Improving the extraction of hemp proteins from hemp seed meals : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand

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The global population growth leads to an increasing demand for proteins. This increasing demand creates pressure on natural resources and the environment, of which the impact of animal protein sources is more significant than that from plant proteins. Besides that, the use of animal proteins might cause some health concerns to consumers. Therefore, alternative sources of proteins, such as plant proteins, are necessary. Oilseed meals (also known as oilseed cakes) are becoming increasingly promising alternative protein sources; they have high protein content and can be a sustainable source. Oilseed cakes are considered a by-product of the oil industry and their use as a protein source would create less impact on the environment. Hemp seeds have a significant content of protein highly digestible and with great nutritional value. However, the application of hemp protein into food products has not been fully exploited because of several technological challenges, such as the poor solubility in water-based foods - a key challenge in the adoption of novel sources of proteins. The aim of this work was to explore different approaches for improving the extractability of hemp protein from hemp seed meals and investigate the effect of processing conditions on the yield of extraction and the characteristics of resulting proteins. Four commercially available hemp seed meals (HSMs) in this study were chosen based on the availability in the New Zealand markets and investigated for physicochemical characteristics to understand the properties of the starting material for the protein extraction process. The microstructure, proximate compositions, particle size, and protein solubility of four HSMs was measured. The effect of reduced particle size by using nitrogen grinding on the solubility of hemp seed protein was studied. The compositions and particle size distributions of solubilised hemp proteins in water were characterised by using SDS-PAGE and dynamic light scattering, respectively. To improve the extractability of hemp seed proteins from HSMs, two approaches were taken in this work. Firstly, the effect of different process variables was investigated; these included heating, pH adjustment, the use of salts, pH cycling and ultrasound. The effect of heating conditions on extractability was investigated using three different temperatures (20, 65 and 95 °C). The pH 7 and pH 9 were used to investigate the effect of pH on protein extractability. Four types of salt (KCl, NaCl, CaCl₂ and MgCl₂) were added at different concentrations (0 to 0.8 M) to determine the effect of salts on the protein extraction yield. Two pH cycles (pH-9-2-9) and (pH 9-12-9) were employed to investigate whether pH cycling improves the yield. In addition to the above, ultrasound was used at different amplitudes (20 and 80%), different temperatures (20, 65 °C), and in different ultrasonic duration times and the impact of these conditions on extraction yield was investigated. These treatment combinations were studied further in the second approach. To investigate the combined effect of ultrasound with pH cycling and the addition of salt, the condition of amplitude 80%, 65 °C, and 15 min was used. The extractability of proteins was derived from the estimation of protein contents on the supernatants obtained after centrifugation of samples (protein extraction yield), and this was analysed to determine the effectiveness of treatments. In addition, the protein compositions, total phenolic and available lysine content, and microstructure of hemp seed meals after protein extraction were analysed to understand the impact of treatment on resulting protein characteristics and food matrix. The commercial HSMs powder contained 48 to 65% protein. However, their corresponding aqueous mixtures (10g powder/90 mL water) had low protein solubility, from 6 to 13%. Reducing the particle size of HSMs by nitrogen grinding improved the solubility of hemp proteins (22% higher than non-grinded samples). The compositions of hemp protein solubilised in the water were not significantly different between the four samples investigated and included two main fractions albumin and globulin. The results of this study reveal that heating at 65 °C and pH 9 are the optimal conditions for hemp protein extraction. The combination of these two optimal conditions led to a yield of approximately 31%. Under these conditions (65 °C, pH 9), the additional treatments showed a further increase in the extraction of hemp proteins. For example, the addition of salt (NaCl) up to 0.8 M resulted in a significant increase in the extraction yield (up to 81.2 %). In comparison, the alkaline pH cycling (pH 9-12-9) increased the yield to approximately 47%. Furthermore, the application of ultrasound treatment (amplitude 80%, 15 min) showed an extraction yield of 37 %. The combination of ultrasound and addition of salt showed a synergistic effect at a low salt concentration (NaCl 0.25 M), but not at higher concentrations. Similarly, ultrasound treatment combined with alkaline pH cycling (pH 9-12-9) increased the yield of extraction by 81%. The SDS-PAGE analysis of extracts showed that samples from all investigated treatments had largely similar protein compositions. The albumin and two globulin fractions (globulins 7s and 11s) were contained in the extracts. Almost all conditions resulted in co-extraction of phenolic compounds but the total phenolic content in the extracts varied as per extraction conditions. The ultrasound treatment extracted most efficiently the phenolic compound in hemp seed meals. Furthermore, the use of alkaline pH cycling reduced the available lysine content per gram extracted protein, while the ultrasound treatment increased this value. Overall, the results of this study have compared the different methods of extraction on the extractability of hemp proteins. This might provide the basis for the design protocols of hemp protein extraction and extend research on the functional properties and applications of hemp proteins.
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