among the most important agricultural producers of hempseeds (Rodriguez‐Leyva & Pierce, 2010). In recent years, some states in the United States, including North Dakota and Kentucky, have also passed legislation approving its production.
Hempseeds, a by‐product obtained after the commercial utilization of fiber, are achieving growing popularity as an excellent source of nutrients. Whole hempseeds contain 25% to 35% oil, 20% to 25% protein, 20% to 30% carbohydrates, 10% to 15% insoluble fibers, and vitamins and minerals such as phosphorus, potassium, magnesium, sulfur, calcium, iron, and zinc (Callaway, 2004; House, Neufeld, & Leson, 2010).
After removal of the hull, the edible portion of the seeds contains, on average, 46.7% oil and 35.9% protein. The concentration of antinutritional compounds, such as phytic acid, condensed tannins, and trypsin inhibitors, is very low in hempseeds (Russo & Reggiani, 2015). The oil extracted from hempseeds is rich in polyunsaturated fatty acids, especially linoleic (ω‐6) and α‐linolenic (ω‐3) acids with a desirable ratio between 2:1 and 3:1 for optimal health (Callaway, 2004; Porto, Decorti, & Natolino, 2015).
The by‐product (hemp cake or meal) after oil extraction is abundant in high‐quality storage proteins. Hempseed protein has been well known for its excellent digestibility and desirable essential amino acid composition (Tang, Ten, Wang, & Yang, 2006; Wang, Tang, Yang, & Gao, 2008). The arginine content in hempseeds, at 12%, is also remarkably high. A recent proteomic characterization of hempseed concluded that hempseed is an underexploited nonlegume, protein‐rich seed (Aiello et al., 2016).
Because of the high nutritional value, hempseed protein has drawn increasing attention in scientific research, and this is well reflected in the progressive increase in the number of scientific publications related to the term “hemp protein” in the title, abstract, and keywords of the publications (Figure 1).
Of particular interest is the purported health benefit of bioactive peptides prepared from hemp protein as well as its technological functionality, such as foaming, emulsifying, gelling, and film‐forming capabilities.
On the other hand, in the food industry, a wide range of products have been developed from hempseed proteins, for example, beverages, functional ingredients, nutritional supplements, and various personal‐care products. The value and application of hemp protein in food products are closely related to the protein structure and functional properties.
Figure 1 Number of publications reported in the Scopus system containing the term “hemp protein” in the title, abstract, and keywords within the years 1960 to 2018 (as of November 17, 2018).
Major Hempseed Proteins
Protein fractions
Hempseed protein consists mainly of globulin (edestin) and albumin. Edestin accounts for approximately 60% to 80% of the total protein content (Odani & Odani, 1998; Tang et al., 2006), while albumin constitutes the rest.
Edestin
The globular edestin is located inside the aleurone grains as large crystalloidal substructures (Angelo, Yatsu, & Altschul, 1968). Using crystallographic techniques, edestin is shown to have a structure similar to that of the hexamer of soy glycinin; it is composed of six identical subunits, each consisting of an acidic (AS) and a basic (BS) subunit linked by one disulfide bond (Patel, Cudney, & McPherson, 1994). The molecular weight (MW) of edestin is estimated to be approximately 300 kDa (Wang et al., 2008). The AS is approximately 34.0 kDa and relatively homogeneous, while BS consists mainly of two subunits of about 20.0 and 18.0 kDa (Figure 2).
A fine characterization of edestin (globulin) from a Korean variety has been reported by Kim and Lee (2011). The authors isolated the edestin protein and analyzed the N‐terminal amino acid sequence of the first seven and six amino acid residues of the AS and BS, respectively, by the automated Edman degradation method. The seven amino acid residues in AS had a sequence of Ile‐Ser‐Arg‐Ser‐Ala‐Val‐Tyr in the N‐terminus, while two constituents of BS showed an identical N‐terminus of Gly‐Leu‐Glu‐Glu‐Thr‐Phe. The 11S‐rich and 7S‐rich hemp protein isolates (HPIs) have been prepared by Wang et al. (2008) using a similar extraction method for 7S and 11S fractions of soy protein isolate (SPI).
SDS–PAGE analysis indicates that the main component in HPI‐11S is edestin, and the BS of edestin, along with a subunit of about 4.8 kDa, makes up the HPI‐7S. Further analysis shows that the 7S polypeptide has no thermal transition, while the 11S protein exhibits a similar denaturation temperature as HPI at 91.9 °C, indicating the HPI thermal property was due mainly to the 11S component (Wang et al., 2008).
Docimo, Caruso, Ponzoni, Mattana, and Galasso (2014) isolated seven cDNAs encoding for edestin, suggesting that they were divergent forms of two edestin types (CsEde1 and CsEde2) based on the sequence similarity. Both edestin types exhibit high amounts of arginine (11% to 12%), but CsEde2 is particularly rich in methionine (2.36%), which is even higher than the methionine‐rich 2S albumin (8 Met) isolated from hempseed (Odani & Odani, 1998), and also exceeds the methionine contents in soybean glycinins (Docimo et al., 2014). Ponzoni, Brambilla, and Galasso (2018) identified a type3 edestin gene, CsEde3, which shows approximatively 65% and 58% sequence homology when compared to the genomic forms of CsEde1and CsEde2, respectively.
Albumin
The albumin fraction constitutes about 25% of hempseed storage protein. Malomo and Aluko (2015a) isolated the globulin and albumin fractions by dialysis of the salt extract of hempseed meal against water.
