Bacteria used for dietary foods

Phenylketonuria (PKU) is one of the most common inherited metabolic disorders in Germany. The disease prevents affected individuals from breaking down the amino acid phenylalanine due to a missing or reduced-activity enzyme. As a result, phenylalanine accumulates in the body at high concentrations. This accumulation is neurotoxic and can lead to irreversible brain damage.

Phenylalanine is an essential amino acid for humans and is present in almost all animal-based and protein-rich plant foods. Therefore, affected individuals must adhere to a lifelong low-protein diet, primarily consisting of natural foods with low protein content, such as fruits and vegetables. In addition, they use substitute products in the form of phenylalanine-free amino acid mixtures, which provide the essential amino acids they cannot produce themselves and cannot obtain sufficiently from their diet.

These amino acid mixtures are usually produced synthetically and have several disadvantages, such as limited solubility. Moreover, their organoleptic properties—taste, appearance, smell, and color—often leave room for improvement. Consequently, research and industry are continuously seeking new dietary approaches for PKU patients that offer improved taste and more versatile applications in food preparation.

The Fraunhofer IME is therefore conducting research on phenylalanine-low or phenylalanine-free proteins to compensate for the drawbacks of synthetic production. Instead of using an amino acid mixture, a naturally occurring protein should be identified that best meets the requirements for a phenylalanine-free diet. The desired protein should have a balanced amino acid profile that allows for a high biological value (>100), as such proteins are efficiently converted into body proteins. Additionally, low allergenic potential is an important criterion. For optimal production in bacteria, the protein should be relatively small (<50 kilodaltons).

Using an algorithm that considered these requirements, over 830,000 proteins from animal, plant, and microbial organisms in the UniProt database (www.uniprot.org) were analyzed—specifically those already used in human nutrition or for which products are produced (e.g., lactic acid bacteria for yogurt production). Together with metaX Institut für Diätetik GmbH, the researchers identified a protein from the hay bacterium Bacillus subtilis as most suitable. However, it still contained phenylalanine, and the essential amino acid tryptophan was missing. Using molecular biology techniques, the scientists successfully replaced phenylalanine with tryptophan.

For protein production in the food industry, microbial cell factories such as yeasts or bacteria are used. In this project, the researchers employed the bacterium Pseudomonas fluorescens, which can produce proteins very efficiently, with yields in the double-digit gram-per-liter range. To produce the phenylalanine-free protein, the corresponding gene was introduced into the bacterium. Initial cultivations in shake flasks (2.5 liters, with 0.5–1 liter culture volume) already achieved 2.5 grams per liter. Cultivation in bioreactors (5–350 liters working volume) increased productivity to an average of 20 grams per liter, enabling the production of 3.5 kilograms of protein within a few months. Purification was performed with high yield via affinity chromatography, followed by the removal of excess salts through filtration and drying via freeze-drying and/or spray-drying.

The protein has an almost neutral taste and good solubility, making it suitable for the preparation of dietary foods. Initial tests in mice with PKU showed that the protein normalized phenylalanine levels in the blood. Further studies are now required to make it available for humans with phenylketonuria.