Departments of
Agriculture, Food and Environment
Veterinary Sciences
Master Degree in
Food Biosafety and Quality
STABILITY OF VITAMIN B
12
IN BAKING PROCESS
Author Marco Santin
Supervisors Advisor
Annamaria Ranieri Monica Agnolucci
Vieno Piironen
STABILITY OF VITAMIN B
12
IN BAKING PROCESS
Master Thesis
University of Pisa
gonfi le tue vele. Esplora. Sogna. Scopri.”
Vitamin B12 is an essential macromolecule in mammal metabolism.
However, it can be detected in sufficient amount only in animal-derived foods. This fact exposes vegetarians and vegans to vitamin B12
defi-ciency. Therefore the need to develop plant-based food rich in vitamin B12 is high. However, it is important to know the stability of both
supplemented vitamin B12 forms and naturally produced vitamin B12,
during different food processes. Many attempts have been done during the last decades in order to produce vitamin B12-fortified plant-based
products, whose main aim was to replace vitamin B12 supplements, in
accordance with the increasing demands of natural and healthy prod-ucts. This study aimed to investigate the stability of vitamin B12
during different bread-making processes, firstly adding standard solu-tions, and subsequently mixing the natural vitamin B12 produced by
the fermentation of a selected strain of Propionibacterium freudenre-ichii subsp. freudenrefreudenre-ichii.
Firstly, the stability of different forms of vitamin B12
(methylcobal-amin (Me-Cbl), 5´-deoxyadenosylcobal(methylcobal-amin (Ado-Cbl), cyanocobal(methylcobal-amin (CN-Cbl) and hydroxocobalamin (OH-Cbl) after different times of ex-posure to light was investigated. While Me-Cbl and Ado-Cbl stability was tested only in water, CN-Cbl and OH-Cbl tests were performed at different pH conditions (pH 2.5, 4.5, 7.0) and water. Furthermore, the stability of CN-Cbl and OH-Cbl was studied during three different bread-making methods: straight-dough, sponge-dough and sourdough. Finally, a malt extract medium fermented by a selected strain of P. freudenreichii subsp. freudenreichii was used in the straight-dough baking. The vitamin B12 was quantified by a microbiological assay
(MBA) and the ultra high performance liquid chromatography
PLC).
Ado-Cbl and the Me-Cbl were extremely photolable and they con-verted entirely into OH-Cbl within 30 min, while both CN-Cbl and OH-Cbl showed a greater stability under light depending on the pH conditions: the CN-Cbl was more photo-sensitive at low pHs, while the OH-Cbl was more unstable at higher pHs. In addition, one unidentified peak was observed in the chromatogram originated from OH-Cbl and CN-Cbl due to the exposure to light.
The proofing steps had not effect on the loss of supplemented OH-Cbl and CN-OH-Cbl in straight- and sponge-doughs. Instead, they were lost during the baking in the oven, where the loss of OH-Cbl was higher than CN-Cbl. In straight-dough and sponge-dough processes, the loss of OH-Cbl was 15% and 32% , and the loss of CN-Cbl 6% and 7%, respectively. The sourdough method caused the highest destruction. During the baking, OH-Cbl and CN-Cbl losses were 57% and 42%, respectively.
In straight-dough baking with the fermented malt extract, the loss of naturally produced vitamin B12 was on average 29±12% obtained
with the MBA. By contrast, results analysed with the UHPLC meth-ods showed no loss, and vitamin B12 content in baked breads was still
considerable, on average 106 ng/g dm. This difference between the results may indicate about inactive B12 forms produced by the selected
strain of P. freudenreichii subsp. freudenreichii and also high stability of vitamin B12 produced by such bacterium. Generally, the
supple-mentation of OH-Cbl and CN-Cbl did not effect on appearance and taste of breads. However, the specific volume of the breads baked with fermented malt extract was slightly lower than control breads.
Producing a vitamin B12-fortified bread using fermented malt
ex-tract, as presented in this study, is possible and may represent a great opportunity for vegetarian and vegan people. However, further studies are needed, e.g., to clarify the effects of the compounds in the fer-mented media on the yeast growth, as well as the determination and the quantification of the inactive corrinoids produced by such bacteria during the fermentation step.
La vitamina B12 rappresenta una macromolecola essenziale per il
metabolismo dei mammiferi. Tuttavia, gran parte dei vegetariani e dei vegani ne ´e carente, dal momento che essa `e presente in quantit`a tali da sopperire al fabbisogno giornaliero raccomandato solamente in prodotti di origine animale. Per questo motivo vi `e una sempre mag-giore necessit`a di alimenti di origine vegetale ricchi in vitamina B12,
ottenibili attraverso diversi metodi di arricchimento. Inoltre, con la crescente richiesta da parte dei consumatori di alimenti sani, genui-ni e con propriet`a nutraceutiche, l’interesse nei confronti di alimenti arricchiti in vitamina B12 che potessero sostituire farmaci ed
integra-tori `e notevolmente aumentato. Prima di produrre alimenti con un incrementato contenuto di tale vitamina, per`o, `e necessario studiarne la stabilit`a in diverse preparazioni alimentari sia in forma cristallina, sia prodotta naturalmente da microrganismi selezionati. Questo lavoro si propone quindi di investigare la stabilit`a della vitamina B12 durante
diversi processi di panificazione: in un primo momento aggiungendo una soluzione acquosa in cui `e stata disciolta la vitamina B12, e
succes-sivamente mescolando all’impasto la vitamina B12 naturalmente
pro-dotta dalla fermentazione di un ceppo selezionato di Propionibacterium freudenreichii subsp. freudenreichii.
Inizialmente `e stata studiata la stabilit`a di diverse forme di vitami-na B12 (metilcobalamina (Me-Cbl), 5´-deossiadenosilcobalamina
(Ado-Cbl), cianocobalamina (CN-Cbl) e idrossicobalamina (OH-Cbl)) a di-versi intervalli di esposizione alla luce. Mentre la fotostabilit`a di Me-Cbl e Ado-Me-Cbl `e stata testata solo in soluzione acquosa, lo studio su CN-Cbl e OH-Cbl `e stato condotto in soluzioni a diversi pH (2.5, 4.5, 7.0) e in acqua. In un secondo momento, la stabilit`a di CN-Cbl e
OH-Cbl `e stata testata in tre diversi metodi di panificazione: metodo diretto, metodo indiretto con Poolish e metodo indiretto con lievito naturale. Successivamente, un estratto di malto opportunamente fer-mentato da un ceppo selezionato di Propionibacterium freudenreichii subsp. freudenreichii, produttore di vitamina B12, `e stato impiegato
nella panificazione secondo il metodo diretto. La vitamina B12 `e stata
quindi quantificata sia attraverso un saggio microbiologico (MBA), sia utilizzando l’ultra high performance liquid chromatography (UHPLC). Per quanto riguarda i test di stabilit`a alla luce, Ado-Cbl e Me-Cbl si sono rilevate estremamente fotosensibili e venivano convertite in OH-Cbl in meno di 30 min. Al contrario, CN-Cbl e OH-Cbl hanno dimostrato una stabilit`a considerevolmente maggiore in funzione del pH: mentre CN-Cbl `e risultata pi`u fotosensibile a bassi pH, OH-Cbl `e emersa essere pi`u instabile ad alti pH. Inoltre, un picco non identificato `e stato osservato nei cromatogrammi relativi a OH-Cbl e CN-Cbl in seguito all’esposizione alla luce.
Nella panificazione secondo metodo diretto e indiretto con Poolish, lo step di lievitazione non ha provocato alcuna perdita nella OH-Cbl e CN-Cbl aggiunte all’impasto. Al contrario, una certa percentuale di degradazione di tali forme di vitamina B12`e stata riscontrata durante
la cottura, dove la perdita di OH-Cbl `e stata maggiore di quella di CN-Cbl. Nei metodi diretto e indiretto con Poolish, la perdita di OH-Cbl `e stata del 15% e del 32%, mentre quella di CN-Cbl `e stata del 6% e 7%, rispettivamente. La degradazione maggiore `e stata riscontrata nel metodo indiretto con lievito naturale. Durante la cottura, le perdite di OH-Cbl e CN-Cbl sono state del 57% e del 42%, rispettivamente.
