Modification, characterization and application of hemp (Cannabis sativa L.) protein
Creators: name: Liu, Xinye
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 300607
subject: Food technology

description: Hemp (Cannabis sativa L.) protein is an emerging alternative protein. It is known to possess high nutritional value (availability of most essential amino acids), better digestibility and some health-promoting functions (e.g., lowering blood pressure). In recent years, emerging proteins (e.g., hemp protein) are attracting increased attention as animal proteins are more expensive to produce and come with greater carbon footprint. The functional (e.g., solubility, emulsifying and encapsulating) properties of proteins are particularly important for their application in the food industry. However, the functional properties of hemp protein are inferior to many commonly used plant proteins (e.g., soybean protein and pea protein), which limits its application in food industry. To address this limitation, this study aimed to improve solubility, emulsifying properties, encapsulating and antimicrobial properties of hemp protein. To achieve these aims, physical (ultrasound treatment), chemical (complexation with polysaccharides and polyphenols), and enzymatic hydrolysis were applied to improve the functional properties. 

These modification methods were followed with evaluation of their effectiveness in producing oil-in-water emulsions, microencapsulation of essential oils and preparation and application of edible film. Time, temperature and salt ion stability of emulsion, antioxidant property of the microcapsules and the ability of the peptide-based film to extend the shelf life of a common perishable fruit (blue berry) were used to assess the effectiveness of above-mentioned physicochemical modifications.

The first experimental chapter (Chapter 3) focused on enhancing the solubility and emulsifying properties of hemp protein isolate (HPI) through ultrasound treatment. To determine the underlying mechanism for this improvement, particle size, surface hydrophobicity, and molecular structure were analyzed. The optimal ultrasound condition was identified as 400 W for 12 minutes at which an increase of solubility by 3.7-fold, emulsion activity index (ESI) by 1.7-fold and emulsion stability index (ESI) 1.5-fold was achieved. Compared to commercial pea protein, ultrasound treated HPI showed better solubility, EAI and ESI indicating to its potential in food industry.

The second experimental chapter (Chapter 4) aimed to improve the stability of HPI-stabilized emulsions through the formation of complex coacervates between HPI and commonly available polysaccharides (gum Arabic, sodium alginate, and pectin). The optimum parameters for the formation of complex coacervates (protein-polysaccharide complexes formed through electrostatic attraction) were found to be HPI-to-gum Arabic ratio of 4:1 at pH 3.5, HPI-to-sodium alginate ratio of 16:1 at pH 3.5 and HPI-to-pectin ratio of 12:1 at pH 4.0. To determine the effectiveness of these complex coacervates as emulsifier, pH, temperature, and ionic stability of emulsions stabilized by them was evaluated. Complex coacervates were synthesized using in-situ (where complex coacervation occurred instantly during emulsification and covered the oil droplets) ex-situ (where pre-formed complex coacervates were used as emulsifiers) methods to come up with a better protocol to achieve better stability. The results indicated that the emulsions prepared by in-situ method had better storage stability at cold temperature (4°C), while the emulsions prepared by the ex-situ method were more stable against change in temperature and ionic strength.

The third experimental chapter (Chapter 5) studied the application of HPI-polysaccharide complex coacervates as encapsulating shell material for essential oil. The HPI-gum Arabic complex coacervates were utilized to encapsulate essential oils of mustard, lemon, grapefruit, and oregano. The study evaluated the effects of the type (nature) of essential oil on encapsulation efficiency, dispersibility in aqueous phase, thermal stability, microstructure, release pattern, antioxidant and antibacterial properties. The results showed that among the microcapsules, those loaded with oregano essential oil exhibited the highest encapsulation efficiency (57%), better dispersion in aqueous phase, more compact structure, slow rate of release, higher thermal stability, and antioxidant activity. Encapsulation also improved the antibacterial activity of the essential oils, with the exception of oregano essential oil, which showed higher antibacterial activity in its free form.

The fourth experimental chapter (Chapter 6) focused on improving the above-mentioned properties of HPI by covalently conjugating it with polyphenols. Two easy-to-implement methods, i.e., alkali treatment and ultrasound-assisted alkali treatment, were used to synthesize HPI-polyphenol (gallic acid and catechin) conjugates. The effectiveness of these two methods on the degree of conjugation, particle size, surface charge density, secondary and tertiary structures, surface hydrophobicity, solubility and emulsifying properties, antioxidant activities of the resulting conjugates were determined. The effectiveness of the conjugates on emulsion stability (storage, temperature, and salt stability) was also evaluated. The conjugation with polyphenol significantly improved the solubility, emulsifying properties of HPI, especially in the case of HPI-gallic acid conjugate. The conjugation of HPI with these two polyphenols also improved its antioxidant activities. Emulsions stabilized by HPI-polyphenol conjugates also exhibited higher storage, temperature, and salt stability.

The fifth experimental chapter (Chapter 7) focused on application of HPI in producing edible films intended to be used to extend the shelf life of perishable common fruit (blue berry). Peptides of HPI were produced through enzymatic hydrolysis. Then, carboxymethyl chitosan (CMCh)-peptide conjugates were produced by grafting CMCh (through Maillard reaction) with peptides obtained from HPI, maize and casein. Nanoemulsions of Camellia essential oil were produced using these conjugates as emulsifiers and then used to develop active films. The physicochemical properties of resulting films were evaluated. The film prepared with peptide-peptide conjugate showed better water vapor and oxygen barrier properties and mechanical properties compared to those of the film produced using CMCh. The peptide-CMC film extended the shelf life of blueberry to 12 days.

This research expands the knowledge base which helps to expand the application of hemp protein. The methods and the associated underpinning science developed and documented in this thesis helps to substantially improve the solubility, emulsifying, encapsulating active film forming properties of HPI and broadens its scope of application in food industry. By helping to expand its application (emulsions, microcapsules, active film) of this less commonly used plant protein, it contributes to goal of sustainable development. This research also contributes to expanding the science associated with ultrasound, complex coacervation, covalent conjugation paving the way for their optimum use.