Hemp: A Sustainable Plant with High Industrial Value in Food Processing

The major protein in hemp kernel is edestin, accounting for around 70% of hemp protein. Edestin is a hexamer of identical subunits and belongs to the globulin family [57]. Each subunit consists of the acidic (−34 kDa) and the basic (18–20 kDa) chains [58]. An original single protein is cleaved into these two chains post-translationally at the Asn-Gly site [57]. An edestin subunit has five cysteine residues, two of which form a single intermolecular disulfide bond between basic and acidic subunits. The acidic subunit has one intramolecular disulfide bond. The remaining cysteine has a free sulfhydryl (SH) group. Edestin is less soluble in water or buffer with neutral or acidic pH, but soluble in a basic buffer [59]. As solubility in water and content of free SHs increases by sonication or pH adjustment of the edestin solution, control of the protein structure/function may be possible in food processing [60]. Despite its less solubility, edestin is known for its high digestibility [61,62].

The second major hemp protein is albumin, which has fewer disulfide bonds compared to edestin (globulin), thus having a flexible structure with higher protein solubility and foaming capacity [59]. Another noteworthy protein is rich in Met and Cys, and 20 mole % of the total amino acids contain sulfur [63]. It consists of two subunits made of 27 and 61 amino acid residues, respectively, which are held together by two intermolecular disulfide bonds [63].

Recently, reverse micelles (RMs) technology has been applied for protein extraction as it is convenient and cost-effective [64]. Protein extraction by RMs is achieved via two steps: “forward and backward” extractions. In the forward extraction, soluble proteins are encapsulated in the inner aqueous core of RMs, which are then recovered in the backward extraction by disrupting the RMs. Protein extraction from hemp flour using RMs did not require prior defatting in terms of extraction performance [65]. Hemp protein isolates obtained from non-defatted and defatted hemp flour shared similarities in proximate composition, subunit structure, as well as principal properties of protein including solubility, isoelectric point, emulsifying/foaming abilities, and thermal stability. Meanwhile, protein from non-defatted flour exhibited higher β-sheet content and surface hydrophobicity. Furthermore, higher least gelling concentration and higher gel strength were observed in the gel formed by protein from non-defatted hemp flour [65].

In vitro digestion tests demonstrated that hemp protein has a high degree of digestibility [61]. Moreover, most hemp allergens, such as the major thaumatin-like protein and lipid transfer protein, were eliminated in the protein-isolation or digestion processes. Mamone et al. [61] concluded that hemp protein is usable as an ingredient for hypoallergenic foods.

Hemp protein has a unique feature in view of the disulfide structure. It shows much higher free sulfhydryl content than soy protein [66]. The cysteine content of hemp protein is 1.6–1.4 g cysteine/100 g protein [32,67], higher than pea (1.0 g cysteine/100 g protein) and broadbean or soybean (1.1–1.3 g cysteine/100 g protein) [68]. Moreover, at pH 8, while the free sulfhydryl content of soy protein is 1.6 × 10⁶ mol/g protein, the counterpart of hemp is as high as 3.9 × 10⁶ mol/g protein [66]. Tang et al. [66] discussed that the poor functional properties of hemp protein seems mostly ascribed to the formation of intermolecular disulfide bonds between individual proteins and subsequent aggregation at neutral or acidic pH, because it has a high free sulfhydryl content. Meanwhile, exchange reactions between disulfide (S-S) and free SH play critical roles in the cereal biology [69] as well as food processing [70,71]. In the preparation of microparticles made from soy protein isolate and egg white, the egg white protein serves as a free sulfhydryl “donor” to accelerate intermolecular S-S linkage with the soy protein [72]. Thus, hemp protein with a high free SH content should have unexplored unique processing characteristics. Heat-dependent formation of the envelope made of hemp and soy protein is just a glimpse of this attribute [73,74].

Hemp cake, a residual material of oil expression from the hemp seed, has long been utilized for livestock feeds [100,101]. It is a highly nutritive as well as sustainable feed stuff for cows [102], quails [103], cockerels [104], pigs [105], and broilers [106]. Now, research is in progress to utilize hemp protein “directly” as a material for plant meat.