Nel metodo di panificazione diretto utilizzando estratto di malto fermentato, la diminuzione di vitamina B12 naturalmente prodotta `e
stata in media del 29±12%, secondo i risultati ottenuti dall’MBA. Al contrario, i dati ottenuti dall’UHPLC hanno evidenziato una sostan-ziale invarianza della concentrazione di vitamina durante il processo, quantificabile mediamente in 106 ng/g dm. Questa differenza nei ri-sultati ottenuti dai due metodi potrebbe indicare sia la presenza di forme inattive di vitamina B12 prodotte dal ceppo selezionato di P.
freudenreichii subsp. freudenreichii, sia una grande stabilit`a della vi-tamina B12prodotta da tale microorganismo. Generalmente, l’aggiunta
di OH-Cbl e CN-Cbl non ha influenzato l’aspetto e il sapore del pane ottenuto. Comunque, il volume specifico del pane ottenuto impiegando l’estratto di malto fermentato `e risultato leggermente inferiore rispetto al pane di controllo.
Produrre un pane arricchito in vitamina B12utilizzando estratto di
malto fermentato, come esposto in questo lavoro, `e possibile e potreb-be rappresentare una grande opportunit`a soprattutto per vegetariani e vegani. In ogni caso, studi pi`u approfondi sono necessari allo scopo di comprendere, ad esempio, gli effetti dei diversi composti presenti nell’ estratto di malto fermentato sulla crescita dei lieviti impiegati nella pa-nificazione, cos`ı come l’identificazione e la quantificazione dei corrinoidi inattivi prodotti dal ceppo di P. freudenreichii subsp. freudenreichii durante lo step di fermentazione.
1 Introduction 1
2 Literature Review 5
2.1 Vitamin B12 Chemistry and Structure . . . 5
2.1.1 Brief History of Vitamin B12 . . . 5
2.1.2 Structure and Forms of Vitamin B12 . . . 6
2.1.3 Stability of Different Vitamin B12 Forms . . . 7
2.2 Analysis of Vitamin B12 . . . 10
2.3 Biosynthesis and Production of Vitamin B12 . . . 12
2.3.1 Biosynthesis by Microorganisms . . . 12
2.3.2 Propionibacteria . . . 16
2.3.3 Vitamin B12 Industrial Production . . . 17
2.3.4 Production of Vitamin B12 in Plant-derived Foods . . . 18
2.4 Vitamin B12 Content in Foods and Recommended Intakes . . . 19
2.5 Vitamin B12 and Bread-making . . . 21
2.5.1 Fortification of Cereal Products with Vitamin B12 . . . 21
2.5.2 Bread-making Processes . . . 22
2.5.3 Stability of Vitamin B12 in Bread-baking . . . 23
3 Experimental Research 25 3.1 Aims . . . 25
3.2 Materials and Methods . . . 25
3.2.1 Chemicals, Reagents and Equipment . . . 25
3.2.2 Stability Tests with the Vitamin B12 Forms in Laboratory . 27 3.2.3 Baking Processes with OH-Cbl and CN-Cbl . . . 27
3.2.4 Freeze-drying of Dough and Bread Samples . . . 32
3.2.5 pH Measurement of Dough and Bread Samples . . . 32
3.2.6 Moisture Content Measurement of Dough and Bread Samples 34 3.2.7 Baking with Fermented Malt Extract . . . 34
3.2.8 Vitamin B12 Analysis . . . 36
3.2.9 Analysis of Bread Characteristics . . . 40
3.2.10 Statistical Analyses . . . 41
3.3 Results . . . 42
3.3.1 Stability Tests with the Standard Compounds of Vitamin B12 42 3.3.2 Stability of Vitamin B12 During different Baking Processes . 46 3.3.3 Straight-dough Baking with Fermented Malt Extract . . . . 50
3.3.4 Characterization of Doughs and Breads . . . 53
3.4 Discussion . . . 58
3.4.1 Stability of different Vitamin B12 Forms in different Light and pH Conditions . . . 58
3.4.2 Stability of OH-Cbl and CN-Cbl Standard Solution in dif-ferent Bread-Making Processes . . . 59
3.4.3 Stability of Vitamin B12 synthetized by P. freudenreichii subsp. freudenreichii during the Straight-Dough Process . . 61
3.4.4 Characteristics of the OH-Cbl and CN-Cbl-fortified and Nat-ural Vitamin B12-fortified Breads . . . 63
4 Conclusions 65
Bibliography 66
2.1 General structure of cobalamin; varying the group as upper ligand,
different vitamin B12 forms can be obtained ([71]). . . 7
2.2 Vitamin B12 structure and, on the right, characteristic moieties of some inactive corrinoids, which differs from vitamin B12: (1) vita-min B12or cyanocobalamin; (2) pseudovitamin B12 or factor IV; (3) factor A (2-methyladenylcobamide); (4) factor S (2-methylmercapto-adenylcobamide); (5) factor IIIm (5-methoxybenzimidazolylcobamide); (6) factor III (5-hydroxybenzimidazolylcobamide); (7) BIA (benz-imidazolylcobamide); (8) pCC (p-cresolylcobamide) ([118]). . . 11
2.3 Microbial biosynthesis of cobalamin through both anaerobic and aerobic pathways (modified from [71]). . . 15
2.4 Macrocyclic tetrapyrroles whose biosynthetic pathways have a com-mon intermediate, uroporphyrinogen III (modified from [86]). . . 16
3.1 The straight-dough process. . . 29
3.2 The sponge-dough process. . . 31
3.3 The sourdough process. . . 33
3.4 Growth curve of Lb. delbrueckii ATCC 7830 culture used as test microorganism for MBA, with respective OD measurements. . . 38
3.5 UHPLC chromatograms of acqueous solutions of OH-Cbl, CN-Cbl, Ado-Cbl and Me-Cbl as soon as they were prepared. . . 42
3.6 Combined chromatogram of OH-Cbl, CN-Cbl, Ado-Cbl and Me-Cbl aqueous solution injected in the UHPLC after 30 min of exposure to light. . . 43
3.7 Peak areas of CN-Cbl detected during exposure to light at different pHs, combined with the peak area of OH-Cbl and of an unknown compound. (A) pH 2.5; (B) pH 4.5; (C) pH 7.0; (D) Milli-Q water. 45 3.8 Peak areas of OH-Cbl detected during exposure to light at different
pHs, combined with the peak area of an unknown compound. (A) pH 2.5; (B) pH 4.5; (C) pH 7.0; (D) Milli-Q water. . . 46 3.9 The OH-Cbl and CN-Cbl content (ng/ g dm) of straight-dough
baking samples determined with the MBA and with the UHPLC method. MBA data represent the mean of 3 replicates± SD, while UHPLC data represent the mean of 2 replicates± SD. Statistically significant differences among MBA data and between UHPLC data were evaluated by one-way ANOVA (P < 0.05) and Student’s t-test (P < 0.05), respectively. Different letters indicate significantly different values according to Tukey–Kramer test. . . 47 3.10 The OH-Cbl and CN-Cbl content (ng/g dm) of sponge-dough
bak-ing samples determined with the MBA. Data represent the mean of 2 replicates ± SD. Statistically significant differences were eval-uated by one-way ANOVA (P < 0.05) and different letters indicate significantly different values according to Tukey–Kramer test. . . 48 3.11 The average OH-Cbl and CN-Cbl content (ng/g dm) of sourdough
baking samples determined with the MBA. . . 49 3.12 The average of OH-Cbl content (ng/g dm) of sourdough baking
samples determined with the MBA and with the UHPLC method. Data represent the mean of 2 replicates ± SD. Statistically signifi-cant differences were evaluated by one-way ANOVA (P < 0.05) and different letters indicate significantly different values according to Tukey–Kramer test. . . 50 3.13 The average B12 content (ng/g dm) of straight-dough baking
sam-ples baked in fermented (168 h) malt extract inoculated with a strain of P. freudenreichii subsp. freudenreichii, and analysed with the MBA or UHPLC. Data represent the mean of 3 replicates ± SD. Statistically significant differences were evaluated by one-way ANOVA (P < 0.05) and different letters indicate significantly dif-ferent values according to Tukey–Kramer test. . . 51
3.14 Breads supplemented with OH-Cbl and CN-Cbl standard solutions. 53 3.15 Baked breads made with the straight-dough method adding
fer-mented malt extract (batches 1, 2, and 3) inoculated with a strain of P. freudenreichii subsp. freudenreichii. The picture are taken frontally: (A) the outside, and (B) a section of the inside. . . 53
2.1 Test organisms used for vitamin B12 microbiological assays (MBA)
([67]). . . 12 2.2 Techniques and methods used for vitamin B12 detection, their
sen-sitivity and their concrete applications ([67]). . . 13 2.3 Vitamin B12 content and bioavailability in some animal and
non-animal foods ([117]) . . . 21
3.1 Bread recipe used in the straight-dough method. . . 28 3.2 Bread recipe used in the sourdough method. . . 30 3.3 Content of malt extract, according to the manufacturer information
(Laihian Mallas Ltd., Finland), and calculated content referred to the 10% malt extract medium. . . 35 3.4 SV (mean ± SD) of straight-dough and sponge-dough: control