The albumin fraction was found to contain fewer disulfide‐bonded proteins and hence a less compact structure with greater flexibility than the globulin fraction. This was also confirmed by both intrinsic fluorescence and circular dichroism analysis which illustrated greater exposures of tyrosine residues when compared with globulin.
Furthermore, albumin exhibited significantly higher solubility and foaming capacity (FC) than globulin, while no differences in emulsion‐forming ability were observed between the two protein fractions (Malomo & Aluko, 2015a).
Additionally, a methionine‐ and cystine‐rich seed protein (10 kDa protein, 2S albumin) has been isolated from hempseed (Odani & Odani, 1998). The protein contains 18% (w/w) sulfur amino acids and consists of two polypeptide chains (small and large) with 27 and 61 amino acid residues, respectively. The two polypeptide chains are held together by two disulfide bonds.
This sulfur‐rich protein has no inhibitory activity against trypsin and could serve as a rich thiol source to formulate highly nutritious foods, since various plant food proteins, especially legumin proteins from soybean, pea, and beans, are deficient in sulfur.
The gene families encoding the precursor polypeptides of 2S albumin have recently been identified by Ponzoni et al. (2018), and two genomic isoforms for 2S albumin were obtained, namely, Cs2S‐1 and Cs2S‐2. The alignment of the deduced gene with the mature 2S protein sequence published in the literature (Odani & Odani, 1998) showed that Cs2S is 97% identical to the mature 2S protein.
Protein processing
Hemp protein meal
The oil extraction by‐product of crushed hempseeds is commonly referred to as hemp protein meal (HPM). The protein content in HPM ranges from 30% to 50% in dry matter depending on the variety of hemp used and the oil extraction method (cold‐pressing or solvent) and efficiency (Malomo, He, & Aluko, 2014). Pojić et al. (2014) separated HPM into four fractions by particle size (>350, >250, >180, and <180 µm).
The two cotyledon‐containing fractions (>180 and <180 µm) were found to be significantly richer in protein and higher in free radical‐scavenging capacity compared with the hull‐containing fractions (>350 and >250 µm). Antinutrients (trypsin inhibitors, phytic acid, glucosinolates, and condensed tannins) were mostly located in the cotyledon fractions. Moreover, Russo and Reggiani (2015) showed that the dioecious varieties have lower contents of antinutritional compounds than monoecious varieties.
Hemp protein concentrate
Hemp protein concentrate (HPC) is prepared from dehulled and defatted hempseed or HPM by removing most of the water‐soluble, nonprotein constituents. HPC contains at least 65% protein (N × 6.25) on a dry weight basis. Malomo and Aluko (2015b) obtained HPC by enzymatic digestion (carbohydrase and phytase) of fiber coupled with membrane ultrafiltration that enriched protein content up to 70%. Protein digestibility of the HPC was significantly higher than that of HPM and traditional isoelectric protein isolate.
Hemp protein isolate
The most purified and enriched form of commercial protein product, HPI (>90% protein), is prepared to meet food processing needs that entail minimal influence of unwanted nonprotein components. Depending on the method of extraction employed, the final HPI could vary in protein content, composition, solubility, and, when applied as a functional ingredient, the reactivity with food additives.
Alkaline extraction followed by isoelectric precipitation is the most common method for the preparation of HPI (Lu et al., 2010; Malomo et al., 2014; Orio et al., 2017; Raikos, Duthie, & Ranawana, 2015; Ren et al., 2016; Tang et al., 2006; Wang et al., 2008).
The method can produce an isolate with a purity up to 94% depending on the specific extracting conditions, for example, pH, temperature, extraction time, and centrifugal force.
The alkaline extraction pH is generally 9 to 10, higher than that for legume protein extraction (pH 8), because native hempseed proteins are tightly compacted, and may be closely integrated with other components, for example, phenolic compounds.
To maximize the yield, some researchers use elevated extracting temperatures, for example, 35 to 40 °C, to improve the protein solubility. It should be noted that adverse chemical reactions, such as the conversion of cysteine and serine residues to nephrotoxic lysinoalanine compounds, can occur under highly alkaline and heating conditions.
As reported by Wang, Jin, and Xiong (2018), HPI extracted at pH 10 and room temperature had a low level of lysinoalanine (0.8 mg/100 g protein). However, if the extracted HPI was held at pH 12 for as short as 5 min at 40 °C, the lysinoalanine content would increase to 4 mg/100 g protein. Corresponding to alkaline extraction, acid extraction was also adopted to prepare HPI (Teh, Bekhit, Carne, & Birch, 2014).
The yield of protein extracted at acidic pH was lower than that extracted at alkaline pH. Compared to alkaline‐extracted HPI, this protein isolate had a lower lightness (L* value), higher redness (a* value), and lower yellowness (b* value). Another method, known as “salt extraction with micellization”, has been described for HPI preparation in a recent report (Hadnađev et al., 2018a). HPI obtained by this method has a very high purity (98.9% protein, on a dry basis) and significantly greater colorimetric values (L*, a*, and b*), but lower recovery yield, in comparison with the common alkaline extraction‐isoelectric precipitation method.
The color difference is because the alkaline condition employed to extract HPI favors the coextraction of phenolics from hempseed meal, resulting in the development of dark green to brown color of protein isolates from the exposure to molecular oxygen. Based on the SDS–PAGE profiles, it appears that the salt extraction minimally influences the subunit composition of hemp protein, differing from the high pH‐isoelectric precipitation method that could cleave disulfide bonds between some subunits.
