stability of vitamin b12 with the protection of whey

Accepted Manuscript

Stability of Vitamin B12 with the Protection of Whey Proteins and Their Effectson the Gut Microbiome

HuanhuanWang Yikai Shou Xuan Zhu Yuanyuan Xu Lihua Shi ShashaXiang Xiao Feng Jianzhong Han

PII S0308-8146(18)31798-9DOI httpsdoiorg101016jfoodchem201810033Reference FOCH 23694

To appear in Food Chemistry

Received Date 5 October 2017Revised Date 28 September 2018Accepted Date 6 October 2018

Please cite this article as HuanhuanWang Shou Y Zhu X Xu Y Shi L Xiang S Feng X Han J Stabilityof Vitamin B12 with the Protection of Whey Proteins and Their Effects on the Gut Microbiome Food Chemistry(2018) doi httpsdoiorg101016jfoodchem201810033

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1

Stability of Vitamin B12 with the Protection of Whey Proteins and Their Effects on the

Gut Microbiome

Running title Stability and Effect of Vitamin B12Whey Complexes

HuanhuanWangadagger huanvalhznueducn

Yikai Shouadagger 403278497qqcom

Xuan Zhub zhuxuanmailzjgsueducn

Yuanyuan Xub 362120957qqcom

Lihua Shib 624872970qqcom

Shasha Xiangb xsscherishqqcom

Xiao Fengb 249259608qqcom

Jianzhong Hanb jzhan99zjgsueducn

aSchool of Medicine Hangzhou Normal University Hangzhou 310018 China

bSchool of Food Science and Bioengineering Zhejiang Gongshang University Hangzhou

310018China

daggerThese authors contributed equally to this work

Corresponding author Dr Xuan Zhu Email zhuxuanmailzjgsueducn Phone

+8657128008902 School of Food Science and Bioengineering Zhejiang Gongshang

University Xuezheng Str 18 Hangzhou 310018 China

Abstract

Cobalamin degrades in the presence of light and heat which causes spectral changes and loss

of coenzyme activity In the presence of beta-lactoglobulin or alpha-lactalbumin the thermal-

and photostabilities of adenosylcobalamin (ADCBL) and cyanocobalamin (CNCBL) are

increased by 10-30 Similarly the stabilities of ADCBL and CNCBL are increased in the

presence of whey proteins by 197 and 22 respectively when tested in gastric juice for 2

h Due to the limited absorption of cobalamin during digestion excess cobalamin can enter

the colon and modulate the gut microbiome In a colonic model in vitro supplementation with

cobalamin and whey enhanced the proportions of Firmicutes and Bacteroidetes spp and

reduced those of Proteobacteria spp which includes pathogens such as Escherichia and

Shigella spp and Pseudomonas spp Thus while complex formation could improve the

stability and bioavailability of cobalamin these complexes might also mediate gut

microecology to influence human nutrition and health

2

Keywords cobalamin whey protein gut microbiome stability

3

1 Introduction

Cobalamin (vitamin B12) is a naturally occurring organometallic compound containing

cobalt that serves as an important water-soluble vitamin for human health The recommended

daily intake for cobalamin is 24 microg (Rucker Suttie McCormick amp Machilin 2001) This

vitamin functions as a cofactor for two classes of human enzymes namely isomerases and

methyltransferases Consequently cobalamin deficiency can cause disturbances in cell

division leading to neuropathy nervous system disease and pernicious anemia (Allen 2010)

Cobalamin has four bioactive forms and many analogues which different in their upper

andor lower functional groups For example the adenosyl can be replaced by a methyl

hydroxyl or cyano group to form methyl- hydroxo- or cyano-cobalamin respectively

Cyanocobalamin (CNCBL) is not found in nature but is used as a supplement for humans and

animals Cobalamin is not stable during food processing or storage as it is photo- and

heat-labile (Ahmad Hussain amp Fareedi 1992) Moreover the aerobic photodecomposition of

cobalamin processes more rapidly than the anaerobic photodecomposition (Demerre amp