breads, OH-Cbl breads, CN-Cbl breads. . . 54 3.5 Baking loss (mean± SD) of straight-dough, sponge-dough and
sour-dough: control breads, OH-Cbl breads, CN-Cbl breads. . . 55 3.6 SV and baking loss calculated separately on the breads obtained
with fermented malt extract from each single dough (mean ± SD). . 55 3.7 Average pH values of straight-dough, sponge-dough and sourdough
samples supplemented with OH-Cbl (n=2). . . 56 3.8 Average pH values of samples from the straight-dough method using
fermented MEM (n=2). The pH values are reported separately for each fermented MEM batch. . . 57
Ado-Cbl 5´-deoxyadenosylcobalamin
AH2 Ascorbic acid
ALA 5-aminolevulinic acid
ANOVA Analysis of variance
ATP Adenosine triphosphate
Cbi Cobinamide
Cbl Cobalamin
CN-Cbl Cyanocobalamin
DMBI 5,6-Dimethylbenzimidazole
FW Fresh weight
GDP Guanosine di- or pyrophosphate
GMP Guanosine monophosphate
GRAS Generally recognized as safe
K2HPO4 Dipotassium phosphate
KH2PO4 Monopotassium phosphate
L Lactic acid
LAB Lactic acid bacteria
LC Liquid chromatography
MBA Microbiological assay
ME Malt extract
ME+L Malt extract medium with lactate (8 g/L)
ME+L+T Malt extract medium with lactate (8 g/L) and tryptone (5 g/L)
Me-Cbl Methylcobalamin
MS Mass spectrometry
OD Optical density
OH-Cbl Hydroxocobalamin
PAB Propionic acid bacteria
QPS Qualified presumption of safety
RDA Recommended dietary allowance
RH Relative humidity
RT Room temperature
T Trifluoroacetic acid
UHPLC Ultra high performance liquid chromatography
USDA United State Department of Agriculture
Introduction
Vitamin B12can be undoubtedly considered as one of the most important, but
also complicated and captivating, molecules in food and medicine. Firstly identi-fied during the early 1920s, its rich saga was characterized by several Nobel prizes due to many brilliant discoveries focused especially on its ability to cure pernicious anemia. After its isolation, it quickly became clear that vitamin B12was chemically
more sophisticated than anything that had previously been deduced. Its popular-ity as the most enigmatic complex molecule of all cofactors and coenzymes, as well as the largest one in term of molecular weight (1335-1580 Da) [71, 106], increased year after year thanks to its fascinating chemistry and its unique biological fea-tures. One of these is the presence of the rare metal cobalt, which characterizes the core of its structure. The central planar corrin ring, composed of four pyrrole units, contains four ligands for the central cobalt ion, whose coordination sphere is then completed by a lower ligand and an upper ligand. Moreover, it exists in Nature in three different forms: methylcobalamin (Me-Cbl), 5’-deoxyadenosylcobalamin (Ado-Cbl) and hydroxocobalamin (OH-Cbl), while another one, cyanocobalamin (CN-Cbl), can be obtained synthetically by the addition of cyanide [71].
Vitamin B12 is necessary in the animal and human metabolism because it
is required as a cofactor by two enzymes: methionine synthase and (R)-methyl-malonyl-CoA mutase. Methionine synthase is involved in the conversion of ho-mocysteine in methionine by transferring a methyl group, a reaction needed in activating folate and, by consequence, in the DNA synthesis. On the other hand, (R)-methyl-malonyl-CoA mutase takes part in the catabolism of odd-chain fatty
acids as it catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA, a key molecule of the tricarboxylic acid (TCA) cycle. This enzyme is also involved in the metabolism of certain amino acids and cholesterol [26]. Furthermore, it has been demonstrated that vitamin B12 is produced only by some bacteria and
archaea, although it is required also by animals and protists [97]. However, it seems that plantae and fungi neither synthetize nor require it. By consequence of it, vitamin B12 can be detected mainly in animal-derived products, as it derives
both from the fermentation of vitamin B12-producing microorganisms located in
the digestive tract, and from the vitamin B12-containing feed. Although animal
food represents the most important source of vitamin B12 for humans, traces of
cobalamin can be detected also in plant-derived products, which are contaminated or fermented by such bacteria.
Vegetarians and vegans, and elderly people are the two groups of people which can be considered at high risk of vitamin B12 deficiency, due to an inadequate
dietary intake and malabsorption of cobalamin from food, respectively. This could lead to serious health problems related to the blood cells formation, such as mega-loblastic anemia. Moreover, when the vitamin B12 deficiency reaches high levels,
more severe disease may emerge that include nervous disorders like neurophaties, dementia and Alzheimer’s [82]. To prevent these problems and ensure the vitamin B12 daily requirement, the consumption of pharmaceutical supplements in pills or
tablets, as well as vitamin B12 fortified foods, is highly recommended. To meet
the increasing interest of people towards a healthier and more sustainable lifestyle, and consequently the increasing requests of genuine foods, many studies have been carried out during the last decades to improve the vitamin B12production directly
in the plant-based food by fermentation of inoculated vitamin B12-producing
bac-teria. This in situ fortification system of food provides many advantages, such as a more environmental-friendly approach and a less economic impact on companies then the chemical process of vitamin B12 production, which needs several
addi-tional steps like the extraction, the purification and the crystallization ones. To provide this natural vitamin B12enrichment of food, only three microorganisms are
used by food and pharmaceutical companies: Bacillus megaterium, Pseudomonas denitrificans and Propionibacterium [12, 93, 56]. Many researches have been fo-cused on understanding the biosynthetic pathways which lead to the formation of the complete vitamin B12 molecule, in order to characterize the vitamin B12
production by such bacteria. Once elucidated that, several studies have been car-ried out to investigate the requirement of different precursors and different cereal matrices in the vitamin B12 biosynthesis, in order to optimize and increase the
cobalamin production.
Moreover, several studies have been focused on testing the stability of the vita-min B12 forms in many different physical conditions of light, pH, and temperature.
It has been found out that the two natural vitamin B12 forms synthetized by
bac-teria, Me-Cbl and Ado-Cbl, are very light-sensitive and they convert into OH-Cbl within seconds [63]. On the contrary, results show that CN-Cbl, which is used for the production of pharmaceutical vitamin B12 supplements, is the most stable in
light. In addition, although some studies have demonstrated that microwave can lead to a significant loss of biologically active vitamin B12 [114], it has been
re-ported that it is stable at high temperature and during several cooking processes. However, the stability tests so far have been focused mostly on meat and dairy products, and not on fortified cereal products such as bread, whose production technologies and temperatures are completely different.
To have a proper and complete background about vitamin B12 and its
im-portance, the literature review focused on the description of its structure and its different forms, followed by its presence and availability in foods. Afterwards, the review focused on the stability of vitamin B12forms in different light, temperature
and pH conditions, considering also different cooking processes. The biosynthesis of vitamin B12, the possible formation of inactive corrinoids and how to detect
them were also explained, followed by a description of Propionibacteria, their uti-lization as vitamin B12-producing bacteria and recent studies about optimizing
vitamin B12 production using plant-derived food and waste media. The last part
of the literature review focused on the main differences amongst the most common bread-making methods and the attempts that have been done so far in order to increase the vitamin B12 content in bread.
