Fermentation fortification of active vitamin B12 in food matrices using Propionibacterium freudenreichii : Analysis, production and stability

Vitamin B12 (later B12) intake is insufficient in developing countries, and globally, vegetarians and vegans are also at risk of B12 deficiency. Occurring naturally only in foods of animal origin, new affordable and sustainable dietary sources of B12 are needed to ensure sufficient intake. The only known food-grade producers of active B12, Propionibacterium freudenreichii strains, however, are yet to be exploited to enrich plant-based foods with B12. The B12 production capacity of P. freudenreichii depends on the strain, and the availability of the B12 lower ligand (5,6-dimethylbenzimidazole, DMBI) is a key factor for the production of active B12. Bread can be considered as a potential food for B12 fortification; yet the stability of in situ-produced B12 incorporated during breadmaking processes is not known. Current analytical methods such as the microbiological assay (MBA) lack the required specificity and the existing high-performance liquid chromatography (HPLC) methods are only capable of measuring higher B12 levels in fortified foods and supplements. The determination of active B12 in fermented foods, however, needs sensitive and selective methods. An ultra-HPLC (UHPLC) method was developed and validated to measure the active B12 contents. The identity of the B12 form was confirmed with an ion-trap or quadrupole time-of-flight mass spectrometry (MS). The B12 production capacity of 27 P. freudenreichii and 3 Propionibacterium acidipropionici strains was first studied in whey-based medium (WBM), and three of these P. freudenreichii strains were chosen to study B12 production in three aqueous cereal matrices prepared from malted barley (BM; 33% w/v), barley flour (BF; 6% w/v) and wheat aleurone (AM). Riboflavin (RF) and nicotinamide (NAM) as food-grade replacements for DMBI were investigated in WBM and cereal matrices. The stability of in situ-produced B12 and added cyanocobalamin (CNCbl) and hydroxocobalamin (OHCbl) during straight-dough, sponge-dough and sourdough breadmaking was studied. The developed UHPLC method employing an Acquity high-strength silica (HSS) T3 column showed excellent separation of active B12 from its analogues. A low limit of detection (0.075 ng/inj) enabled the measurement of the B12 levels in cell extracts directly and following immunoaffinity purification in extracts of fermented cereal matrices and B12-fortified baking samples. Analysis with UHPLC MS confirmed the production of active B12 by all 27 P. freudenreichii strains in WBM and 3 P. freudenreichii strains in cereal matrices. P. acidipropionici strains, however, produced an inactive form (pseudovitamin B12), thus making them unsuitable for active B12 fortification in foods. The level of B12 production in WBM varied considerably between the strains (0.45‒3.35 µg/mL), which increased up to 4-fold in 12 of the 27 P. freudenreichii strains following supplementation with RF and NAM. In many of these strains, the B12 yield was higher with RF and NAM co-supplementation than with DMBI. In cereal matrices without supplementation, the produced levels of active B12 (9‒37 ng/g) with P. freudenreichii strains were nutritionally significant. The B12 production increased many-fold, reaching up to 430 ng/g in BM, 39 ng/g in BF and 114 ng/g by adding cobalt (Co) and reached 712 ng/g in BM and 180 ng/g in AM with RF and NAM co-supplementation with Co. The incorporated in situ-produced B12 was retained during straight-dough breadmaking and the loss of 29% during sourdough baking was similar to the losses observed for relatively stable CNCbl. However, the added OHCbl decreased by 21%, 31% and 44% respectively in straight-dough, sponge-dough and sourdough breadmaking. These results showed that B12 produced in situ and incorporated during breadmaking was well retained in the bread prepared by the conventional breadmaking processes. This thesis shows that UHPLC combined with MS allows for the accurate identification and quantitation of low levels of active B12 in fermented food matrices (≥1 ng/g). P. freudenreichii strains could be utilised for in situ production of active B12 in cereal matrices and WBM. The availability of RF and NAM could considerably improve B12 production. The produced levels could easily fulfil the recommended dietary allowance set for B12 (e.g. 2‒2.4 µg/day for adults), and could be well retained in bread in the commonly used breadmaking processes.