For salt extraction, extreme alkaline or acidic pH and temperature elevation are not necessary, as compared with acid and alkaline extraction. Protein extraction occurs at a slightly acidic pH (5.5 to 6.5), although Aiello, Lammi, Boschin, Zanoni, and Arnoldi (2017) and Zanoni et al. (2017) also isolated hemp protein by salt extraction under a slightly alkaline pH to maximize protein extractability.
Nutrition and Health Benefits of Hempseed Protein
Nutritional quality
Amino acid composition
The dietary requirements of humans are not for protein per se, but for specific amounts of indispensable or essential amino acids (building blocks of protein). Hemp protein provides all the essential amino acids with a balanced amino acid profile (Table 1). The essential amino acids are comparable to other high‐quality proteins, such as casein and soy protein (Tang et al., 2006), and are sufficient for the Food and Agriculture Organization (FAO)/World Health Organization (WHO) suggested requirements for 2‐ to 5‐year‐old children.
Hemp protein contains an exceptionally high amount of arginine and glutamine (Lu et al., 2010). Arginine accounts for approximately 12% of hempseed protein when compared with less than 7% for most other food proteins, including the proteins from potato, wheat, maize, rice, soy, rapeseed, egg white, and whey (Callaway, 2004). Arginine is a precursor of nitric oxide, the vasodilating agent that enhances blood flow and contributes to the maintenance of normal blood pressure (Wu & Meininger, 2002).
The Arg/Lys ratio is a determinant of the cholesterolemic and antherogenic effects of a protein. The Arg/Lys ratio of hemp protein, at 3.0 to 5.5, is remarkably higher than that of SPI (1.41) or casein (0.46), making hemp protein particularly valuable as a nutritional and bioactive ingredient for the formulation of foods that promote cardiovascular health. Furthermore, hemp protein products (HPM, HPC, and HPI) contain excellent amounts of the sulfur amino acids cysteine and methionine (Callaway, 2004; Russo & Reggiani, 2013).
Total sulfur‐containing amino acids are in the range of 3.5% to 5.9%, which is close to the reference protein profiles established by FAO/WHO/United Nations University (UNU) as requirements for infants and preschool children 2‐ to 5‐year‐old (Table 1). House et al. (2010) calculated the respective amino acid scores of hemp protein, identifying that lysine (score 0.5 to 0.62) was the first‐limiting amino acid in hemp protein, followed by leucine and tryptophan.Table 1. Amino acid composition (%, w/w) of hemp protein products and selective nonhemp proteins for comparison
| Hempseed | Meal | Concentrate | Protein isolate | Protein fraction | Other protein | FAO/WHO/UNU | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Amino acids | Whole seeda | Dehulled seeda | HPM1a | HPM2b | HPCb | HPI1b | HPI2c | HPI3d | HPI4d | HPI5c | Albumine | Globuline | SPIf | Caseing | Infant | Child2–5 y |
| Isoelectric precipitation | Acid extraction | Micellization (salt extraction) | ||||||||||||||
| Asx | 11.23 | 10.68 | 10.69 | 10.78 | 11.17 | 11.31 | 10.06 | 9.38 | 9.38 | 10.27 | 7.93 | 9.47 | 11.47 | 6.30 | ||
| Thr | 4.75 | 3.70 | 3.94 | 3.75 | 3.77 | 3.42 | 3.24 | 4.76 | 4.81 | 3.27 | 4.63 | 2.60 | 3.98 | 3.70 | 4.3 | 3.4 |
| Ser | 5.59 | 4.96 | 5.05 | 5.65 | 5.55 | 5.58 | 5.00 | 5.48 | 5.59 | 5.16 | 5.12 | 5.73 | 5.32 | 4.60 | ||
| Glx | 17.58 | 18.17 | 17.61 | 18.45 | 17.53 | 19.10 | 16.92 | 15.99 | 16.41 | 17.60 | 20.37 | 21.48 | 20.67 | 19.00 | ||
| Pro | 4.23 | 4.73 | 4.64 | 4.37 | 4.98 | 4.30 | 3.64 | 4.00 | 3.81 | 3.79 | 3.82 | 3.87 | 5.13 | N/A | ||
| Gly | 4.98 | 4.70 | 4.85 | 4.67 | 4.86 | 4.31 | 3.68 | 4.64 | 4.57 | 3.73 | 8.26 | 4.10 | 3.74 | 1.60 | ||
| Ala | 4.51 | 4.43 | 4.70 | 4.19 | 4.25 | 3.81 | 3.76 | 4.14 | 3.91 | 4.09 | 3.91 | 2.84 | 3.72 | 2.70 | ||
| Cys | 1.93 | 1.90 | 2.04 | 1.52 | 1.62 | 1.25 | 1.46 | 1.64 | 1.42 | 1.49 | 3.20 | 3.32 | 0.05 | 0.04 | ||
| Val | 5.36 | 5.19 | 5.58 | 4.69 | 5.34 | 4.66 | 5.33 | 5.80 | 5.59 | 5.67 | 2.90 | 3.41 | 4.28 | 6.00 | 5.5 | 3.5 |
| Met | 2.63 | 2.74 | 2.57 | 2.28 | 1.94 | 1.92 | 2.02 | 4.23 | 2.33 | 2.12 | 1.74 | 4.07 | 0.92 | 2.60 | ||
| Ile | 3.76 | 3.76 | 4.23 | 3.59 | 3.81 | 3.63 | 3.64 | 4.46 | 4.47 | 4.66 | 2.02 | 2.86 | 4.35 | 4.90 | 4.6 | 2.8 |