The experimental part of this thesis aimed to investigate the stability of vitamin B12 in laboratory conditions and in baking processes. Firstly, the stability of the
vitamin B12 standard compounds was studied at different pH conditions and in
different times of exposure to light. Secondly, their stability was tested during different baking processes. Finally, the stability of natural vitamin B12produced by
Literature Review
2.1
Vitamin B
12Chemistry and Structure
2.1.1
Brief History of Vitamin B
12The discovery of vitamin B12 dates back to 1920s in order to find a cure to an
ultimately fatal disease firstly described in 1855 by the physician Thomas Addison, the pernicious anemia. A cure for this disease in dogs was found firstly in 1925 by the American physician George Hoyt Whipple, and the following year two American physicians, George Richards Minot and William Parry Murphy, showed the effectiveness of it also in humans. They demonstrated that a diet rich in raw pig liver was able to restore the normal haemoglobin level in blood [119, 90], thanks to the high iron content. Later on, a new molecule isolated from the liver -the extrinsic factor - was found to be able to cure the pernicious anemia, and it was used as a treatment of this disease. In 1934, Whipple, Minot and Murphy received the Nobel Prize in Physiology or Medicine. After 20 years, some researches succeeded in purification and crystallization of a new reddish vitamin isolated from the liver juice that was shown to be able to recover from pernicious anemia. From that moment on, the anti-pernicious anemia factor has been named vitamin B12. Furthermore an X-ray crystallographer, Dorothy Crowfood Hodgkin,
managed to elucidate the three-dimensional structure first of vitamin B12
(CN-Cbl), and 5 years later of coenzyme B12 (Ado-Cbl) [49, 50, 51, 68]. She was
awarded the Nobel prize in Chemistry in 1964, together with the work focused on
elucidating the molecular structure of penicillin and insulin. After a work that lasted for 11 years, Robert Woodward and his team succeeded in synthetizing the vitamin B12 chemically, which involved more than 100 reactions. Woodward
received the Nobel Prize in Chemistry in 1965. In the following years a lot of new discoveries concerning the identification of new forms of vitamin B12, their
biological role, their metabolic reactions, the methods used to analyze them and others were made, giving a deeper knowledge on this molecule.
2.1.2
Structure and Forms of Vitamin B
12Vitamin B12 is a water soluble vitamin, whose term is used to indicate all
the cobalt-containing corrinoids with the biological activity of CN-Cbl [24]. It has the largest molecular weight (1355.4 g/mol) and the most complex chemical structure amongst all the vitamins [117]. The core structure has a central planar corrin ring with a central cobalt ion coordinated by four pyrrole units (A, B, C, D). These pyrrols are joined to each other by carbon-methyl or carbon-hydrogen methylene bridges, except two neighboring pyrrole rings which are directly linked to each other. The central cobalt ion is linked to the four nitrogen atoms of each pyrrole unit. The two other ligands related to the cobalt ion, which complete its octahedral coordination sphere, are located above the plane of the main corrin structure, and that is the reason why they are also called the lower (alpha) and the upper (beta) ligands [71]. The lower ligand in human active B12 forms is
5,6-dimethylbenzimidazole (5,6-DMBI), which is connected to the ring at N-7 atom. The other nitrogen of DMBI molecule is joined to a ribose unit, which is in turn attached to a phosphate group. This nucleotide is covalently linked to the corrin ring through the propionic acid side chain of the pyrrole unit D. The upper ligand can vary, and according to this, the different vitamin B12 forms are named (Figure
2.1). The biological forms consist of the upper ligand either as 5´-deoxyadenosyl group, naming the molecule 5’-deoxyadenosylcobalamin (Ado-Cbl), or a methyl group, thus the name methylcobalamin (Me-Cbl). It is known that Ado-Cbl and Me-Cbl are very photosensitive, and even a short exposure to light in aqueous solutions is able to convert them into a new vitamin B12 form called
hydroxo-cobalamin (OH-Cbl), by replacing the upper ligand with a –OH group. It is also possible that a new form, called aqueocobalamin, is generated by the attachment
Figure 2.1: General structure of cobalamin; varying the group as upper ligand, different vitamin B12 forms can be obtained ([71]).
of a water molecule at the upper ligand of the molecule [98]. When vitamin B12
forms are exposed to cyanide, they are completely converted into cyanocobalamin (CN-Cbl), where the –CN group replaces the other upper ligands [71]. CN-Cbl is not naturally found in biological systems. However, it is the most stable cobalamin amongst the other forms, and it is the primary form manufactured by industries for pharmaceutical purposes [37].
2.1.3
Stability of Different Vitamin B
12Forms
Me-Cbl and Ado-Cbl are well known to be very light sensitive compounds and they photodegrade in aqueous solutions and food products [25]. The photostabil-ity of the different cobalamins depends on the β-ligand linked to the cobalt ion, which means that the stability depends on the different vitamin B12form studied.
Juzeniene and Nizauskaite in 2013 [63] investigated the photodegradation rate of CN-Cbl, Me-Cbl, Ado-Cbl and OH-Cbl under ultraviolet A (UVA) rays exposure
in aqueous solutions at physiological pH. They found that Me-Cbl and Ado-Cbl are the most sensitive to UVA radiation, and they convert to OH-Cbl with a pho-toconversion rate of only a few seconds. Me-Cbl was found to be 3-fold more sensitive to UVA radiation than Ado-Cbl [53, 21, 2]. Furthermore, the study by Juzeniene and Nizauskaite [63] showed that OH-Cbl was the most stable cobalamin under the identical conditions. Ahmad et al. in 1992 [2] reported that CN-Cbl photoconverts to OH-Cbl, albeit with a lower rate than Ado-Cbl and Me-Cbl, and its photodegradation rate is around 5-fold greater than that of OH-Cbl both in weak acid and aqueous solutions. However, Gentili et al. in 2008 [40] showed that, if exposed to light and in the presence of cyanide, all forms of cobalamin converts to CN-Cbl. As CN-Cbl is the mostly used form in pharmaceutical preparations and food supplementations, many studies have been carried out to test its sta-bility at different light exposures: under daylight [109, 14], sunlight [14, 33], and artificial light [54, 110]. These studies show that the exposure to different lights degrades CN-Cbl into OH-Cbl. Ahmad et al. in 1993 [3] tested the photodegra-dation of two CN-Cbl solutions (950 µg/mL and 95 µg/mL) at pH 4.0, 5.5 and 7.0 after exposure to sunlight (emission at 295-899 nm) and to artificial light. It was observed that the highest loss of CN-Cbl under sunlight occurred in pH 4.0 (21.0% in the 950 µg/mL solution, and 81.5% in the 95 µg/mL solution), followed by the formation of OH-Cbl (15.2% in the 950µg/mL solution, and 78.9% in the 95 µg/mL solution). The CN-Cbl photodegradation was higher in pH 4.0 also under artificial light: 19.4% in the 950 µg/mL solution, and 27.7% in the in the 95 µg/mL solution, while the formation of OH-Cbl was 19.3% in the 950 µg/mL solution, and 27.8% in the 95 µg/mL solution. According to these results, it is clear that CN-Cbl is much more sensitive to light at lower pH (pH 4.0), probably because of the lower stability of the protonated molecule [3], which leads to the dissociation of the cyano group and its replacement with the hydroxyl group. In addition to that, Raju et al. in 2012 [84] observed that CN-Cbl degrades and converts into OH-Cbl proportionally with the exposure to the artificial light of the laboratory. On the contrary, if samples are kept in completely dark conditions, CN-Cbl remained stable at least up to a month. Voigt and Eitenmiller in 1978 [112] observed that CN-Cbl is stable at pH region of 4.5 to 5.0, and solutions of CN-Cbl at pH from 4.0 to 7.0 can be autoclaved at 120 °C for 20 min without a significant degradation of the molecule, according to study by Lindemans [69].
Further, CN-Cbl can be inactivated by strong alkaline or acid solutions, UV, in-tense visible light, and oxidizing agents [112]. In addition, some other conditions have been demonstrated to increase the degradation of vitamin B12 in aqueous
solution. Belitz et al. in 2004 [15] showed that reducing agents such as ascorbic acid (AH2), may inactivate vitamin B12. This was first observed by Herbert and
Jacob (1974), when they added vitamin C to homogenized meals followed by an incubation at 37 °C for 30 min to simulate the digestive tract conditions. Using a radioassay method, they found that the adding of AH2 led to destruction of
vitamin B12. The incompatibility and the interactions between vitamin B12 and
AH2 were observed also by more recent studies [57, 5]. Further, Kuehl et al. [66]
and Connors et al. [29] showed that OH-Cbl degrades more rapidly than CN-Cbl in the presence of AH2, leading to the formation of irreversible corrin ring
cleav-age oxidation products such as 3,3-dimethyl-2,5-dioxopyrrolidine-4-propionamide and 3,3-dimethyl-2,5-dioxopyrrolidine-4-propionic acid. Ahmad et al. in 2014 [5] investigated the degradation of CN-Cbl and OH-Cbl in presence of AH2 at pH
1-8. They showed that the destruction of CN-Cbl and OH-Cbl occurred with its maximum rate at pH 5.0 due to the increased presence of the ascorbate monoan-ion (AH−). At higher pHs, the monoanion is oxidized and, by consequence, the CN-Cbl or OH-Cbl destruction rate decreased, while at lower pH the protonated vitamin B12 molecule showed a higher stability towards AH2. The stability of
vitamin B12 can be decreased also by the presence of vitamin B2 [4]. Moreover,
an appreciable loss of vitamin B12 activity can be detected also in the presence of
other strong reducing agents, such as sulfite and iron(II) salts, but also if subjected to high concentration of oxidizing agents. Ahmad et al. in 2012 [4] showed that the addition of vitamin B2to CN-Cbl solutions promotes the conversion of CN-Cbl
into OH-Cbl at a very wide pH range (2.0-12.0).
