Vitamin B12 and folate enrichment of kefir by Propionibacterium freudenreichii and Streptococcus thermophilus strains

Abstract

In South Africa malnutrition exists due to inadequate dietary intake of micronutrients which is one of the major causes of vitamin deficiencies leading to disease. The treatment of malnutrition over the past years has been a considerable burden on the South African economy. Therefore, food fortification is one of the current strategies used to minimize malnutrition by increasing the nutritional value of staple foods. Commercial dairy products and pharmaceutical nutritional products (food supplements) in South Africa have been developed and produced for affluent consumers. Hence the need to develop an affordable fortified dairy product for the majority of South Africans prompted this study aimed at using a “naturally” fortified kefir beverage with vitamin B12 and folate to increase B-vitamins levels. Since Propionibacterium freudenreichii and Streptococcus thermophilus are known to be good producers of vitamin B12 and folate, respectively, and propionibacteria has the ability to grow symbiotically in the presence of lactic acid bacteria, the inclusion of these organisms with the kefir grains was an achievable objective. In order to conduct the analysis of vitamin B12 and folate in the samples, sample extraction and HPLC assay techniques were developed. The extraction of vitamin B12 and folate were achieved by using KCN extraction buffer and the trienzymatic method, respectively. The samples were also subjected to purification and concentration using solid phase extraction for optimum results. All standards and samples were flushed with nitrogen gas and stored for a maximum of 2 weeks at –20°C to prevent B-vitamin deterioration. The HPLC assembly for the vitamin B12 analysis included a Luna C18 column and a diode array detector (DAD) for the detection and quantification. For the folate analysis it included a Zorbax SB-C18 and Luna C18 columns in tandem and the fluorescence detector (FLD) was used for the detection and quantification of THF, 5-CH3-THF and 5-CHO-THF, while the DAD was used for PGA and pteroyltri-γ-L-glutamic acid concentration in the samples.