Characterisation of antibiotic-resistant psychrotrophic bacteria in raw milk

Dynamics of raw milk associated bacteria during cold storage of raw milk and their antibiotic resistance was reviewed, with focus on psychrotrophic bacteria. This study aimed to investigate the significance of cold storage of raw milk on antibiotic-resistant bacterial population and analyse the antibiotic resistance of the Gram-negative antibiotic-resistant psychrotrophic bacteria isolated from the cold-stored raw milk samples. Twenty-four raw milk samples, six at a time, were obtained from lorries that collected milk from Finnish farms and were stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Antibiotics representing four classes of antibiotics (gentamicin, ceftazidime, levofloxacin and trimethoprim-sulfamethoxazole) were used to determine the antibiotic resistance of mesophilic and psychrotrophic bacteria during the storage period. A representative number of antibiotic-resistant Gram-negative isolates retrieved from the cold-stored raw milk samples were identified by the phenotypic API 20 NE system and a few isolates by the 16S rDNA gene sequencing. Some of the isolates were further evaluated for their antibiotic resistance by the ATB PSE 5 and HiComb system. The initial average mesophilic counts were found below 105 CFU/mL, suggesting that the raw milk samples were of good quality. However, the mesophilic and psychrotrophic population increased when stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Gentamicin- and levofloxacin-resistant bacteria increased moderately (P < 0.05) while there was a considerable rise (P < 0.05) of ceftazidime- and trimethoprim-sulfamethoxazole-resistant population during the cold storage. Of the 50.9 % (28) of resistant isolates (total 55) identified by API 20 NE, the majority were Sphingomonas paucimobilis (8), Pseudomonas putida (5), Sphingobacterium spiritivorum (3) and Acinetobacter baumanii (2). The analysis by ATB PSE 5 system suggested that 57.1% of the isolates (total 49) were multiresistant. This study showed that the dairy environment harbours multidrug-resistant Gramnegative psychrotrophic bacteria and the cold chain of raw milk storage amplifies the antibioticresistant psychrotrophic bacterial population

Niacin contents of cereal-milling products in food-composition databases need to be updated

The niacin content of cereal raw materials reported in food-composition databases often differs considerably. One major reason for this discrepancy is the analytical method used for its measurement is that a significant part of the niacin in cereals exists in bound form. In this study, we compared the niacin content of some representative cereal raw materials analysed with a sensitive and validated ultra-high performance liquid chromatography-fluorescence method against the values found in five national food-composition databases. We used established extraction methods that are assumed to liberate niacin available for absorption (acid hydrolysis mimicking human digestion) or total niacin (strong acid-alkaline hydrolysis). The niacin content (mg/100 g dry weight) obtained with acid hydrolysis ranged from a low level in corn flour (0.26), white wheat flour (0.45) and oat flakes (0.48), to a higher level in wholegrain flours (rye: 0.79, barley: 0.99, wheat: 0.88), wheat bran (2.7) and wheat germ (2.7). The niacin content with the acid-alkaline hydrolysis, however, was 1.9-11-fold the value measured after extraction with acid hydrolysis. In general, the niacin content found in the databases is closer to the results obtained after the acid-alkaline extraction, suggesting that the niacin values reported in the databases may not reflect actual bioaccessible niacin but total niacin.Peer reviewe

Fermentation of cereal, pseudo-cereal and legume materials with Propionibacterium freudenreichii and Levilactobacillus brevis for vitamin B12 fortification

The present study investigated the in situ production of vitamin B12 in eleven cereal, pseudo-cereal and legume materials by fermentation with Propionibacterium freudenreichii DSM 20271 and Levilactobacillus brevis (formerly Lactobacillus brevis) ATCC 14869. P. freudenreichii was used as the vitamin producer and L. brevis was selected to improve the consistency and microbial safety of the process. The study showed that more than 300 ng/g dw of vitamin B12 (daily requirement: 2.4 ug) were produced during fermentation in most of the studied brans and legumes. The highest vitamin B12 production was observed in the fermentation of the rice bran (ca. 742 ng/g dw), followed by the fermentation of buckwheat bran (ca. 631 ng/g dw). Furthermore, partial least squares (PLS) regression analysis suggested that the production of vitamin B12 was greatly influenced by the nutrient composition of the fermented raw materials. Meanwhile, L. brevis was found to effectively inhibit the growth of Enterobacteriaceae during fermentation. These results demonstrated that fermentation of cereal, pseudo-cereal and legume materials with P. freudenreichii and L. brevis is effective in fortifying plant-based food with vitamin B12.Peer reviewe