Stability of vitamin B12 in different cooking treatments has also been studied,
in order to understand the effect of processing on loss and thus on bioavailability of vitamin B12. Generally, vitamin B12 is considered a very stable vitamin at high
temperature [40]. Stability studies on non-animal foods such as fortified cereal products are limited [35]. Experiments with beef showed that technological pro-cesses can influence the concentration of vitamin B12in the final product, although
no significant loss was found in roasted and grilled meat. A destruction of 49% of vitamin B12 was observed in a poorly conducted frying process [30]. Another
study by Riccio et al. in 2006 [88] demonstrated that, in samples of homogenized boiled ham enriched with 25 µg/g of thiamin (vitamin B1), pyridoxine (vitamin
B6) and CN-Cbl, after a boiling process of 5 min, the loss of vitamin B12was 51%
and, after 20 min at 120 °C, most of the vitamin B12 was destroyed (84.4%).
Ac-cording to the same study, the degradation in fortified boiled ham and cow meat was less during a grilling process: 42% and 17%, respectively. Furthermore, the stability of vitamin B12 after microwave heating was tested. Watanabe et al. in
1998 [114] found a considerable loss (40%) of vitamin B12 in homogenized beef and
pasteurized cow’s milk, with the formation of both identified and still unidentified degradation products.
2.2
Analysis of Vitamin B
12Some bacteria synthetize vitamin B12 analogues with the lower ligand other
than DMBI, which are not active for humans [46]. These inactive corrinoids are barely absorbed by mammalian intestine, as they show a lower affinity for the intrinsic factor, responsible for the absorption of vitamin B12 [102]. These
com-pounds, which are involved as coenzyme in bacterial reactions, share the same architecture as cobalamin, with the same corrin structure and the cobalt ion chelated at the centre of the molecule, but they show lower ligands other than DMBI [94, 118] (Figure 2.2). The most common example of this is the pseudovi-tamin B12, in which N7-linked adenine replaces DMBI as the α-axial ligand [52].
Firstly reported by Pfiffer et al. in 1952 [32] and then confirmed by Santos et al. in 2007 [94], it has been demonstrated that Lactobacillus reuteri CRL1098 under anaerobic condition is able to produce pseudovitamin B12, where DMBI is
substi-tuted by adenine. On the contrary of humans, bacteria and algae do not need the whole cobalamin structure as coenzyme, but they require only the corrin structure [46] for their enzymatic reactions. Watanabe et al. in 2013 [118] reported that commercially available tablets containing the cyanobacteria Spirulina sp., contain a high amount of vitamin B12 (127-244 µg/100 g). After identification, it was
Figure 2.2: Vitamin B12 structure and, on the right, characteristic
moi-eties of some inactive corrinoids, which differs from vitamin B12: (1)
vitamin B12 or cyanocobalamin; (2) pseudovitamin B12 or factor IV;
(3) factor A (2-methyladenylcobamide); (4) factor S (2-methylmercapto-adenylcobamide); (5) factor IIIm (5-methoxybenzimidazolylcobamide); (6) factor III (5-hydroxybenzimidazolylcobamide); (7) BIA (benzimidazolylcobamide); (8) pCC (p-cresolylcobamide) ([118]).
The presence of these inactive corrinoids in food or pharmaceutical products can represent a problem mostly due to the detection methods used, especially be-cause in the past, with the traditional methods, it was impossible to distinguish them from the active vitamin B12. The determination of vitamin B12 is
tradition-ally carried out by a microbiological assay (MBA) using Lactobacillus delbrueckii ATCC 7830 as test organism, which is also the referential method of AOAC [7]. However, it is possible to utilize other vitamin B12-depending bacteria as test
or-ganisms for MBA (Table 2.1). It has been observed that some bacteria do not grow properly in a medium without vitamin B12, and this is the basic principle of MBA.
Table 2.1: Test organisms used for vitamin B12 microbiological assays (MBA)
([67]).
As their growth is proportional to the vitamin B12 concentration, it is possible to
calculate the vitamin B12 content on the basis of the turbidimetric measurement
of the media [67]. Normally the results obtained from MBA are overstimated by 5-30% due to the poor specifity of this assay [10]: Lb. delbrueckii is able to use also inactive corrinoids and other compounds, e.g. nucleic acids, other than cobalamin, as coenzyme [10].
To overcome the lack of specificity, it is needed to use techniques that can separate the compounds basing on their chemical structure. The most common approach is the HPLC-based method, which have been used for the determination of vitamin B12 in fortified and non-fortified foods, infant formula and vitamin
sup-plements [25, 28, 48, 70] (Table 2.2). Most of these HPLC methods were coupled with UV as detection system, although Pakin, Bergaentzl´e, Aoud´e-Werner, and Hasselmann in 2005 [81] developed a method based on fluorescence detection after the conversion of vitamin B12 into a fluorescent compound, α-ribazole. Recently,
Chamlagain et al. in 2015 [27] developed a UHPLC/UV method, which can sep-arate also inactive corrinoids. Analyses carried out on fermented rye and barley matrices showed that the MBA gave higher (about 40%) vitamin B12content than
by UHPLC.
2.3
Biosynthesis and Production of Vitamin B
122.3.1
Biosynthesis by Microorganisms
For the entire de novo biosynthesis of vitamin B12 by microorganisms, more
than 30 genes are required, which amounts to about 1% of a typical bacterial genome [92]. Vitamin B12biosynthesis in microorganisms takes place according to
(aer-Table 2.2: Techniques and methods used for vitamin B12detection, their sensitivity
and their concrete applications ([67]).
obic), found in Pseudomonas denitrificans, and the oxygen-independent pathway (anaerobic), which exists in Salmonella typhimurium, Bacillus megaterium and Propionibacterium freudenreichii [20, 24, 75, 93] (Figure 2.3). These two routes differ from each other basically in the requirement of oxygen and insertion of cobalt. The vitamin B12 pathways can be divided into three stages: 1) the synthesis of
cobinamide (Cbi), a vitamin B12 precursor missing the DMBI molecule, 2) the
synthesis of the lower ligand, and 3) the incorporation of the lower ligand to the Cbi ring [78].
The anaerobic pathway begins with the 5-aminolevulinic acid (ALA) synthe-sis, which is the first general precursor of all tetrapyrroles, and it is derived from glutamic acid of succinyl-CoA and glycine [86, 91, 78]. Two molecules of ALA are condensed by ALA dehydratase, forming a pyrrolic molecule, porphobilinogen. In turn, porphobilinogen deaminase catalyses the polimerization and rearrange-ment of four porphobilinogens, forming preuroporphyrinogen, a linear tetrapyrrole whose synthesis is accompanied with the release of four ammonia units. Preu-roporphyrinogen is then linked with the D-ring pyrrole unit with the help of uroporphyrinogen III synthase, forming uroporphyrinogen III. From decarboxyla-tion of this molecule, precursors of the chlorophyll and hemes are formed (Figure 2.4). For the cobalamin biosynthesis, uroporphyrinogen III is then methylated at C-2 and C-7 by S-adenosyl-L-methionine uroporphyrinogen III methyltrans-ferase, forming precorrin-2 [75]. This dimethylated dipyrrocorphin has a crucial
role also in the coenzyme F430 and siroheme synthesis. The methyl groups are given by S-adenosyl-L-methionine. From precorrin-2, the aerobic and anaerobic pathway diverge [85]: in the oxygen-dependent route, another methyltransferase catalyzes the methylation of precorrin-2 at C-20 giving the precorrin-3A, while the oxygen-independent route is characterized by the insertion of a cobalt atom to give cobalt-precorrin-2. This chelation reaction, in the aerobic pathway, occurs only nine reaction steps after and, on the contrary of the anaerobic pathway, it requires ATP instead of high-energy equivalents [71]. Other main differences be-tween the two routes are the enzymes involved in the reaction steps: that is mainly because, in the anaerobic pathway, the intermediates are cobalt-complexes, while in the aerobic pathway they are metal-free intermediates. The two routes combine again with the synthesis of adenosyl-cobyric acid, whose propionic acid side-chain of ring D is then linked to an aminopropanol residue to give Cbi. The cobinamide is converted to α-ribazole by the transfer of phosphoribosyl moiety of nicotinic acid mononucleotide to DMBI, giving the lower nucleotide loop. Finally, the at-tachment of GDP-activated adenosylcobinamide to α-ribazole, accompanied with the release of GMP, results in the complete synthesis of coenzyme B12 molecule
Figure 2.3: Microbial biosynthesis of cobalamin through both anaerobic and aer-obic pathways (modified from [71]).