| Leu | 7.00 | 6.24 | 6.86 | 6.87 | 6.88 | 6.46 | 6.54 | 7.23 | 7.02 | 6.53 | 4.05 | 5.57 | 6.79 | 8.40 | 9.3 | 6.6 |
| Tyr | 3.20 | 3.73 | 3.36 | 3.04 | 2.79 | 3.61 | 3.64 | 3.49 | 3.88 | 3.67 | 2.02 | 3.41 | 3.61 | 5.50 | 1.7 | 1.1 |
| Phe | 4.84 | 4.17 | 4.73 | 4.68 | 4.74 | 4.72 | 4.23 | 5.41 | 5.53 | 4.20 | 1.32 | 3.27 | 5.14 | 4.50 | ||
| His | 2.58 | 2.83 | 2.72 | 3.26 | 3.48 | 3.15 | 2.71 | 2.52 | 3.19 | 2.81 | 3.68 | 3.87 | 2.81 | 2.70 | 2.6 | 1.9 |
| Lys | 4.04 | 3.68 | 3.86 | 3.94 | 4.21 | 2.73 | 2.89 | 2.77 | 2.82 | 2.87 | 7.37 | 3.69 | 5.23 | 7.10 | 6.6 | 5.8 |
| Arg | 10.71 | 13.27 | 11.42 | 13.20 | 11.98 | 15.06 | 10.02 | 13.98 | 15.24 | 9.98 | 12.82 | 16.12 | 7.35 | 3.30 | ||
| Trp | 1.08 | 1.11 | 1.14 | 1.06 | 1.05 | 1.00 | N/A | 0.09 | 0.03 | N/A | 0.16 | 0.34 | N/A | N/A | ||
| Arg/Lys | 2.65 | 3.61 | 2.96 | 3.35 | 2.85 | 5.52 | 3.47 | 5.05 | 5.40 | 3.48 | 1.74 | 4.37 | 1.41 | 0.46 | ||
| Met+Cys | 4.56 | 4.64 | 4.61 | 3.80 | 3.56 | 3.17 | 3.48 | 5.87 | 3.75 | 3.61 | 4.94 | 7.39 | 0.97 | 2.64 | 4.2 | 2.5 |
| Phe+Tyr | 8.04 | 7.90 | 8.09 | 7.72 | 7.53 | 8.33 | 7.87 | 8.90 | 9.41 | 7.87 | 3.34 | 6.68 | 8.75 | 10.00 | 7.2 | 6.3 |
| E/T | 0.36 | 0.33 | 0.36 | 0.34 | 0.35 | 0.32 | 0.34 | 0.37 | 0.36 | 0.38 | 0.29 | 0.29 | 0.35 | 0.48 |
- N/A, not available; E/T, the proportion of essential amino acids to total amino acids; Asx, Glx, sum of asparagine and aspartic acid, and glutamine and glutamic acid, respectively. Data are from
- a House et al., 2010.
- b Malomo and Aluko, 2015b.
- c Hadnađev et al., 2018a.
- d Teh et al., 2014.
- e Malomo and Aluko, 2015a.
- f Wang et al., 2008.
- g Tang et al., 2006.
When the amino acid composition of whole hempseed or meal is compared with protein products (Table 1), the influence of protein composition (types) of the final product can be observed. As addressed by Malomo and Aluko (2015b), the contents of essential amino acids and branched‐chain amino acids of HPC were higher than HPM and isoelectric precipitated HPI, which could contribute to better muscle metabolism and maintenance of protein homeostasis (Herman, She, Peroni, Lynch, & Kahn, 2010).
Isoelectric precipitated HPI has a higher Arg/Lys ratio than HPM and HPC. Hadnađev et al. (2018a) have demonstrated that HPI prepared by micellization (salt extraction followed by membrane filtration) has a similar amino acid pattern to HPI obtained by isoelectric precipitation (Table 1). However, acid‐extracted hemp protein was slightly different in amino acid composition from alkali‐extracted (isoelectric precipitated) HPI, especially for methionine, histidine, and arginine (Teh et al., 2014).
It is not a surprise that different fractions of HPI (albumin and globulin) possess different amino acid compositions (Malomo & Aluko, 2015a).
The globulin fraction has a higher content of sulfur amino acids, especially methionine, and is also higher in hydrophobic and aromatic amino acids when compared to albumin. A higher Arg/Lys ratio (4.37) in globulin, when compared with albumin (1.74), suggests strong potential for globulin utilization in the formulation of cardiovascular health‐promoting food products.
Digestibility
The degree of digestion of dietary proteins depends on enzyme accessibility, which is affected by the molecular structure as well as other components associated with proteins. House et al. (2010) measured the protein digestibility‐corrected amino acid score (PDCAAS) of whole hempseed, dehulled hempseed, and HPM using a rat bioassay for protein digestibility and the FAO/WHO amino acid requirement of children (2 to 5 years of age) as reference. The protein digestibility of dehulled hempseed, depending on the sources, was 90.8% to 97.5%, almost comparable to 97.6% for casein. The PDCAAS value for hemp protein, depending on the source, was 0.48 to 0.61.
These values are within the range of major pulse proteins, for example, from beans, but are above those of cereal grain products, for example, whole wheat. Lysine and tryptophan are the main limiting amino acids in hempseeds, which presumably contribute to the relatively low PDCAAS score. Removal of the hull improved the protein digestibility and the corresponding PDCAAS. It is worth noting that, currently, there is no literature report comparing digestibility and PDCAAS for HPIs prepared with different methods.
