Co-culturing Bifidobacteria and propionic acid bacteria in soy whey can boost vitamin B12 levels in plant-based products

This method offers a natural approach to managing vitamin B12 deficiency that could arise from a plant-based diet.

Vitamin B12 is predominantly found in animal products. To address this nutritional gap, the food industry has been relying on synthetic fortification.

Ricco Tindjau, a PhD candidate in the Department of Food Science and Technology at the National University of Singapore, proposes a natural alternative with health benefits that synthetic fortification cannot offer.

“The synergy between these probiotic strains leads to promising health benefits in the form of postbiotic metabolites. These include short-chain fatty acids and phenolic compounds that protect against oxidative stress,”​ said Tindjau at our Growth Asia Summit held in Singapore from 16 to 18 July.

Furthermore, these probiotics improve the body’s ability to absorb minerals from soy whey by breaking down anti-nutrients like soyasaponin, phytate, and oxalate, which typically hinder the absorption of crucial minerals like calcium and iron.

Tindjau's research focuses on enhancing the vitamin B12 content in soy whey, a byproduct of tofu and soy protein isolate production.

As soy whey is plant-based, it aligns with the growing demand for vegan and vegetarian products. It is also rich in beneficial phytochemicals like isoflavones.

Moreover, utilising soy whey in vitamin B12 production turns what was once a manufacturing side-stream into a valuable nutritional resource, reducing waste and supporting sustainability in the food industry.

The research spanned four separate studies, where researchers began by testing various bacterial strains to assess their ability to produce vitamin B12 in eight days.

Findings from the first and second studies​

In the first​ and second​ studies, six strains were tested: Bifidobacterium longum ​subsp. longum (B. longum)​, Bifidobacterium animalis ​subsp. lactis A (B. lactis A)​, Bifidobacterium animalis ​subsp. lactis B (B. lactis B), B. lactis C​, B. lactis D​, and B. breve​.

B. longum​ is a human residential bifidobacteria (HRB), which are naturally occurring in the gut.

B. lactic ​strains are not HRB, but they have similar benefits as B. longum​. It can also utilise plant-based oligosaccharides, while B. longum​ prefers HMOs.

Out of the six strains, only three could grow in supplemented soy whey: B. longum​, B. lactis A​, and B. lactis B​.

B. lactis A​ could produce 2.06–4.56 μg/day vitamin B12 in 48 hours when soy whey was supplemented with glucose, cysteine, and yeast extract – a process that is both costly and resource-intensive.

For B. lactis longum​, the B12 levels were only at 1.6 μg/day when supplemented with glucose, cysteine, and yeast extract. This is much lesser than the recommended intake of 2.4μg/day.

The vitamin B12 growth for B. lactis B ​was not significant.

Results from the third study​

To source for another alternative, the researchers conducted a third study​ with propionic acid bacteria (PAB), a non-probiotic strain commonly used in the fermentation of Swiss cheese.

PAB could grow and produce vitamin B12 in soy whey without the need for supplements.

PAB also has probiotic-like characteristics, such as bacteriocin production and the ability to attach to the mucosal lining of the gut.

Two PAB strains were tested – Propionibacterium freunderuchii ​and Propionibacterium shermanii. ​

The Vitamin B12 content in soy whey with Propionibacterium shermanii ​was comparable to B. lactis_A at ​around 2.1 μg/day, but this was still below the recommended daily intake of 2.4 μg/day.

Exploring co-culturing probiotics​

This led the researchers to explore another strategy in a fourth study​: co-culturing of B. lactis A​ with PAB.

“So we hypothesised that Propionibacterium and Bifidobacterium could work together, and the co-culturing approach yielded promising results,”​ said Tindjau.

Propionibacterium supported the growth of Bifidobacterium in unsupplemented soy whey by breaking down plant oligosaccharides into simple sugars, which led to acid production by both cultures.

At the same time, Bifidobacterium produced precursors of vitamin B12, which Propionibacterium then utilised to produce vitamin B12.

“In our experimental design, we explored various approaches to optimise vitamin B12 production, first working with monocultures, then moving on to sequential cultures, followed by simultaneous co-inoculation,”​ said Tindjau.

The researchers first tested monocultures, where they separately grew Bifidobacterium and Propionibacterium in unsupplemented soy whey. This did not yield any growth for vitamin B12.

Then, they tried sequential cultures, where they first cultured Bifidobacterium followed by Propionic bacteria, and vice versa. The growth for vitamin B12 was not significant.

Finally, they tested a simultaneous co-culture where Bifidobacterium and Propionic bacteria were grown together from the start and observed a significant growth in vitamin B12.

“The growth of P. shermanii was not negatively impacted by the presence of B. lactis A. This suggests that B. lactis A was able to grow in unsupplemented soy whey when co-cultured with PAB, possibly due to growth factors provided by PAB,” said Tindjau.​

Simultaneous co-culturing showing higher vitamin B12 levels after 8 days of fermentation compared to the other methods.

The amount of vitamin B12 reached a maximum of 8μg/L by the eighth day, indicating that 300ml of fermented substrate could meet the recommended daily intake of 2.4 µg.

“This increase in vitamin B12 production was only seen in the simultaneous co-culture, likely due to the enhanced growth of Bifidobacterium and the possible production of B12 precursors, ​

The B12 precursors are intermediate compounds that are necessary for the synthesis of vitamin B12. When Bifidobacterium produces these precursors, they can be utilised by Propionibacterium to complete vitamin B12 synthesis.

However, the same results could not be seen when using B. longum​, a human resident bifidobacteria (HRB), which is naturally occurring in the gut.

While Bifidobacteria growth improved and followed a similar trend to B. lactis A​, the vitamin B12 production was less promising. The maximum level of vitamin B12 was reached on the sixth day at around 7.5 μg/L, and there was no significant difference in B12 content by the eighth day.

This could be due to precursor cross-feeding, where the bacteria use the B12 precursors for their own metabolic needs, or a cobalt bottleneck – a limitation in the availability of cobalt, which is crucial for B12 synthesis.

“Co-inoculation provides synergistic effects. However, B12 enhancement is species dependant. Co-culturing B. lactis A and PAB has shown a significant growth in vitamin B12 levels. Furthermore, there is an added benefit of an improvement in organic acids and phytoestrogen content through probiotic fermentation,” concluded Tindjau.​