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Vitamin B12 fortification with PAB by In-situ fermentation

Are you aware about In-situ fortification? In-situ fortification is basically fermentation of foods having Vitamin B12 with microorganisms over a time period. To combat with malnutrition or nutrition deficiency, we are in urgent need to adopt fortification in our day to day culture.

Fortification of cereal varieties having vitamin B12 with Propionic acid bacteria (PAB) during dough fermentation is proving an economically feasible solution for mass production. Usually, biotechnological process produces the fortification of cereal.

What is Vitamin B12?

Vitamin B12 also known as cobalamin is a micronutrient that is predominantly present in foods of animal origin. According to Vitamin B12 chemical structure, it is a group of corrinoids having a cobalt ion in the centre of the corrin ring and two axial ligands. And, the lower and upper ligand coordinated to the cobalt ion.

It is basically restricted in certain forms of micro-organisms and resides in higher level animals. Some of its sources are beef, pork, lamb, goat, chicken, turkey, duck, egg, cow milk, yoghurt, fish, oyster, cheese, clamp, shrimp, Tempeh, Natto, and dry nori.

Deficiency in vitamin B12 may result in various symptoms, such as fatigue,
anemia, distal sensory impairment, cognitive impairment, memory loss, stupor, and psychosis, depending on the degree and duration of the deficiency.

Importance of fortification

Here, in-situ fortification refers to fermentation of foods having Vitamin B12 with microorganisms over a period at considering internal and external factors.

Vitamin B12 is usually found deficient in people who do not consume animal-based food. It is made by biotechnological processing, where the chemical synthesis is very cost effective and quite complex process.  It marks the significance of producing plant-based nutrition rich Vitamin B12 rich foods for consumption.  Malabsorption and inadequate intake are primary reasons for this vitamin deficiency, where the research shows that smoking, alcoholism, long term usage of certain drugs, and immune system damaging diseases leads to inability of vitamin absorption.

Protagonist: Microbes

Here, comes the Propionibacterium freudenreichii! Propionic acid bacteria (PAB) are a Gram positive, mesophilic, and aerotolerant Actinobacteria with a peculiar metabolism, which produces the propionic acid as the main metabolic end-product. 


It is the only food grade microbe, which shows the positive ability in producing Vitamin B12. An important point to note that the genus Lactobacilli produce pseudo-Vitamin B12 to combat with malnutrition or nutrition deficiency of millions of people, which created an urgent need to adopt fortification in our day to day culture. This selected micro-organism has inherent characteristics of benefits like low growth rate, sensitivity to decreased pH conditions, and so on.


Interesting results of this study:

In this Insitu fortification, vitamin B12 in native grain materials are fermented by P. freudenreichii and adds up to following De Novo biosynthesis process.

Fermentation of wheat flour, whole-wheat flour, and wheat bran with P. freudenreichii stemmed in a physiologically noteworthy level of vitamin B12 (up to 155 ng/g dw) after 7 days.

Whole-grain, wheat flour, and wheat bran have a higher content of vitamin B12 than refined wheat flour.

The fermented 11 types of grain materials demonstrates the wider applicability of established co-culture. Where, the fermenting grains include cereals, pseudo cereals, and legumes, with P. freudenreichii and L. brevis.

The observed results after fermentation process were:

Variety Amount of B12
Rice bran ca. 742 ng/g dw
Buckwheat Bran ca. 631 ng/g dw


Importance of co fermentation:

The prior screening establises an effective co-culture of Lactobacillus brevis and P. freudenreichii to certify microbial safety due the propagation of Enterobacteriaceae. During co-fermentation in wheat bran, P. freudenreichii produced a high level of vitamin B12 (ca. 183 ng/g dw on day 3).

Moreover, controlling pH during fermentation could greatly enhance the vitamin B12 production.

Co-fermentation is a common method in the production of cheese between lactic acid producer and propionic acid producing bacteria. But, unlike the tolerance level to increased acidic environment in cheese production, it is not possible in case of Insitu fermentation for Vitamin B12 fortification.


Controlling pH and increasing availability of cobalt can enhance in situ production of vitamin B12 during fermentation. The fermentation of grain materials with P. freudenreichii and an appropriate co-culture, such as L. brevis, is a promising way to provide vitamin B12 in non-sterilized grain-based materials without conceding microbial safety.

Read more about Microbiological need of cream: all you need to know 

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