However, a digestibility study for HPIs (HPI, 7S, and 11S) and SPI using an in vitro digestion model that measures nitrogen release has been published (Wang et al., 2008). During the pepsin digestion, edestin (including AS and BS) was rapidly degraded within 1 min, similar to the digestion of the AS and BS of soy glycinin, releasing oligo‐peptides with MWs less than 10 kDa.
Although HPIs displayed a similar digestibility to SPI when only digested by pepsin, the total digestibility (pepsin plus trypsin digestion) of HPIs (88% to 91%) was distinctly higher than that of SPI (71%), hinting that HPI is an efficient source of protein nutrition for human consumption. Mamone, Picariello, Ramondo, Nicolai, and Ferranti (2019) also showed evidence that hemp flour and HPI had a high degree of digestibility. A very limited number of peptides survived in the simulated intestinal digestion, whereas some free aromatic amino acids Tyr, Phe, and Trp were also detected.
This finding supports, on a molecular basis, the claim of good digestibility with the ready release of bioaccessible amino acids from HPI (Wang et al., 2008).
Health benefits
Allergenicity
Hempseeds are considered to be a low‐allergen food material. The allergenicity associated with hemp, including allergens from its seeds, roots, fibers, leaves, and flowers, has been comprehensively reviewed by Decuyper et al. (2017).
Five allergens have been identified in the hemp plant, including C. sativa 3/ns‐LTP (nonspecific lipid transfer proteins, 9 kDa), profilin (14 kDa), oxygen‐evolving enhancer protein (23 kDa), thaumatin‐like protein (38 kDa), and ribulose‐1,5‐biphosphate carboxylase/oxygenase (RuBisCo, 50 kDa). Of these allergenic proteins, only RuBisCo has been detected in hempseeds through proteomic analysis (Aiello et al., 2016; Park, Seo, & Lee; 2012).
A systematic evaluation of HPI by Mamone et al. (2019) concluded that all known hemp allergens, including the major thaumatin‐like protein and LTP, were entirely eliminated by the HPI production process. Neither fragments of the proteins were present after gastrointestinal digestion. Although there are limited reports on immunological responses to hempseed proteins, the presence of allergen‐homolog proteins (for example, serpins) in HPI and their digestion‐resistant peptide fragments warrants further research to establish the potential health impact.
Bioactivity
Aside from their nutritive values, proteins from both plant and animal sources have been hydrolytically converted to peptides that exhibit a wide range of bioactivity both in vitro and in vivo, for example, antioxidant, antihypertensive, antimicrobial, antithrombotic, hypocholesterolemic, mineral‐binding, immunomodulatory, and cytomodulatory properties (Mine, Li‐Chan, & Jiang, 2010; Sánchez & Vázquez, 2017; Sharma, Singh, & Rana, 2011).
Enzymatic digestion of proteins can break down long polypeptide chains into short fragments that fit in, hence, disrupt the specific active site of the disease‐related metabolic enzymes. The peptides may also bind to nonactive sites on the target disease‐causing enzymes through hydrophobic or electrostatic interactions.
Hemp protein has been explored for its feasibility and potential in the production of bioactive peptides during the past decade. The various health benefits and pharmaceutical value of peptides generated by controlled hydrolysis of hemp proteins are summarized in Table 2. The specific health‐promoting activities include angiotensin I‐converting enzyme (ACE) inhibition, renin inhibition, acetylcholinesterase (AChE) inhibition, metal‐binding capacity, antioxidant activity, hypocholesterolemic effect, and serum glucose regulation.
The bioactivity is elicited through the hydrolysis with gastrointestinal enzymes (pepsin, pancreatin, and trypsin) (Aiello et al., 2017; Girgih et al., 2014d,b; Girgih, Alashi, He, Malomo, & Aluko, 2014a; Girgih, He, & Aluko, 2014c; Girgih, Udenigwe, & Akuko, 2011a, 2013; Girgih, Udenigwe, Li, Adebiyi, & Aluko, 2011b; Zanoni, Aiello, Arnoldi, & Lammi, 2017) or exogenous proteases such as alcalase, papain, flavourzyme, neutrase, and thermolysin (Hadnađev et al., 2018b; Lu et al., 2010; Malomo & Akuko, 2016; Malomo, Onuh, Girgih, & Aluko, 2015; Tang, Wang, & Yang, 2009; Wang, Tang, Chen, & Yang, 2009). Peptides with ACE inhibitory activity have also been produced from hemp protein with acid hydrolysis (Orio et al., 2017).Table 2. Reported bioactivity of peptides derived from hempseed protein
| Bioactivity | Study details | Reference |
|---|---|---|
| Angiotensin I‐converting enzyme(ACE) inhibition, in vitro and in vivo | Peptide fractions (˂1 and 1 to 3 kDa) had less potency against ACE than the original hempseed protein hydrolysate (HPH) produced from pepsin–pancreatin hydrolysis. Small‐size peptides (˂1 kDa) were more effective than larger size peptides (1 to 3 kDa) as ACE inhibitors. | Girgih et al., 2011b |
| Plasma ACE activity was remarkably suppressed in hempseed meal protein hydrolysate (HMH)‐fed rats compared to control rats. | Girgih et al., 2014a | |
| HT (bacterial) produced the highest ACE‐inhibitory activity of alkali‐ and acid‐extracted HPI among all microbial and plant proteases (AFP, HT, protease G, actinidin, and zingibain). | Teh et al., 2016 | |
| Four peptides (GVLY, IEE, LGV, and RVR) were identified from HPH by extensive chemical hydrolysis (6 M HCl). GVLY possessed the highest ACE‐inhibitory activity, while IEE was almost inactive in inhibiting ACE. | Orio et al., 2017 | |
| Renin inhibition | Peptide fractions (˂1 and 1 to 3 kDa) exhibited moderate renin inhibitory activity, which was lower than HPH. | Girgih et al., 2011b |
| Plasma renin level was decreased for HMH‐fed rats compared to rats fed only casein and HPI. | Girgih et al., 2014a | |
| Novel peptides WVYY, WYT, SVYT, and IPAGV were generated from an enzymatic digest of hempseed protein. WYT and SVYT had similar renin‐inhibitory activity, which was significantly better than that of IPAGV. | Girgih et al., 2014c | |