Wilson 1956) meaning common food processing can enhance degradation All forms of

cobalamin are irreversibly inactivated under irradiation However some enzymes requiring

adenosylcobalamin (ADCBL) and methylcobalamin can also protect these compounds from

decomposition (Demerre amp Wilson 1956)

Whey protein typically consists of beta-lactoglobulin alpha-lactalbumin bovine serum

albumin and immunoglobulins The main protein (65) in bovine whey is beta-lactoglobulin

(18 kDa) a globular protein with 162 amino acid residues Beta-lactoglobulin belongs to the

lipocalin family and is able to bind small hydrophobic molecules in the internal cavity of its

β-barrel Because of multiple binding sites beta-lactoglobulin can bind a variety of ligands

including fatty acids polyphenols and vitamins such as cobalamin (Sawyer Brownlow

Polikarpov amp Wu 1998) Alpha-lactalbumin (14 kDa) the second most prevalent protein is a

small globular protein containing 123 amino acid residues with a large alpha-helical domain

and a small beta-sheet domain (Cawthern Narayan Chaudhuri Permyakov amp Berliner 1997)

The two domains are separated by a deep cleft and linked by a calcium binding loop It has

been reported that alpha-lactalbumin may function as a carrier of hydrophobic lipids vitamins

and metabolites

Ligand-binding proteins such as lactoglobulins and lactalbumins have been used to bind

low-molecular weight molecules for their protection and delivery (de Wolf amp Brett 2000)

The maximum cobalamin binding capacities of beta-lactoglobulin alpha-lactalbumin casein

blood serum album and proteose-peptone were determined to be 850 690 98 80 and 370

μgg respectively (Gizis Kim Brunner amp Schweigert 1965) Beta-lactoglobulin and

alpha-lactalbumin have also been reported to bind vitamin B12 and protect it from

4

decomposition (Gizis Kim Brunner amp Schweigert 1965 Johns et al 2015) Some

researchers have suggested that lactoferrin can dramatically increase the photostability and

solubility of cobalamin (US Pat 6500472B2 2002)

Due to limited absorption of cobalamin during digestion excess cobalamin can enter the

colon and modulate the gut microbiome Cobalamin is the only vitamin produced exclusively

by bacteria and archaea but it is used by all domains of life Synthesis of cobalamin is

energetically costly requiring nearly 30 different enzymes (Martens Barg Warren amp Jahn

2002) and only 20 of prokaryotes have the capacity to produce cobalamin (Degnan Barry

Mok Taga amp Goodman 2014) Cobalamin is a precious commodity and has been suggested

to be important not only as a nutrient but also as a signaling molecule for the spatial and

functional organization of gut microecology

In this study we examined whether beta-lactoglobulin or alpha-lactalbumin could

enhance the stability of cobalamin during food processing and positively affect the

composition of a model human gut microbiome The results obtained from this research

provide insights into possible applications of ligand-binding proteins as carriers of cobalamin

in the development of functional foods and pharmaceuticals

2 Method and Materials

21 Chemical reagents

Beta-lactoglobulin (purityge90) and alpha-lactalbumin (purityge85) were purchased

from Beingmate Company (Hangzhou China) and used without further purification ADCBL

and CNCBL were purchased from Sigma-Aldrich Chemical Company (St Louis US) and

used without further purification

22 Sample preparation

Stock solutions of beta-lactoglobulin alpha-lactalbumin ADCBL and CNCBL were

prepared at a final concentration of 10 μM in 1 mM phosphate buffer at pH 60

Cobalamin-whey protein complexes were prepared by mixing different concentrations of

protein (025 05 075 μM) with the ligand (025 μM) in phosphate buffer All samples were

prepared and incubated for approximately 1 h at room temperature in 200 mL flasks

(DURAN no 2121654 Schott GmbH Mainz Germany) while shaking (50 rpm) in the dark

23 Photodecomposition and heat treatment procedures

Briefly 200 mL flasks containing the cobalamin-whey protein complexes were placed

under a UV LED bulb (9 W) (Leiman Co Ltd Shenzhen China) with a light intensity of ca

100 μmol m-2 s-1 Each flask was placed 10 cm from the light source Samples were analyzed