Riboflavin, niacin, folate and vitamin B12 in commercial microalgae powders

This study aimed to investigate the riboflavin, niacin, folate and B12 content in microalgae powders. Riboflavin was determined with an ultra-high-performance liquid chromatographic (UHPLC) method after extraction and a two-enzyme treatment. Niacin analysis involved mild acidic hydrolysis, with niacin quantitated as the sum of nicotinic acid and nicotinamide using a UHPLC method. Both a microbiological (MBA) and a UHPLC method was used for vitamin B12 quantification as a cyanocobalamin (CNCb1) and UHPLC-mass spectrometry was used to confirm the vitamin B12 form. Total folate was determined both with MBA and as a sum of the vitamers with a UHPLC method after extraction and tri-enzyme treatment. The riboflavin and niacin content varied from 21 to 41 mu g/g and 0.13-0.28 mg/g, respectively, in Chlorella sp., Spirulina (Arthrospira sp.) and Nannochloropsis gaditana powders. Chlorella powders were, on average, richer in total folate (19.7 mu g/g) than Spirulina powders were (3.5 mu g/g). The sum of the folate vitamers determined with UHPLC matched better with the microbiological total folate content in Chlorella than in Spirulina powders. Pseudovitamin B12 was the predominant form over active vitamin B12 in Spirulina powders, whereas Chlorella sp. and N. gaditana powders solely contained active vitamin B12 up to 2.1 mu g/g.Peer reviewe

Ultra-high performance liquid chromatographic and mass spectrometric analysis of active vitamin B12 in cells of Propionibacterium and fermented cereal matrices

A sensitive and selective method is needed to analyse in situ produced vitamin B12 in plant-based materials, potential new dietary sources of vitamin B12. A UHPLC/UV method was developed and validated for the determination of human active vitamin B12 in cell extracts of Propionibacterium freudenreichii subsp. shermanii and after immunoaffinity purification in extracts of cereal matrices fermented by P. freudenreichii. An Acquity HSS T3 C18 column resulted in a baseline separation, a calibration curve of excellent linearity and a low limit of detection (0.075 ng/5 μL injection). As confirmed by UHPLC–MS, the active vitamin B12 could be separated from pseudovitamin B12. The recovery of vitamin B12 from purified spiked cereal matrices was good (>90%; RSD < 5%). A nutritionally relevant amount of active vitamin B12 was produced by P. freudenreichii in cereal malt matrices (up to 1.9 μg/100 g) in 24 h at 28 °C.Peer reviewe

In situ fortification of vitamin B12 in wheat flour and wheat bran by fermentation with Propionibacterium freudenreichii

Vitamin B12 is a micronutrient naturally existing in animal products. A growing interest and need to replace animal protein with plant protein sources have resulted in increased attention to developing vitamin B12-fortified plant-based food. Natural fortification by Propionibacterium freudenreichii is a promising alternative to chemical fortification, as P. freudenreichii can synthesize active vitamin B12. In this work, we studied vitamin B12 production in non-sterile matrices prepared from three raw materials of wheat: durum flour, wholewheat flour and wheat bran. Viable cell counts, pH, total titratable acidity and concentration of acids were determined. After seven days of fermentation, vitamin B12 levels reached 33 +/- 4, 87 +/- 10 and 155 +/- 17 ng/g dry weight in durum flour, wholewheat flour, and wheat bran, respectively. While durum flour supported the growth of P. freudenreichii to higher cell densities and more efficient propionic acid production compared with the other two matrices, wholewheat flour and wheat bran were found to be the most promising of the three matrices for in situ production of vitamin B12. (C) 2018 Elsevier Ltd. All rights reserved.Peer reviewe

Stability of added and in situ-produced vitamin B12 in breadmaking

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Letter to the editor on ‘Enhancing vitamin B12 content in soy-yogurt by Lactobacillus reuteri, IJFM. 206:56–59’

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Food-Like Growth Conditions Support Production of Active Vitamin B12 by Propionibacterium freudenreichii 2067 without DMBI, the Lower Ligand Base,or Cobalt Supplementation

Propionibacterium freudenreichii is a traditional dairy bacterium and a producer of short chain fatty acids (propionic and acetic acids) as well as vitamin B12. In food applications, it is a promising organism for in situ fortification with B12 vitamin since it is generally recognized as safe (GRAS) and it is able to synthesize biologically active form of the vitamin. In the present study, vitamin B12 and pseudovitamin biosynthesis by P. freudenreichii was monitored by UHPLC as a function of growth in food-like conditions using a medium mimicking cheese environment, without cobalt or 5,6-dimethylbenzimidazole (DMBI) supplementation. Parallel growth experiments were performed in industrial-type medium known to support the biosynthesis of vitamin B12. The production of other key metabolites in the two media were determined by HPLC, while the global protein production was compared by gel-based proteomics to assess the effect of growth conditions on the physiological status of the strain and on the synthesis of different forms of vitamin. The results revealed distinct protein andmetabolite production, which reflected the growth conditions and the potential of P. freudenreichii for synthesizing nutritionally relevant amounts of active vitamin B12 regardless of the metabolic state of the cells.Peer reviewe