The commercially available vegan meat products, represented by high moisture meat analogues (HMMAs), are made mostly from soy. In the production of HMMA, protein-rich materials, such as soy protein isolate, are subjected to a twin screw co-rotating extruder. Thermomechanical stresses are applied to the material at a high-water content (>40% w/w) followed by forcing through a cooling die [107]. Soy-based HMMAs have a desirable chewy meat-lite texture. However, cultivating soy in colder climates such as in northern Europe is challenging. Thus, Zahari et al. [108] sought to investigate whether and to what extent soy protein isolate could be replaced by hemp protein concentrate in the production of HMMAs. A rapid visco analyzer (RVA) and differential scanning calorimeter (DSC) were used to investigate pasting features and melting temperature of the raw materials. They found that hemp protein absorbed less water and requested higher temperature for denaturation compared to soy protein. However, replacement of soy protein with hemp protein was possible up to 60% to yield layered and fibrous meat-like extruded products. Based on the DSC and RVA results, a higher cooking temperature and longer retention time are recommended for the extrusion of hemp/soy meat, as hemp protein needs a higher temperature for denaturation. Furthermore, to develop a more laminar fiber structure, the interior structure of the extruder should be equipped with more complicated kneading elements such as screws to hold the material longer in the extruder. Zahari et al. [108] concluded that while future studies are needed to optimize the condition of the extrusion process and the formulation matrix, it is possible to substitute soy protein with hemp protein without sacrificing the quality in meat analogue formulation.

To develop plant-based meat with a realistic “meaty” texture, anisotropy and fibrousness are among the most critical factors [109,110]. Anisotropy is the property of a material expressing different behaviors depending on the directions from which the external pressure is applied [111]. Meat structure is highly directional. On the molecular scale, actin and myosin form well-ordered and parallel arrays of filaments. On the macroscale, those filaments form into the muscle fiber bundles [112].

During a high-moisture extrusion of plant protein-based materials, the dynamics of protein aggregation and phase separation are the keys for the formation of meaty fibrous structures. The fibrousness is expressed during migration from the die to the cooling zone through a “sub-layer transformation” cross-linking [113]. The desired anisotropic structure of plant-based meat analogues has been accomplished by extrusion at high water content (>40%) and at elevated temperatures (>100 °C) followed by passing through a cooling die which prevents expansion of the matrix at the ejection from the extruder [114]. Interestingly, there are two distinct hypothetical mechanisms to explain how the anisotropic structure is made in the extruded plant meat. One is the “cross-linking” mechanism in which the anisotropic structure is explained as being formed by the alignment of protein and the subsequent stabilization at the molecular level. Protein molecules are unfolded and align along the direction of flow, followed by stabilization of the aligned proteins by way of interactions between/among proteins newly developed by disulfide or hydrophobic interactions.

The other is the “multiphase” mechanism in which formation of the anisotropic structure is explained due to the existence of multiphase systems [114]. Thermodynamic immiscibility of the biopolymers involved triggers the occurrence of phase separation in the extrusion process. The dispersed phase is deformed in the extruder die, then directed along the flow. The subsequent cooling process solidifies the material resulting in the anisotropic structures of the plant-meat products. In the case of extrudates using soy protein isolate solely as a protein source, the formation of anisotropic structures are derived from a multiphase system. Cryo-imaging and X-ray analysis of the extrudates revealed a water-rich dispersed phase surrounded by a continuous protein-rich phase with less moisture. Meanwhile, significant changes of protein–protein interactions were not observed [114]. Thus, in this model system, multiphase systems rather than cross-linking of proteins seemed to be the primary factor of the anisotropic structure.