5

every 15 min for up to 120 min Similarly the 200 mL flasks containing cobalamin-whey

protein complexes were placed into a water bath at 80 degC (Sonorex TK 52 ultrasonic water

bath Bandelin Electronics Berlin Germany) Samples were analyzed every 15 min for up to

120 min in the dark to avoid light degradation All experiments were performed in triplicate

24 In vitro stomach digestion

The stability of cobalamin in gastric fluid (SGF) was determined by subjecting samples

to simulated digestion in the presence of pepsin (pH 13 37 degC 2 h) SGF was prepared as

described previously (Tokle Lesmes Decker amp McClements 2012) with slight modifications

Briefly SGF was prepared by dissolving 1 (wv) pepsin 3 g of sodium chloride and 9 ml

of HCl in 1 L of water the pH was adjusted to 13 using 1 M HCl Cobalamin-whey protein

complexes (01 g) were incubated in 100 mL of SGF with shaking (100 strokesmin) in a

water bath at 37 degC for 2 h to simulate stomach conditions

25 Cobalamin determination

To extract cobalamin from 10 mL samples of B12-whey complexes 50 mL of sodium

acetate buffer (pH 60) was added in the presence of sodium cyanide (1) and the solution

was heated in a water bath (Type 1004 water bath GFL Gesellschaft fuumlr Labortechnik

Burgwedel Germany) for 40 min at 90 degC The pH was adjusted to 7 and 10 mL hexane was

added before the mixture was centrifuged for 15 min at 4010 g (Varifuge 30 Heraeus

centrifuge Heraeus Instruments Hanau Germany) The aqueous layer was collected and

passed through a solid phase extraction column (SPE) (CEC181M6 United Chemical

Technologies Bristol PA USA) which had been prewashed with 3 mL of methanol and 3

mL of Milli-Q water with the help of a pump (AL 15 Knf Neuberger Hamburg Germany)

to control the flow (1 drop per second) The column was washed three times with Milli-Q

water and 3 mL of methanol was used to elute cobalamin The solvent was evaporated to

dryness and the residue was subsequently dissolved in 1 mL of Milli-Q water before being

passed through a membrane filter (02 μm) (Macherey-Nagel Duumlren Germany) and analyzed

by HPLC using a RP-18 column (2504 mm ID 5 microm Merck Darmstadt Germany)

Cobalamin was detected using a modified HPLC method that was previously reported

(Gu Zhang Song Li amp Zhu 2015) All chromatographic separations were performed at

room temperature The mobile phases consisted of a mixture of methanol with 01 formic

acid (A) (Merck Darmstadt Germany) and ultra-purified water with 01 formic acid (B)

which was degassed by an ultrasonic water bath the flow rate was 05 mL per min The

gradient elution was programmed as follows 0-2 min 20 A 2-3 min 20-25 A 3-11 min

25-35 A 11-19 min 35-20 A 20-22 min 100-100 A 22-26 min 100-20 A and

6

26-36 min 20 A The injection volume was 100 μL and the column eluate was monitored

by a Diode Array Detector (Waters US) at 361 nm

26 In vitro intestinal digestion

Colonic fermentation of the cobalamin-whey complex was conducted according to

Macfarlane et al (2006) with a slight modification Fecal samples from a three-year-old child

who had not received antibiotic treatment in the previous three months were collected and

maintained in anaerobiosis until bacterial immobilization on gellan-xanthan beads as

previously described (Cinquin Le Blay Fliss amp Lacroix 2006) Fecal microbiota was

immobilized under anaerobic conditions in 1-2 mm gel beads composed of gellan (30 wv)

xanthan (03 wv) and sodium citrate (03 wv) The gel beads (70 mL) were transferred

immediately to a fermentation reactor containing 200 mL of nutritive medium The nutritive

medium contained the following (gL of distilled water) rice starch (8) peptone (3) tryptone

(3) bile salts (005) mucin (4) yeast extract (25) hemin (001) Tween 80 (1) salts (NaCl

45 KCl 45 MgSO47H2O 125 CaCl2 015 K2HPO4 05 NaHCO3 15 FeSO47H2O

0005) filter-sterilized vitamin solution (2) (mgL riboflavin 360 pyridoxine hydrochloride