Figure 2.4: Macrocyclic tetrapyrroles whose biosynthetic pathways have a common intermediate, uroporphyrinogen III (modified from [86]).
2.3.2
Propionibacteria
Propionic acid bacteria (PAB) have gained a lot of commercial importance in food industry, especially in dairy industry. PAB are Gram-positive, non-spore-forming, microaerophilic bacteria with a peculiar metabolism, characterized by the formation of propionic acid as main fermentation end-product [105]. PAB can be divided into two main categories: dairy and cutaneous [105]. The most important species belonging to the dairy group are P. freudenreichii and P. acidipropionici, and are recognized as safe for use. P. freudenreichii can be classified into two subspecies, freudenreichii and shermanii, based on its lactose metabolism. They
have been used for many years in the Swiss-type cheese manufacturing thanks to their ability to influence positively the cheese flavor and to contribute to the “eye” formation, mostly due to propionic acid and acetic acid, and CO2 [104, 8].
Moreover, it has been suggested that PAB exhibit probiotic effects due to their production of important metabolites, bacteriocins, growth stimulation of beneficial LAB and resistance to gastric digestion [89, 60]. In addition to the enhancement of nutritional values, PAB are able to inhibit harmful and pathogenic microorganisms and extend the shelf life of the food products by inhibiting or at least slowing down the growth of moulds and fungi [9].
2.3.3
Vitamin B
12Industrial Production
Since chemical synthesis of vitamin B12 is challenging and highly expensive, it
was not possible to be adopted by companies. Thus, vitamin B12 is entirely
pro-duced through fermentation processes using genetically optimized bacterial strains. Some species known to be able to synthetize vitamin B12 belong to the following
genera: Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostrid-ium, CorynebacterClostrid-ium, FlavobacterClostrid-ium, Micromonospora, MycobacterClostrid-ium, Nor-cardia, Propionibacterium, Protaminobacter, Proteus, Pseudomonas, Rhizobium, Salmonella, Serratia, Streptomyces, Streptococcus and Xanthomonas [83]. Among all of these species, the ones for the industrial production of vitamin B12are
Bacil-lus megaterium, Pseudomonas denitrificans and Propionibacterium freudenreichii [12, 93, 56]. The genus Propionibacterium is nowadays the most used for the indus-trial production of vitamin B12due to its high yield. The vitamin B12productivity
of the strains can be increased by treating them with mutagenic agents like UV light, ethyleneimine, nitrosomethyluretane or N-methyl-N’-nitrosoguanidine. Af-ter that, the most suitable strains for the vitamin B12 production are selected
by paying attention to their productivity, genetic stability, reasonable growth rate and resistance to high concentrations of toxic intermediates present in the medium [71]. Using the best growing conditions for P. freudenreichii and P. shermanii, the production has been reached up to 206 and 60 mg/L, respectively [71].
The industrial production of vitamin B12by PAB consists of two stages. During
the first three days of fermentation, the culture is grown anaerobically in order to synthetize the Cbi. After that, PAB are transferred to aerobic conditions for 1-3
days to complete the vitamin B12structure by allowing the synthesis of the DMBI
moiety and linking it to Cbi [71]. PAB need oxygen to synthetize the DMBI moiety. According to Eggersdorfer (1996) [36], it is necessary to neutralize progressively the propionic acid which is produced by PAB during the fermentation process, in order to maintain the culture at pH 7. Furthermore, to optimize and increase the vitamin B12 production, it has been suggested to add precursors, such as DMBI
and cobalt [17, 71, 55].
2.3.4
Production of Vitamin B
12in Plant-derived Foods
The de novo biosynthesis of natural vitamin B12by some microorganisms could
be utilized in order to produce vitamin B12-enhanced foods. Especially vegans
and vegetarians would benefit from increased vitamin B12 content in plant-based
foods, instead of having vitamin B12 supplements. Although the most common
microorganism used for optimizing in situ cereal fermentations is P. freudenre-ichii, recently it has been also reported that LAB strains isolated from fermented products would have the ability to synthetize the cobalamin molecule. Taranto et al. in 2003 [103] suggested that Lb. reuteri CRL1098, isolated from sour-dough, would be able to produce a compound with vitamin B12activity. However,
it has been demonstrated by Santos et al. in 2007 [94] that the corrinoid-like molecule sinthetized by such microorganism was the pseudovitamin B12, where
the 5,6-DMBI is replaced with adenine. Another LAB which has been also re-garded as a cobalamin-synthetizing bacteria is Lb. sanfranciscensis. In particular, the strain TMW1.1304 was recently recognized as a vitamin B12 producer due to
its gene which has been fully characterized (cobyrinic acid a, c-diamide synthase, EC 6.3.5.11; LSA 2900630) [111]. However, it was observed that the corrinoid produced was a pseudovitamin B12, thus inactive in human metabolism. Waste
products have been used as the substrate in the vitamin B12 production. It is
possible to use cheese whey, prefermented vegetables from pickled vegetables and spent media (MRS medium) to produce biomass of P. freudenreichii and vitamin B12 [17, 72, 39].
2.4
Vitamin B
12Content in Foods and
Recom-mended Intakes
The synthesis of vitamin B12 is restricted solely to a certain species of
bacte-ria and archaea [97]. On the other hand, plants and fungi neither synthetize nor require it [71]. As vitamin B12 is synthetized only by some prokaryotes, it may be
found in plant foods which have been contaminated or fermented by such organ-isms. Animal products are the main vitamin B12 source for humans, as animals
can accumulate vitamin B12 in several ways: by ingesting feed supplemented with
vitamin B12 or feed materials contaminated with vitamin B12-producing
microor-ganisms, and some animals such as ruminants can absorb and utilize the vitamin B12 produced by some vitamin B12-synthetizing bacteria residing in the
gastroin-testinal tract [13]. However, the contribution of gut microbiota to human vitamin B12 status is not well known [99].
The Recommended Dietary Allowance (RDA) for vitamin B12 is 2.4 µg/day
[113]. Bor et al. in 2006 [22] reported that a daily intake of 6 µg appears to be sufficient to maintain a steady-state concentration of plasma vitamin B12 and
vitamin B12–related metabolic markers.
Table 2.3 shows the vitamin B12 content in some animal and non-animal foods.
The highest amount of vitamin B12can be found in organ meats such as kidney and
especially liver, but the meat in general is considered one of the most important dietary sources for vitamin B12. According to the USDA data, the amount of
vitamin B12 content in beef liver is 83 µg/100 g, although considerable losses
(27-33%) of this nutrient occur in cooking [16]. Furthermore, it is found in significant amount in eggs (0,9-1,4 µg/100 g) , milk and dairy products (0,3-0,4 µg/100 g) [117]. Amongst the seafood, vitamin B12 can be found in high amounts (more
than 10 µg/100 g fw) in shellfishes such as clams, oysters, mussels [117, 47], with a great variability among different species. In addition, the vitamin B12-producing
bacteria filtrated by such bivalves are also able to synthetize corrinoids which are inactive in human metabolism, as they have a different lower ligand than 5,6-DMBI. Amongst fishes, the vitamin B12 content can vary from 3.0 to 8.9 µg/100
g in certain fish species such as salmon, sardine, trout and tuna [117].
with B12-producing bacteria [24]. It is observed that it is possible to increase
the vitamin B12 content of spinach leaves up to 0.14 µg/100 g fw adding organic
fertilizer such as cow manure [77], but it is still insufficient to provide the RDA for adult humans. Furthermore, Bito et al. in 2012 [18] showed that most of corrinoids detected in organic fertilizers are inactive for humans. Bito et al. in 2013 [19] recently tried to produce a vitamin B12-enriched lettuce (Lactuca sativa)
by treating it for 24 h with various concentrations of CN-Cbl in hydroponic nutrient solution. They showed that the CN-Cbl concentration in the leaves reached up to 164.6 ng/g fw, without interfering with other compounds. A wide amount of different vitamin B12 forms has been found especially in edible algae or blue green
algae, but it was also shown that those compounds are inactive in mammals [116]. However, vegetarians and especially vegans have a lower intake and a low serum concentration due to their diet. The deficiency may lead to nervous disorders, such as multiple sclerosis [99, 74, 6].