| Antihypertensive effect | Oral administration of HPH to spontaneously hypertensive rats led to significant reductions in systolic blood pressure, while the antihypertensive effect of peptide fractions (˂1 and 1 to 3 kDa) was significantly lower than that of HPH. | Girgih et al., 2011b |
| WVYY and PSLPA showed maximum systolic blood pressure (SBP) reduction in spontaneously hypertensive rats among 23 identified short‐chain (≤5 amino acids) peptides. | Girgih et al., 2014d | |
| HMH had stronger hypotensive effects in spontaneously hypertensive rats when compared to HPI and casein. | Girgih et al., 2014a | |
| The 1% alcalase HPH was most effective in reducing systolic blood pressure among different HPHs produced with 2% pepsin, 4% pepsin, 1% alcalase, 2% alcalase, 2% papain, and 2% pepsin + pancreatin, while the pepsin HPHs produced longer lasting effects. | Malomo et al., 2015 | |
| Acetylcholinesterase(AChE) inhibition | The pepsin–HPH was the most active ACE inhibitor when compared to HPHs obtained by papain, thermoase, flavourzyme, alcalase, and pepsin + pancreatin. | Malomo and Aluko, 2016 |
| Antioxidation, in vitro and in vivo | HPHs by six proteases (alcalase, flavourzyme, neutrase, protamex, pepsin, and trypsin) exhibited varying antioxidant properties indicated by DPPH radical scavenging and Fe2+ chelating abilities and reducing power, depending on the yield of trichloroacetic acid (TCA)‐soluble peptides and surface hydrophobicity. | Tang et al., 2009 |
| HPH by Neutrase® exhibited antioxidant activities (DPPH radical scavenging ability, reducing power and Fe2+ chelating ability) to various extents, depending on TCA‐soluble peptide yields. | Wang et al., 2009 | |
| HPH separated with macroporous adsorption resin after alcalase hydrolysis of HPI displayed improved DPPH radical scavenging activity. The active fraction was further separated by gel filtration and RP‐HPLC to obtain two purified peptides, namely NHAV and HVRETALV. | Lu et al., 2010 | |
| HPH exhibited a significantly weaker ferric‐reducing power and hydroxyl radical scavenging activity in comparison with the fractionated peptides. HPH and peptide fractions significantly inhibited linoleic acid oxidation compared to HPI. | Girgih et al., 2011a | |
| Eight peptide fractions were separated by reverse‐phase (RP)‐HPLC from HPH produced through simulated gastrointestinal tract. Partially purified HPH peptides demonstrated stronger antioxidant properties by scavenging free radicals, and the antioxidant activity was closely associated with the amino acid composition and hydrophobicity. | Girgih et al., 2013 | |
| WVYY and PSLPA were the most active antioxidant peptides of 23 identified short‐chain (≤5 amino acids) peptides, with metal chelation activity of 94% and 96%, respectively. | Girgih et al., 2014d | |
| HMH diets reduced the rate of lipid peroxidation in spontaneously hypertensive rats with evidently higher plasma superoxide dismutase and catalase levels and lower total peroxide levels. | Girgih et al., 2014b | |
| HPH produced by protease N exhibited comparatively superior radical scavenging activity in DPPH system compared to HPHs by protease A and papain. | Das, 2015 | |
| Among all hydrolysates (prepared by AFP, HT, protease G, actinidin, and zingibain), HT hydrolysates exhibited the highest oxygen radical absorbance capacity (ORAC) and DPPH scavenging activity in the shortest hydrolysis time (2 hr). | Teh et al., 2016 | |
| The hydrolysates obtained using pancreatin exhibited stronger DPPH antiradical activity as well as reducing power than that derived by alcalase treatment. | Hadnađev et al., 2018b | |
| Metal binding | Zn2+‐binding capacity of HPHs was lower than that of HPI, but the water‐soluble Zn2+–HPH complex was more abundant in HPHs. Peptides by flavourzyme possessed the highest Zn2+‐binding activity, while those with pepsin exhibited the maximum solubility. | Wang et al., 2018 |
| Cell growth | Purified peptides from alcalase HPH by gel filtration and RP‐HPLC possessed protective effects for PC12 cells against oxidative stress‐mediated (H2O2) injuries. | Lu et al., 2010 |
| Hypocholesterolemic effects | Pepsin and trypsin HPHs showed higher inhibitory activity of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGCoAR) than pancreatin HPH. | Aiello et al., 2017 |
| HPH can inhibit the catalytic activity of HMGCoAR and modulate the HMGCoAR regulation by the activation of the phospho‐5’‐adenosine monophosphate‐activated protein kinase (AMPK) pathway. Applying HepG2 cells as model system, HPH induced upregulations of the protein levels of regulatory element binding proteins 2 (SREBP2), low‐density lipoprotein receptor (LDLR), and HMGCoAR, and increased the LDL‐uptake and the proprotein convertase subtilisin/kexin type 9 (PCSK9) levels. | Zanoni et al., 2017 | |
| Serum glucose regulation | HPH yielded higher dipeptidyl peptidase IV (DPP‐IV) inhibitory potency than the intact protein when subjected to simulated gastrointestinal digestion. | Nongonierma and FitzGerald, 2015 |
| High α‐glucosidase inhibitory activity was obtained from HPH by alcalase at the degree of hydrolysis 27.2%. Two novel α‐glucosidase inhibitory oligopeptides were isolated and identified having the sequences of Leu‐Arg (287.2 Da) and Pro‐Leu‐Met‐Leu‐Pro (568.4 Da). | Ren et al., 2016 |
To increase the efficacy, peptides in the protein hydrolysates can be fractionated, purified, and enriched. Girgih, Udenigwe, and Aluko (2011a,b) subjected HPI to the sequential action of pepsin and pancreatin and applied membrane ultrafiltration to separate the protein hydrolysate into different MW fractions. Three peptide fractions with MWs of 1 to 3, 3 to 5, and 5 to 10 kDa exhibited significantly greater radical scavenging activity but lower metal‐chelation activity, than the whole protein hydrolysate (HPH, that is, mixed peptides) (Girgih et al., 2011a).
