Researchers at DTU have developed a new method that can reduce the time needed to find new bacteria for fermentation. They have now identified a bacterium that can be used both for acidification and to increase the vitamin B2 content of soya drinks.

A new method developed by researchers at the DTU National Food Institute can speed up the process of identifying bacteria that can be used both for fermentation and to increase the vitamin B2 content in soya drinks. In the study, the microbiome (the complete bacterial community) from bumblebees was examined and tested in microscopic droplets for its ability to produce vitamin B2.

“Our research shows that it is possible to screen entire microbial communities directly and rapidly, and that promising bacteria can be identified from environmental samples without prior isolation and analysis of individual bacteria. This can make the development of new starter cultures faster and more targeted,” says Associate Professor Claus Heiner Bang-Berthelsen from the DTU National Food Institute.

The research has been published in the scientific journal LWT – Food Science and Technology.

Researchers have discovered promising bacteria in bumblebees

The study addresses a problem common to many plant-based dairy alternatives: they contain fewer vitamins and minerals than cow’s milk. One of the nutrients typically lacking is vitamin B2 (riboflavin).

The researchers therefore set out to quickly identify bacteria that could both thrive in soya drinks and produce vitamin B2 themselves during fermentation. They used bacteria from bumblebee guts as a starting point for screening potential vitamin B2-producing strains.

“Bumblebees live close to plants, and their guts contain many microorganisms that are already adapted to plant-based environments. That is why it was interesting for us to test whether we could find bacteria in bumblebees capable of producing vitamin B2 in soya drinks,” says Postdoc Hang Xiao from the DTU National Food Institute.

Tested the bacteria in microscopic droplets

In the study, the researchers applied an existing technology known as ‘droplet screening’ in a new way.

“Unlike conventional agar plate-based methods for microbial cultivation and screening, we encapsulated the bee gut bacteria in microscopic droplets so that each droplet contained only one bacterium and acted as an enclosed culture chamber. In this way, the individual bacterium could be analyzed at ultra-high speed by using our microfluidics screening platform, enabling us to screen millions of bacterial cells within just a few hours,” says Hang Xiao.

Ordinary soya drinks are often cloudy and full of particles, which can interfere with measurements. To make droplet screening compatible with soy-based drinks, the researchers developed a highly transparent soy medium.

“By making the soya liquid transparent, we were able to both screen the bacteria in an environment resembling their future application and, at the same time, obtain more stable droplets and more precise measurements,” says Claus Heiner Bang-Berthelsen.

The bacteria were first exposed to roseoflavin, a substance structurally like riboflavin that can promote the growth of the bacteria best suited to producing vitamin B2. The researchers then selected the droplets that glowed the brightest, as high fluorescence indicates high vitamin B2 production.

“This droplet-based microbial screening approach saved months of work and significantly reduced the resource use compared with conventional screening methods,” says Claus Heiner Bang-Berthelsen.

A particular Lactococcus lactis strain stood out

Among the bacteria identified, one lactic acid bacterium proved particularly interesting. When the researchers tested the bacterium in real plant-based products, it proved especially effective in soya drinks:

“The results suggest that the bacterium works not only under laboratory conditions, but also in actual foods containing a significant amount of protein,” says Hang Xiao.

It turned out that the bacterium continued to produce vitamin B2 in soya drinks even at high levels of added (fortified) vitamin B2, demonstrating robust and stable production.

The bacterium was also found to be capable of utilizing many different types of sugar. This makes it an interesting candidate for use as a starter culture in plant-based fermentation processes, as it is not restricted to a single, very narrow substrate.

It generally performed less well in rice and oat drinks, as well as in some almond drinks, which the researchers attribute to their low protein content. Their interpretation is that the bacterium requires a certain level of fermentable protein to grow well and produce vitamin B2 effectively.

“The exciting thing about the method is that it can not only identify vitamin B2-producing bacteria in soya drinks. It can also be adapted to identify other interesting substances, provided they can be detected using fluorescence. However, the method only works if the medium is transparent and has a low fluorescence background,” says Hang Xiao.

Facts

• New method for identifying bacteria capable of producing vitamin B2 in plant-based products
• The bacteria were sourced from the guts of wild bumblebees
• The researchers screened them in microscopic droplets, one cell at a time
• A special transparent soy medium made the screening possible
• The most promising bacterium was Lactococcus lactis NFICC2835
• The bacterium produces up to 1.23 mg/L riboflavin in soy drinks
• The results were best in soy-based plant drinks
• The results were obtained using the MALDI-TOF Biotyper, which is part of the FOODHAY research infrastructure at the DTU National Food Institute.

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The scientific article 'Droplet microfluidics-based isolation, adaptation, and screening of riboflavin-producing lactic acid bacteria for fermenting plant-based dairy alternatives' has been published in the scientific journal LWT – Food Science and Technology.

The research was supported by, among others, Novonesis and the Innomission research project REPLANTED. Additional support was provided by DTU and FoodHay