On the other hand, during high-moisture extrusion processing of meat analogues made of pea protein and fatty acids, protein–protein interactions played key roles in the product structure [115]. Micromorphology analysis demonstrated that formation of anisotropic fibrous structures in the cooling die was disturbed by the coalescence of fatty acids of an unsaturated type, such as oleic and linoleic acids. Meanwhile, saturated stearic acid dispersed uniformly in the protein matrix, facilitating formation of disulfide bonds and promoting the generation of anisotropic fibrous structures along the extrusion direction [115]. Moreover, in the case of plant meat made by high-moisture extrusion processing of pea protein, amylopectin, and stearic acid, its anisotropic fibrous structures have been explained by the “anchor orientation and flexible cross-linking” mechanism [116]. In the cooling zone, stearic acid played the role of anchors, preventing the unfolded protein structure from refolding. In contrast, amylopectin facilitated the rearrangement, disulfide formation, and polymerization of the protein molecules. Thus, amylopectin and stearic acid synergistically mitigated the interaction between proteins. These aggregates with loose and flexible structures aligned along the extrusion direction and successfully formed anisotropic and fibrous structures in the extruded products.

Nasrollahzadeh et al. [67] compared the structure of plant meat made of hemp protein with the counterpart made of pea proteins. The proteins were respectively mixed with maize starch and were subjected to high moisture extrusion. The extruded pea meat was soft and isotropic while hemp meat was hard and anisotropic. The protein structure was investigated using SDS-PAGE in the presence or absence of a reductant, dithiothreitol (DTT). In the case of the hemp meat sample, some protein bands derived from edestin appeared only in the presence of DTT. In contrast, less-intense protein bands of pea meat appeared regardless of the presence or absence of the reductant. The results demonstrate a higher contribution of disulfide cross-linking in the polymerization of hemp protein during the extrusion process than in the case of pea protein. As mentioned above, hemp protein contains more cysteine (1.6–1.4 g cysteine/100 g protein) than pea protein (1.0 g of cysteine/100 g protein). Thus, in the case of hemp meat, protein–protein interactions played critical roles in the formation of anisotropy and fibrous-like mesoscale structures. More recent studies support the view by demonstrating that addition of cysteine controls the texture of plant meat. Addition of cysteine changed both the physical and chemical properties of extrudates made from soy protein isolate and wheat gluten [117]. The SH-containing amino acid promoted the fiber structure formation and affected the degree of texturization and rheological properties, as well as microstructures of the extrudates by rearranging the disulfide-mediated cross-linking among protein molecules [117]. Meanwhile, addition of L-cysteine or L-ascorbic acid on the material of the pea protein/wheat gluten blend altered the fibrousness and the mechanical properties of the meat analogue obtained by the high-temperature shear cell [118]. Cysteine accelerated protein polymerization through the disulfide–sulfhydryl exchange reactions in the heating process, yielding a continuous protein network upon cooling.

Conclusively, the alternative mechanism of the generation of fibrousness and anisotropy in the plant meat, whether cross-linking or multiphase, depends on the protein species, extraction process of the protein, and the subsidiary materials such as starches and fatty acids. In the case of hemp protein, its high content of free sulfhydryl groups is expected to produce the unique “meaty” texture of the products.

Recently, it was reported that a dough made of egg white and soy protein isolate was baked into a high-protein, low-carbohydrate bread [73]. Egg white protein and soy protein formed heat-dependent elastic air-cells which work to confine enlarging gas in the baking process. Intermolecular S-S bonds contribute to the formation of the cell membranes in which egg white protein works as a donor of SHs to soy proteins [72,73]. Similarly, a dough made of hemp protein/soy protein is baked into a hollow ball (Figure 3) [74].

The outer shell is made of the mixture of soy/hemp proteins which are linked with intermolecular disulfide bonds. Dietary fiber, isolated from the protein complex, is located in the bottom of the ball. Thus, the ball has a low center of gravity and behaves like a “roly-poly” wiggle ball. In this case, hemp protein, rich in free SHs [63,66], is considered to work as a SHs donor, as egg white protein worked as a donor of free SHs in the bread made of egg white/soy protein isolate [73]. Electron microscopic observation of the protein-rich outer shell (Figure 3a) and fiber-rich inner bottom part (Figure 3b) reveals distinct structures. The former has a dense structure while the latter has a reticulated structure with small void spaces. Contribution of the unique structure to the ball’s texture is being investigated by sensory evaluation in our lab. Conclusively, application of hemp protein on food processing will broaden the variation of the food products.