Non-dairy milk alternatives (or milk analogues) are water extracts of plants and have become increasingly popular for human nutrition. Over the years, the global market for these products has become a multi-billion dollar business and will reach a value of approximately 26 billion USD within the next 5 years. Moreover, many consumers demand plant-based milk alternatives for sustainability, health-related, lifestyle and dietary reasons, resulting in an abundance of products based on nuts, seeds or beans. Unfortunately, plant-based milk alternatives are often nutritionally unbalanced, and their flavour profiles limit their acceptance. With the goal of producing more valuable and tasty products, fermentation can help to the improve sensory profiles, nutritional properties, texture and microbial safety of plant-based milk alternatives so that the amendment with additional ingredients, often perceived as artificial, can be avoided. To date, plant-based milk fermentation mainly uses mono-cultures of microbes, such as lactic acid bacteria, bacilli and yeasts, for this purpose. More recently, new concepts have proposed mixed-culture fermentations with two or more microbial species. These approaches promise synergistic effects to enhance the fermentation process and improve the quality of the final products. Here, we review the plant-based milk market, including nutritional, sensory and manufacturing aspects. In addition, we provide an overview of the state-of-the-art fermentation of plant materials using monoand mixed-cultures. Due to the rapid progress in this field, we can expect well-balanced and naturally fermented plant-based milk alternatives in the coming years.

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The Serine Protease Inhibitor (serpin) protein has been suggested to play a key role in the interaction of bifidobacteria with the host. By inhibiting intestinal serine proteases, it might allow bifidobacteria to reside in specific gut niches. In inflammatory diseases where serine proteases contribute to the innate defense mechanism of the host, serpin may dampen the damaging effects of inflammation. In view of the beneficial roles of this protein, it is important to understand how its production is regulated. Here we demonstrate that Bifidobacterium longum NCC 2705 serpin production is tightly regulated by carbohydrates. Galactose and fructose increase the production of this protein while glucose prevents it, suggesting the involvement of catabolite repression. We identified that di- and oligosaccharides containing galactose (GOS) and fructose (FOS) moieties, including the human milk oligosaccharide Lacto-N-tetraose (LNT), are able to activate serpin production. Moreover, we show that the carbohydrate mediated regulation is conserved within B. longum subsp. longum strains but not in other bifidobacterial taxons harboring the serpin coding gene, highlighting that the serpin regulation circuits are not only species- but also subspecies- specific. Our work demonstrates that environmental conditions can modulate expression of an important effector molecule of B. longum, having potential important implications for probiotic manufacturing and supporting the postulated role of serpin in the ability of bifidobacteria to colonize the intestinal tract.

Within the brewing industry, malted barley is being increasingly replaced by raw barley supplemented with exogenous enzymes to lessen reliance on the time-consuming, high water and energy cost of malting. Regardless of the initial grain of choice, malted or raw, the resultant bulk spent grains are rich in proteins (up to 25% dry weight). Efficient enzymatic solubilization of these proteins requires knowledge of the protein composition within the spent grains. Therefore, a comprehensive proteomic profiling was performed on spent grains derived from (i) malted barley (spent grain A, SGA) and (ii) enzymatically treated raw barley (spent grain B, SGB); data are available via ProteomeXchange with identifier PXD008090. Results from complementary shotgun proteomics and 2D gel electrophoresis showed that the most abundant proteins in both spent grains were storage proteins (hordeins and embryo globulins); these were present at an average of two fold higher in spent grain B. Quantities of other major proteins were generally consistent in both spent grains A and B. Subsequent in silico protein sequence analysis of the predominant proteins facilitated knowledge-based protease selection to enhance spent grain solubilization. Among tested proteases, Alcalase 2.4 L digestion resulted in the highest remaining protein solubilization with 9.2 and 11.7% net dry weight loss in SGA and SGB respectively within 2 h. Thus, Alcalase alone can significantly reduce spent grain side stream, which makes it a possible solution to increase the value of this low-value side stream from the brewing and malt extract beverage manufacturing industry.

Bifidobacteria are amongst the first bacteria to colonize the human gastro-intestinal system and have been proposed to play a crucial role in the development of the infant gut since their absence is correlated to the development of diseases later in life. Bifidobacteria have the capacity to metabolize a diverse range of (complex) carbohydrates, reflecting their adaptation to the lower gastro-intestinal tract. Detailed understanding of carbohydrate metabolism regulation in this genus is of prime importance and availability of additional genetic tools easing such studies would be beneficial. To develop a fluorescent protein-based reporter system that can be used in B. longum NCC 2705, we first selected the most promising fluorescent protein out of the seven we tested (i.e., mCherry). This reporter protein was then used to study the carbohydrate mediated activation of PBl1518 and PBl1694, two promoters respectively predicted to be controlled by the transcriptional factors AraQ and AraU, previously suggested to regulate arabinose utilization and proposed to also act as global transcriptional regulators in bifidobacteria. We confirmed that in B. longum NCC 2705 the AraQ controlled promoter (PBl1518) is induced strongly by arabinose and established that the AraU controlled promoter (PBl1694) was mostly induced by the hexoses galactose and fructose. Combining the mCherry reporter system with flow cytometry, we established that NCC 2705 is able to co-metabolize arabinose and glucose while galactose was only consumed after glucose exhaustion, thus illustrating the complexity of different carbohydrate consumption patterns and their specific regulation in this strain.