Moreover, HPH was faster acting and more effective than the peptide fractions in reducing blood pressure in spontaneously hypertensive rats (Girgih et al., 2011b), indicating concerted actions of peptides present in the HPH. Reverse‐phase HPLC (RP‐HPLC) was subsequently used by these authors to obtain eight less heterogeneous peptide fractions based on the elution time (Girgih, Udenigwe, & Aluko, 2013).
Some of the peptide fractions demonstrated excellent antioxidant activity with higher oxygen radical‐absorbance capacity as well as the ability to scavenge superoxide anion and hydroxyl radicals than HPH. Several amino acids, including Lys, Leu, and Pro, appeared to contribute to the observed radical‐scavenging activity.
Tang et al. (2009) and Wang et al. (2009) also witnessed significant Fe2+‐chelating capability of HPH, reporting a strong correlation between the antioxidant activity and the hydrophobicity (hydrophobic amino acids) and the trichloroacetic acid (TCA)‐soluble peptide contents. It should be noted that some of the previous antioxidant studies also employed DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) in a single electron transfer test. However, because of the poor solubility of peptides in the DPPH ethanol assay solution, caution must be taken when interpreting the radical‐scavenging test results.
Great efforts have been made to identify the specific bioactive peptides from HPH and their sequences using robust analytical tools, such as mass spectrometry. A number of short‐chain peptides (≤5 amino acids) with remarkable bioactivity have been identified and purified. Girgih et al. (2014d) have isolated and sequenced 23 short‐chain peptides from the pepsin and pancreatin HPI digests through RP‐HPLC separation followed by tandem mass spectrometry analysis. Trp‐Val‐Tyr‐Tyr (WVYY) and Pro‐Ser‐Leu‐Pro‐Ala (PSLPA) were found to be the most potent antioxidant peptides, which were also capable of lowering the systolic blood pressure in spontaneously hypertensive rats.
Furthermore, Trp‐Tyr‐Thr (WYT) and Ser‐Val‐Tyr‐Thr (SVYT) isolated from the same HPH showed strong dual inhibition in vitro of ACE activity (89% and 79%, respectively) and renin (77% and 86%, respectively). Four ACE inhibitory peptides, namely, Gly‐Val‐Leu‐Tyr (GVLY), Ile‐Glu‐Glu (IEE), Leu‐Gly‐Val (LGV), and Arg‐Val‐Arg (RVR), have also been identified from acid‐hydrolyzed HPI by Orio et al. (2017), of which GVLY exhibited the lowest ACE‐inhibition IC50 values. Lu et al. (2010) also purified two peptides, Asn‐His‐Ala‐Val (NHAV) and His‐Val‐Arg‐Glu‐Thr‐Ala‐Leu‐Val (HVRETALV), from HPH that exhibited high antioxidant activity (in vitro) and protective effects against oxidative stress. More recently, two α‐glucosidase inhibitory peptides (Leu‐Arg and Pro‐Leu‐Met‐Leu‐Pro) were isolated from alcalase‐hydrolyzed HPI (Ren et al., 2016).
It seems that hydrophobic, acidic, and branched‐chain amino acids, which are abundantly present in these bioactive oligopeptides, positively contribute to the peptides’ function both in vivo and in vitro.
The production of bioactive peptides from hemp protein is obviously dependent upon the type and specificity of the proteases used as well as the degree of hydrolysis (DH). Malomo and Aluko (2016) applied six different proteases (pepsin, papain, thermoase, flavourzyme, alcalase, and pepsin + pancreatin) with concentrations ranging from 1% to 4% to hydrolyze HPI, finding that the resulting HPH prepared with 1% pepsin was the most active AChE inhibitor. The high AChE‐inhibitory effects of pepsin‐generated peptides are ascribed to increased synergistic actions from the broad size range when compared to other proteases used that produce narrower size range peptides.
Recently, our group (Wang & Xiong, 2018) investigated the metal‐binding potential of soluble compared with insoluble peptides obtained from HPH. Our results demonstrated different Zn2+‐binding activities for the hydrolysates produced with pepsin, alcalase, flavourzyme, papain, protamex, or trypsin (Figure 3).
