Co-cultures of Propionibacterium freudenreichii and Bacillus amyloliquefaciens cooperatively upgrade sunflower seed milk to high levels of vitamin B12 and multiple co-benefits

Abstract

Background Sunflower seeds (Helianthus annuus) display an attractive source for the rapidly increasing market of plant-based human nutrition. Of particular interest are press cakes of the seeds, cheap residuals from sunflower oil manufacturing that offer attractive sustainability and economic benefits. Admittedly, sunflower seed milk, derived therefrom, suffers from limited nutritional value, undesired flavor, and the presence of indigestible sugars. Of specific relevance is the absence of vitamin B12. This vitamin is required for development and function of the central nervous system, healthy red blood cell formation, and DNA synthesis, and displays the most important micronutrient for vegans to be aware of. Here we evaluated the power of microbes to enrich sunflower seed milk nutritionally as well as in flavor. Results Propionibacterium freudenreichii NCC 1177 showed highest vitamin B12 production in sunflower seed milk out of a range of food-grade propionibacteria. Its growth and B12 production capacity, however, were limited by a lack of accessible carbon sources and stimulants of B12 biosynthesis in the plant milk. This was overcome by co-cultivation with Bacillus amyloliquefaciens NCC 156, which supplied lactate, amino acids, and vitamin B7 for growth of NCC 1177 plus vitamins B2 and B3, potentially supporting vitamin B12 production by the Propionibacterium. After several rounds of optimization, co-fermentation of ultra-high-temperature pre-treated sunflower seed milk by the two microbes, enabled the production of 17 µg (100 g)−1 vitamin B12 within four days without any further supplementation. The fermented milk further revealed significantly enriched levels of l-lysine, the most limiting essential amino acid, vitamin B3, vitamin B6, improved protein quality and flavor, and largely eliminated indigestible sugars. Conclusion The fermented sunflower seed milk, obtained by using two food-grade microbes without further supplementation, displays an attractive, clean-label product with a high level of vitamin B12 and multiple co-benefits. The secret of the successfully upgraded plant milk lies in the multifunctional cooperation of the two microbes, which were combined, based on their genetic potential and metabolic signatures found in mono-culture fermentations. This design by knowledge approach appears valuable for future development of plant-based milk products.

Microaerobic metabolism of lactate and propionate enhances vitamin B12 production in Propionibacterium freudenreichii

“…Our results suggest it is key to keep oxygen fluxes low in order to maintain the ability to oxidise substrates using oxygen as a terminal electron acceptor. These results are supported by results of Tangyu et al [ 29 ], who report highest B 12 production at specific oxygen regimes in their food product. Since oxygen is required as terminal electron acceptor for oxidation of propionate, constraining oxygen to low levels results in oxygen being the growth rate determining factor.…”