Green Biomasses as a Source of Protein for Human Consumption

This PhD thesis presents the work done during the project ”Green Biomasses as a Source of Protein for Human Consumption”. By now it is well established that climate change is caused by human activities and to ensure food security for the growing population of the future, new climate friendly protein sources are needed. It was found early in the project that alfalfa Medicago sativa was the best suited candidate as one of the sources of new proteins for human consumption. Alfalfa contains all essential amino acids, contains only low amounts of anti nutritional factors, and the plant is already considered safe for consumption by EFSA. One of alfalfa’s main proteins, RuBisCO, is known for it ability to emulsify and foam and the plant alfalfa is known to have low requirements with respect to irrigation and fertilizers. Lastly alfalfa protein concentrate has been approved since 2009, as a food supplement with an daily intake of 10 g pr day, thereby lowering the potential requirements for using it as a food ingredient to meet the food requirements of the future.
In the project an investigation of using screw presses for green protein recovery from alfalfa at laboratory and pilot scale was conducted. It was found that 16% of the total protein was recovered in one pressing, and that after re-hydrating and repressing the alfalfa up to ten times, 48% of the total protein could be recovered with a single screw press in pilot scale. The composition of the generated protein concentrate was analysed with respect to total protein, amino acid profile, protein digestibility, color, ash, fiber and fat content. It was found that repetitive pressings lowered the digestibility of the protein and lowered the total protein concentration due to dilution. In this study it was concluded that pressing should only be repeated once to obtain a protein concentrate with 32% soluble protein and more than 82% digestibility.
Following the investigation of the production of a green protein concentrate at pilot scale, a study was conducted to refine and improve the quality parameters with respect to taste, appearance and digestibility. This resulted in the development of a simplified method for the production of a white alfalfa protein concentrate. The produced concentrate still contained undesired flavours that limited its usage in foods, which was sought solved by including a step with supercritical CO₂ extraction in the proposed setup. Two concentrates were produced at lab scale and pilot scale, with yields of 0.012 g (lab scale) and 0.08 g (pilot scale), of protein per g of total protein introduced from the alfalfa plant in the process. The solubility of the protein produced at lab scale and pilot scale was approximately 30% and 15%, respectively and the digestibility of the pilot scale white protein was found to be above 92%. By including supercritical CO₂ extraction at 220 bar, 45 °C for 75 min, off-flavours were lowered to an acceptable threshold for the consumer. It was found that the supercritical CO₂ extraction did not affect the digestibility or the functionality of the protein, when the protein was included in formulations of meringues and chocolate muffins. However, it was concluded that further studies would be needed to optimize the extraction parameters with respect to the taste.
Two other studies was carried out in the project, with respect to the side-streams generated from white alfalfa protein concentrate production. In the first study the residual pulp was utilized as a bio-polymer in a bio-composite with PLA in concentrations up to 50%. It was found that the properties of the bio-composite was lowered when increasing the concentration of alfalfa pulp (tensile strength from 64.54 MPa (blank) to 38.48 MPa (50%), elongation (from 4.11 % (blank) to 1.63 % (50%)). It was concluded that future studies should consider other pre-treatment methods of the pulp to increase the potential of using alfalfa pulp as a bio-polymer.
The other side-stream, brown juice, was also investigated in this project. Due to high concentrations of lactic acid in the brown juice, derived from the proposed processing method for the production of white alfalfa protein concentrate, an investigation of using the brown juice as a fermentation medium with lactic acid metabolising organisms was conducted. Corynebacterium glutamicum-ATCC-13032 (wildtype) and the α-amylase secreting GMO strain Corynebacterium glutamicum-SB025, was grown in brown juice with or without supplementation of yeast extract and glucose. After 24 h of growth Corynebacterium glutamicum-ATCC-13032 had a final OD600 of 44.6 (μmax 0.55) in brown juice without supplements and an OD₆₀₀ of 45.45 (μ_max 0.70) in the brown juice with yeast extract and glucose. The GMO strain, Corynebacterium glutamicum-SB025, had an final OD₆₀₀ of 29.35 (μ_max 0.47) in the unsupplemented brown juice and an final OD₆₀₀ of 50.8 (μ_max 0.545) in the brown juice with yeast extract and glucose. It was concluded that brown juice is a promising fermentation medium for Corynebacterium glutamicum, but further studies are needed to optimize the process and investigate the metabolism occurring while fermenting.
In the end of the study an application to EFSA was generated applying for an extended usage approval of white alfalfa protein concentrate. The application lists 18 different Foodex2 lvl 7 product that would lead to an average daily intake of 10 g of white alfalfa protein concentrate, thus being within the limit set by EFSA. The application for an extended usage of white alfalfa protein concentrate as a food ingredient, concluded that white alfalfa protein concentrate produced in this project would be safe for humans to consume.