During the last decades, many attempts have been done to fortify plant-based foods with vitamin B12 in order to provide the required dietary intake amongst
vegetarians and vegans. Babuchowski et al. in 1999 [9] demonstrated that there is a possibility to fortify fermented vegetable products with vitamin B12 using
propionic acid bacteria (PAB), which are frequently used by industries as vitamin B12-producing bacteria. They showed that there was a significant increase in
vitamin B12 (measured with microbiological assay, MBA) and folate content. In
addition, the shelf-life and the inhibition of pathogenic and harmful microflora increased.
In the USA, the vitamin B12-fortified foods are quite common, such as
breakfast-cereals, soymilk, and soy meat analogues [82]. These products can provide up to 200% of the RDA for vitamin B12 with the recommended intake of the products.
Additionally, one tablespoon of nutritional yeast contains 100% or more of the RDA of this vitamin.
Table 2.3: Vitamin B12content and bioavailability in some animal and non-animal
foods ([117])
2.5
Vitamin B
12and Bread-making
2.5.1
Fortification of Cereal Products with Vitamin B
12Bread and other cereals products provide a significant proportion of nutrients in human. Especially, many health-promoting effects of whole grain foods have been confirmed in several epidemiological studies [62]. Breads and other cereal foods are included in many sets of national dietary guidelines because they are a good sources of nutrients with fewer calories, more fiber, less salt and fewer additives, resulting in a healthier diet [106, 73]. Increasing knowledge about cereal compounds and new processing methods allowed to add optional ingredients in the bread-making process to improve the nutritional quality of bread [59, 101]. Fortifying cereals with vitamin B12is a popular approach, and the consumption of
vitamin B12 fortified cereal products provide a great proportion of dietary vitamin
B12 intake in the United States [113]. The necessity to fortify cereal foods become
clear especially for vegans, vegetarians and elderly people.
Several factors can influence the vitamin B levels in cereal foods, such as the degree of refining, the milling step, the extensive extraction and the chemical and physical factors like the pH of the dough or the baking temperature, which can
lead to losses of many vitamin B in baked products [11, 87]. However, it has been observed that, during the fermentation step, a slight increase of some vitamins B is detectable compared to the original flour, due to the production of these compounds by the developing microorganisms inside the dough: an example is riboflavin, which increases during the proofing time because it is synthetized by yeast [11].
2.5.2
Bread-making Processes
The bread-making process consists of three major stages: mixing, fermenta-tion and baking [34]. Different combinafermenta-tions of times and temperatures during this stages, as well as the ingredients used, determine the different bread-making methods. The most used bread-making methods are the straight-dough method, the sponge-dough method and the sourdough method.
The straight dough process is the most frequently used worldwide. On the contrary of the other two methods, it involves only one mixing step at the very beginning of the process, during which all the ingredients (flour, water, commercial yeast, salt, sugar and margarine) are mixed at the same time. After that, the dough leavens for 1-4 hours before being cut and molded. An additional proofing step is required to reach the desired size before baking [43]. The bread obtained with the straight-dough process usually has a coarse crumb and bland flavor.
On the contrary of the straight-dough method, the sponge-dough process is characterized by two mixing steps and two fermentation times: a longer pre-proofing stage and, later, a second intermadiate pre-proofing step to allow dough relaxation [43]. The ingredients are the same as the ones used in the straight-dough method, but, at the beginning, only the water, the yeast and half amount of flour are mixed. This results in a semi-liquid dough, which is allowed to pre-proof for a few hours at room temperature. During this time, the yeast starts its enzymatic reactions and grows metabolizing the starch and all the other nutrients in the flour. By consequence, the bread is characterized by a finer cell structure and a more complex flavor.
The sourdough process is a one of the oldest bread-making method, which is different from the previous two methods described. The great importance of sour-dough bread can be explained by its unique taste and flavor, as well as its healthy
properties [23]. The first fermentation step takes ca. 16 hours, in which the to-tal amount of water is mixed with one part of flour. In this stage, the microbial population of the sourdough seed starts metabolizing the nutrients in the dough, giving an acid taste, aroma and increased volume due to gas formation [44]. On the contrary of the straight-dough and the sponge-dough, which are fermented by a commercial selected strain of Saccharomyces cerevisiae, the sourdough is leav-ened by LAB and metabolically active yeasts. These microorganisms may develop from selected natural contaminants in the flour or from a starter culture containing one or more known species of LAB. Such bacteria are mostly heterofermentative strains, which produce lactic acid and acetic acid in the mixture, resulting in a sour taste of the final bread. The LAB:yeast ratio is about 100:1 [80]. Hetero-fermentative Lactobacillus strains are the most frequent LAB in the sourdough, while lactococci, enterococci, and streptococci are rarely found due to their less adaptability to this particular environment. Sourdoughs can be freshly prepared or can be found as commercial suppliers. Most European bakeries still use tradi-tionally fermented sourdoughs which are kept metabolically active by adding fresh flour and water at regular intervals. The strong acidification, which reaches pH values of less than 4.1, leads to a significantly longer shelf-life, other that improving nutritional and organoleptic properties [43].
2.5.3
Stability of Vitamin B
12in Bread-baking
Only a few previous studies about the stability of vitamin B12 in baking were
carried out. One of them is the study by Agnoletti et al. [1] and another is the study by Winkles et al. [120]. According to Agnoletti et al. [1], some steps have to be taken into consideration to obtain a vitamin B12-fortified cereal product.
They observed that there are not notable modifications in organoleptic quality of bread in high concentrations of vitamin B12. Agnoletti et al. [1] demonstrated
that 45% of the supplemented vitamin B12 was destroyed during baking the bread.
According to that, it has been proposed that the correct amount of vitamin B12
to use for fortifying should be 10 µg/100 g. Despite this, the recommendation is to add vitamin B12 20 µg/kg flour. If the consumption of flour is from 75
to 100 g per day, the intake of vitamin B12 would be from 75% to 100% of the
addition, as well as the bioavailability of the vitamin B12 ingested, have still to be
well-monitored. A bread cofortified with vitamin B12 and folic acid was obtained
by Winkels et al. in 2008 [120]. They demonstrated that a consumption of 3-5 slices of this bread ( 33 g each) increased the serum vitamin B12 concentration by
Experimental Research
3.1
Aims
The aim of the experimental part was to study the stability of vitamin B12 in
laboratory conditions and in baking processes. To study this, the preliminary aim was to understand the stability of different vitamin B12 forms under different pH
conditions and in different time of exposure to light. In the second part of the study, the main purpose was to investigate the stability of two forms of vitamin B12,
CN-Cbl and OH-CN-Cbl, during different bread-making processes: straight-dough, sponge-dough and soursponge-dough. Finally, the stability of the natural vitamin B12 produced
by a selected strain P. freudenreichii subsp. freudenreichii was investigated in straight-dough baking process.
