Process Biochemistry 46 (2011) 1063–1070 Contents lists available at ScienceDirect Process Biochemistry journal homepage: www.elsevier.com/locate/procbio Concurrent high production of natural folate and vitamin B12 using a co-culture process with Lactobacillus plantarum SM39 and Propionibacterium freudenreichii DF13 Selina Hugenschmidt, Susanne Miescher Schwenninger, Christophe Lacroix ∗ Laboratory of Food Biotechnology, Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7, 8092 Zurich, Switzerland article info Article history: Received 2 July 2010 Received in revised form 10 January 2011 Accepted 18 January 2011 Keywords: Folate Vitamin B12 Lactobacillus plantarum Propionibacterium freudenreichii Co-culture abstract Sufficient intake of folate is critical worldwide due to low folate concentrations in daily diets. Folate supplementation in food is carried out with success in some countries, but recent data also indicate the importance of maintaining a suitable balance between folate and vitamin B12 intake due to their interdependent metabolisms. In this study, a high folate producer Lactobacillus plantarum SM39 was cultured in combination with a vitamin B12 producer, Propionibacterium freudenreichii DF13. The co-culture process was optimized to produce high levels of both vitamins in a whey permeate medium supplemented with yeast extract (SWP). Addition of 5 mg/L cobalt chloride, 15 mg/L DMBI, and 10 mg/L pABA to SWP medium followed by a two-step fermentation under optimal conditions, i.e. three days anaerobic/four days aerobic, led to high vitamin B12 and folate yields, 751 ± 353 ng/mL and 8399 ± 784 ng/mL, respectively. Folate yields were more than 10-fold higher than maximum values reported for natural fermentations and comparable to levels recently achieved using a genetically optimized strain. The controlled addition of pABA and cobalt offers the possibility to produce a vitamin supplement with a suitable balance of folate and vitamin B12 of 170–1. The fermented medium containing high levels of natural folate and vitamin B12 content could be used to fortify foods with natural vitamins or as a nutritional supplement. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Masking of vitamin B12 deficiency by folate is one of the cen- tral issues of on-going discussions worldwide regarding mandatory fortification of foods with folate [1–3]. Due to a metabolic path- way partially shared by both vitamins, high intakes of folate can correct anemia caused by a vitamin B12 deficiency which could remain unrecognized and eventually progress to irreversible neu- rological damage [4]. The optimal folate–vitamin B12 mass ratio for human consumption is approximately 170–1. Co-fortification of food with a mixture of both vitamins could lead to a simultaneous improvement of folate and vitamin B12 status [5,6]. Two different processes, biotechnological and chemical, are used for the industrial production of vitamin B12 and folate, respec- tively. Vitamin B12 is only produced by microbial fermentation followed by extraction and purification using organic solvents, since chemical synthesis is too expensive and complicated involv- ing more than 70 different steps [7,8]. Propionibacterium sp. and Pseudomonas sp. are among the microorganisms most often used ∗ Corresponding author at: Schmelzbergstrasse 7, ETH Zentrum LFV C20, 8092 Zurich, Switzerland. Tel.: +41 44 632 48 67; fax: +41 44 632 14 03. E-mail address: christophe.lacroix@ilw.agrl.ethz.ch (C. Lacroix). for industrial vitamin B12 production. Yields up to 15 g/mL vitamin B12 were reported for natural strains whereas genetic engineering and selection of high producing mutant strains can further increase fermentation yields up to 300 g/mL [9,10]. In con- trast microbial-produced folate yields are always low. Lactic acid bacteria (LAB), bifidobacteria, and propionic acid bacteria (PAB) are classified as folate-producing food-grade microorganisms, but maximum yields reported range from 0.5 g/mL for natural strains to 7.5 g/mL for genetically engineered strains [5,11,12,28]. Indus- trial folate is therefore produced by chemical synthesis by reacting malondialdehyde derivatives with aminobenzoyl-l-glutamic acid and triaminopyrimidinone derivatives [14]. However, a number of recent studies have reported possible adverse effects due to high intakes of folic acid, the synthetic form of the vitamin, such as reduced natural killer cell cytotoxicity and growth promotion of already existing tumours [15,16]. These adverse effects are likely not present with natural folates which are regarded safe over a wide intake range [17,18]. The interest to increase levels of natu- ral forms of folate by selecting high folate-producing food-grade microorganisms and process optimization is therefore high [19]. The aim of the present study was to develop an optimized process for the concurrent production of natural folate and vita- min B12 using a mixed culture of food-grade high-folate producer Lactobacillus plantarum SM39 and vitamin B12 producer Propioni- 1359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2011.01.021