Site‐specific cleavages played a major role in producing the observed diverse peptide profiles that varied in available Zn2+‐binding groups, that is, N–H, C = O, C–N, and COO–. Of the various protein hydrolysates, flavourzyme‐generated HPH exhibited the highest Zn2+‐binding capacity (88.8%), while pepsin‐HPH had the lowest (61.0%).
Zinc was found to bind to both water‐soluble (small) and insoluble (large) peptides to form complexes. However, the two fractions of peptides possessed different Zn2+‐binding sites, where N–H groups were the dominant site in insoluble peptides and C = O groups were the primary binding site in soluble peptides.
Applications in Food Products
Due to hemp’s well‐recognized nutritional value, food manufacturers have developed a wide range of retail products from hemp, such as nuts, oil, protein flour, energy bars, granola, hemp nut butter, pasta, and ice cream (Leson, 2006).
A recent emphasis has been on hemp protein, which is used not only as a nutritive additive but also as a functional ingredient in formulated foods to enhance the product quality attributes. The low allergenicity of hemp protein when compared with most of other plant proteins also permits it as a substitute for other proteins in some food products.
Table 3 lists food applications of hemp protein products, which are separated into five major categories: bakery products, extruded products, beverages, dairy and infant formula, and processed meat products.
The use of hempseed protein products as an alternative to the commonly used casein, whey, wheat, and soy protein is on a rise. For instance, some studies have shown that hemp protein products can be used as value‐added ingredients in the production of bread with increased protein and macro‐ and microelement contents, and lower baking loss and baking time (Korus, Witczak, Ziobro, & Juszczak, 2017b; Lukin & Bitiutskikh, 2017; Pojić et al., 2014).
The color of hempseed protein products can range from light tan to dark brown, depending on the pH condition used during processing and the temperature involved in the final product drying. The key to keeping hemp protein competitive in the plant protein market is to assure its nutritional value, functionality, safety, and acceptable sensory characteristics.Table 3. Edible uses reported for hemp protein and products
| Application | Reference |
|---|---|
| Bakery products | |
| Hemp flour up to 40% (w/w) can be incorporated into cracker formulation to replace rice flour. | Radočaj et al., 2014 |
| Hemp flour used to replace wheat flour up to 10% (w/w) did not have any negative effect on dough stability or strength, and enhanced nutritional value with increased protein and macro‐ and microelement contents. | Pojić et al., 2015; Apostol et al., 2015 |
| Incorporation of hemp flour resulted in significantly higher specific volume and spread ratio of baked cookies. | Hrušková and Švec, 2016 |
| Baking loss was reduced when adding hemp protein to wheat flour. | Ruban et al., 2016 |
| Replacing a portion of starch with hemp flour resulted in the weakening of dough structure, while HPC reinforced the structure. Hemp protein products, especially HPC, resulted in a significant increase in bread volume and limited hardening of the crumb and recrystallization of amylopectin during storage. | Korus et al., 2017b |
| The partial replacement of corn flour by hemp flour increased the hardness and nutritional value of biscuits but reduced sensory scores. | Korus et al., 2017a |
| Bread production using hemp flour reduced proofing time for the dough, and also the baking time. | Lukin and Bitiutskikh, 2017 |
| Extruded products | |
| Using hemp powder to replace rice flour increased the bulk density and antioxidant activity of extruded energy bars. | Norajit et al., 2011 |
| Defatted hemp cake was added to corn grits to make corn snack products. | Jozinović et al., 2017 |
| Beverages | |
| Bioactive compounds from muscadine grape and blackcurrant juice or muscadinepomace incorporated into hemp flour and HPI provided greater inhibition on L‐dopamine oxidation by tyrosinase and microbial proliferation. | Plundrich et al., 2013 |
| HPI can bind with a broad range of polyphenolic compounds in cranberry juice concentrate. | Grace et al., 2013 |
| Hemp protein added tomuscadine and kale juices significantly suppressed proinflammatory gene expression. | Grace et al., 2014 |
| Dairy and infant formula | |
| Hemp protein added to raw milk before pasteurization decreased syneresis and improved viscosity of yogurt more than pumpkin seed flour, soy and pea protein isolates, and wheat gluten. | Dabija et al., 2018 |
| Various hemp protein products (hulled or dehulled hempseed, HPC, hemp protein powder, and HPI) can be made into infant formula, including hemp milk and nutritional powder. | Wright and Sprague, 2015 |
| Processed meat products | |
| Adding hemp flour to the recipe of cutlets does not adversely affect the physicochemical quality parameters of semifinished meat products. | Naumova et al., 2017 |
Conclusions
Hempseeds, an emerging protein‐rich plant material, are becoming an important alternative protein source in the food and nutraceutical industry.
Since the interest of consumers worldwide for ingredients derived from natural sources is ever‐growing, the demand for hempseed protein is expected to grow rapidly.
though research has made progress in recent years in understanding the chemical composition, nutritional and health benefits, processing properties, and functional behavior of hempseed proteins in food processing, much remains unknown about it.
For example, many of the minor protein components present have not been characterized.
Therefore, it is clear that more systematic research is required to explore the structure–functionality relationship of hemp protein, especially its subunits. In spite of its nutritional value, the poor solubility of native hempseed protein impedes its specific functions and applications in food systems.
Therefore, structure‐modifying technologies must be vigorously explored through scientific research to convert hemp protein into a more soluble and diversely functional protein. Additional research is also needed to investigate the health benefits using molecular, cellular, and animal models. Such basic and applied research is essential to the development of this valuable protein source and broadening its market potential in the food industry.