3.2
Materials and Methods
3.2.1
Chemicals, Reagents and Equipment
Analytical balance (Precisa XT 220A, Precisa Gravimetrics, Dietikon, Switzer-land)
Autoclave (Steris Finn-Aqua 46-E, number 7253, Finn Aqua®, Steris Finn Aqua, Tuusula, Finland)
Blender (Bamix M122, ESGE AG, Mettlen, Switzerland) 25
Centrifuge (RC5C, Dupont Instruments-Sorvall, Wilmington, Delaware, USA) Centrifuge (Hermle Z322, Hermle Labortechnik GmbH, Wehingen, Germany) Combined orbital/linear shaking water bath (Grant OLS 200, Keison
Inter-national, Chelmsford, Essex, UK)
Cuvettes (disposable semimicro PS 1.5 mL, 12,5x12,5x45mm, Brand® GmbH, Wertheim, Germany)
Extensograph (Type DM 90/40, Brabender® OHG, Dulsburg, Germany) Farinograph (Brabender® GmbH & Co, Germany)
Freeze dryer (Christ Alpha 2-4 LD plus, Christ Co., Osterode, Germany) Incubator (Certomat®H, Sartorius Stedim Bioech S.A. Aubagne Cedex,
France)
Knife mill (Grindomix GM 200, Retsch GmbH, Haan, Germany)
Linear shaking water bath (Grant GLS400, Keison International, Chelms-ford, Essex, UK)
Microbalance (Sartorius ME5, Sartorius AG, Goettingen, Germany) Microplate reader (Multiskan EX; Labsystems, Finland)
Mixing machine (Hobbart N50, ITW Food Equipment Group, US)
Molding unit of an extensigraph (type DM 90/40, Brabender OHG, Ger-many)
pH-meter (PHM220, MeterLab, Radiometer Analytical, Lyon, France) Shaker (Reax 2000, Heidolph Instruments, Schwabach, Germany)
Spectrophotometer (Novespec® II, Amersham Pharmacia Biotech, Piscat-away, New Jersey, USA)
Vacuum packaging machine (Multivac, Brand® GmbH, Wolfertschwenden, D¨usseldorf, Germany)
3.2.2
Stability Tests with the Vitamin B
12Forms in
Labo-ratory
All the following operations concerning the preparation of standard solutions were carried out under red light, which is characterized by less energy than the white one, in order to minimize the degradation of vitamin B12 structure. As our
aim was to test the photostability of different vitamin B12 forms under different
pH conditions, three solutions with pH values 2.5, 4.5 and 7.0 were prepared using Milli-Q water. The pH of the solutions was adjusted using weak sodium hydroxide (3%) and weak acetic acid (3%). The aqueous standard solutions (1 mg/mL) were prepared using Ado-Cbl, Me-Cbl, CN-Cbl and OH-Cbl stored at -20 °C and kept in the desiccator for 30 min before weighing them. The stock solutions were diluted 1:200 to obtain a concentration of 0.5 ng/mL and stirred accurately. The solutions were filtered through 0.2µm filters (GHP Acrodisk®, Pall Corporation, USA) into vials (12 x 32, Waters Corporation, USA) before injecting into the UHPLC. To test the stability of the different vitamin B12 forms under light, the
same vials were moved to light at room temperature (RT) for the necessary time before the following injection. If the period of light exposure was extended during the night, the laboratory light was kept on.
3.2.3
Baking Processes with OH-Cbl and CN-Cbl
Straight-Dough Method
The recipe of the straight-dough used for the control bread without adding any vitamin B12 solution is shown in Table 3.1. The optimal mixing time and water
absorption capacity of flours was determined with the farinograph until the mixing curve reached 500 Brabender units (BU). The amount of flours for farinograph was calculated in 14% dry-matter content.
Straight-dough baking was performed as outlined in Figure 3.1. In test doughs, 2 mL water was replaced with 2 mL OH-Cbl or CN-Cbl solution in water (0.25 mg/mL). Firstly, flour, salt and sugar were mixed for 1 min at RT at the lowest speed (speed 1) in a mixer. Fresh yeast was dissolved into water (35°C). Margarine was added to the water-yeast mixture and subsequently mixed with the other ingredients for 4 min at middle speed (speed 2). After mixing, the dough was
Table 3.1: Bread recipe used in the straight-dough method.
Weight Ratio of the ingredients mass Ingredient
(g) to the total flour mass (%)
Wheat flour 400 100 Tap water (35 °C) 238 59.5 Fresh yeast 16 4 Salt 4 1 Sugar 12 3 Margarine 12 3 682
weighed and two samples of 50 g were taken and stored at -20 °C. The dough was allowed to rest for 10 min at RT and then divided into three pieces of 150 g each. Each of them was subjected to the molder of the extensigraph to take the correct shape and dimension. Dough pieces were put into tins, properly sprayed with oil, and put into a proofing cabinet for 90 min at 34°C, RH ca. 80%. After proofing, one dough was divided into two pieces, put in plastic bags and stored at -20°C. The two tins left were immediately baked in the convection oven (20 min at 180 °C). The baked breads were allowed to cool for 60 min at RT and then weighed. The volume of baked breads was measured with the rapeseed displacement method. The two baked breads were cut in small pieces, combined together and stored at -20°C. Straight-dough procedure was repeated three times for each type of adding (without adding any standard, adding OH-Cbl, and adding CN-Cbl).
Sponge-Dough Method
The recipe and the ingredients used in sponge-dough method were the same as for the straight dough method (Table 3.1) and figure 3.2 shows the baking process in outline. Yeast was accurately dissolved into the water (35 °C). The solution was mixed with 200 g of flour by hand for 1 min using a spoon. After that, two samples of 50 g each were taken and stored at -20 °C. The dough was then subjected to a pre-proofing step for 120 min at RT. At the end of it, two samples of 50 g each were taken and stored at -20°C. Sugar, salt, margarine and 200 g of flour were subsequently added to the dough and mixed in the mixer first for 1 min at the lowest speed, and after that for 3 min more at the middle speed. Two pieces
Table 3.2: Bread recipe used in the sourdough method.
Ingredient Weight(g)
Rye dough seed 160
Rye flour 687
Tap water (at RT) 719
Vitamin B12 standard solution (0.25 mg/mL) 3.1
Salt 9.5
1575.5
were taken and stored at -20 °C. The dough was then divided into three pieces of 150 g each. Each of them was moulded, transferred into tins, sprayed with oil, and put into the proofing cabinet for 90 min at 34 °C. After proofing, one dough was divided into two pieces, put in plastic bags and stored at -20 °C. The two tins left were immediately baked in the convection oven (20 min at 180°C). The baked breads were cooled, weighed, and volume was measured like in straight-dough breads. Breads were cut in pieces which were combined together and stored at -20 °C. Sponge-dough procedure was repeated three times for each type of adding (without adding of any standard, adding OH-Cbl, and adding CN-Cbl).
Sourdough Method
The recipe of sourdough is shown in Table 3.2, while the whole bread-making process is represented in Figure 3.3.
A rye-dough starter was provided by a local bakery (Pirjon Pakari, Nurmij¨arvi, Finland). The starter (160 g) was regenerated by mixing it with 100 g of tap water and 60 g of rye flour. Pre-dough was fermented for 5 h at 30 °C. Three separate doughs were prepared by mixing 50 g of regenerated dough with 375 g of rye flour and 619 g of tap water. After the mixing step, two samples of ca. 50 g each were taken and stored at -20 °C. The three separate doughs were fermented for 16 h at 30 °C. After the 16-h fermentation, two samples of 50 g each were taken and stored at -20 °C. 806 g of each fermented sourdough were mixed with 252 g of rye flour and 9.5 g of salt by hand. One dough was retained as control and either OH-Cbl (0.8 mg) or CN-Cbl (0.8 mg) were added to the other two doughs. All the ingredients were mixed by hand for 1 min and then with the mixing machine for
3 min: 2 min at the lowest speed and 1 min at middle speed. After mixing, two samples were taken and stored at -20 °C. The doughs were allowed to proof for 60 min at 30 °C. After intermediate proofing, the doughs were divided into four pieces of 200 g, rounded, molded by hand and put in tins for a second proofing step (30 min at 30 °C). Samples were taken from one dough after this proofing time, and stored at -20°C. Breads were baked (70 min at 180 °C) in the convection oven, and cooled (60 min at RT). The weight and the volume were measured using the same method as for the straight-dough and sponge-dough breads. After that, each bread was cut into small pieces and stored at -20 °C. Sourdough procedure was performed only once for each type of adding (without adding any standard, adding OH-Cbl, and adding CN-Cbl).
3.2.4
Freeze-drying of Dough and Bread Samples
One sample from each sampling step was lyophilized using a freeze-dryer at a chamber pressure under 1 mbar approximately for 24 h. Immediately after freeze-drying, the samples were ground using a laboratory knife mill (1 min at 10000 rpm; the time could be increased up to 30 s more to ensure a proper milling also for harder samples). Each sample, after milling, was split into two samples and packed under vacuum using a laboratory vacuum packaging machine. The freeze-dried samples were stored at -20°C until the further analyses.
3.2.5
pH Measurement of Dough and Bread Samples
The samples were taken out from -20 °C for 90 min before starting the pH determination. 10 g of fresh dough or bread sample was suspended in 100 ml MiliQ water by mixing for 1 min with the blender. An additional milling step (1 min at 10000 rpm) was needed for the fresh bread before the determination. The sample was allowed to rest for 15 min at RT and the pH was measured using the pH-meter. During the whole pH measurement time, the sample was kept under stirring using a magnetic stirrer.