400 pantothenate 300 thiamine hydrochloride 400 nicotinamide 450 biotin 150

p-aminobenzoic acid 20 folate 10) and cysteine (08)

Six parallel reactors inoculated with immobilized gut microbiota were operated for 10

days The reactors were continuously fed nutritive media differing only in their

supplementation with cobalamin-whey complexes or cobalamin only The fermentation was

performed under typical conditions of the proximal colon according to previously described

procedures (Macfarlane Macfarlane amp Gibson 1998 Cinquin Le Blay Fliss amp Lacroix

2006) Fecal beads were first colonized by batch fermentation for 72 h The nutritive medium

was replaced every 12 h During the simulated fermentation the pH was maintained at 60

throughout the experiment with the addition of 2 M NaOH and the temperature was

maintained at 37 degC Anaerobiosis was achieved and maintained by continuously flushing N2

into all reactors and medium vessels

The working reactor volume was set at 200 mL with a continuous inflow (40 mL h-1) of

fresh media resulting in a mean retention time of 5 h Complexes (05 μM) or cobalamin (05

μM) were added to the nutritive media for 10 days during fermentation All six reactors were

sampled daily and samples were frozen at -80 degC for pyrosequencing

27 DNA isolation PCR 16S rDNA analysis

DNA was extracted from the samples using a MicroElute Genomic DNA Kit (D3096-01

Omega Inc USA) according to the manufacturerrsquos instructions The reagent designed to

7

recover DNA from trace amounts of sample has been shown to be effective for most bacteria

Blank samples consisted of unused swabs processed via DNA extraction protocols and tested

Total DNA was eluted in 50 μl of elution buffer using a modified procedure described by the

manufacturer (QIAGEN) and stored at -80 degC until PCR amplification (LC-Bio Technology

Co Ltd Hang Zhou Zhejiang Province China)

We amplified the V3ndashV4 region of the bacterial 16S rDNA using the total DNA of

samples from in vitro colonic stimulation as a template and the primers 319F

5rsquo-ACTCCTACGGGAGGCAGCAG-3rsquo and 806R 5rsquo-GGACTACHVGGGTWTCTAAT-3rsquo

All reactions were carried out in a 25 μL total volume containing approximately 25 ng of

genomic DNA extract 125 μL of PCR premix 25 μL of each primer and PCR-grade water

to adjust the volume PCRs were performed on a Master cycler gradient thermocycler

(Eppendorf Hamburg Germany) set to the following conditions initial denaturation at 98 degC

for 30 seconds 35 cycles of denaturation at 98 degC for 10 seconds annealing at 54 degC 52 degC

for 30 seconds and extension at 72 degC for 45 seconds and a final extension step at 72 degC for

10 min PCR products were confirmed via 2 agarose gel electrophoresis Throughout the

DNA extraction process ultrapure water was used as a negative control to exclude

false-positive results PCR products were normalized using AxyPrep TM Mag PCR

Normalizer (Axygen Biosciences Union City CA USA) which allowed elimination of the

quantification step regardless of the PCR volume submitted for sequencing The amplicon

pools were prepared for sequencing with AMPure XT beads (Beckman Coulter Genomics

Danvers MA USA) and the size and quantity of the amplicon library were assessed using

the LabChip GX (Perkin Elmer Waltham MA USA) and Library Quantification Kit for

Illumina (Kapa Biosciences Woburn MA USA) respectively The PhiX Control library (V3)

(Illumina) was combined with the amplicon library (expected at 30) and clustered to a

density of approximately 570 Kmm2 The libraries were sequenced on 300PE MiSeq runs

except for one library that was sequenced with both protocols using the standard Illumina

sequencing primers which eliminated the need for a third (or fourth) index read

Reads were filtered using QIIME quality filters (Quantitative Insights into Microbial

Ecology httpqiimeorgtutorialsprocessing_illumina_datahtml) The CD-HIT pipeline was

used to select operational taxonomic units (OTUs) by making an OTU table Sequences were

assigned to OTUs at 97 similarity Representative sequences were chosen for each OTU

and taxonomic data were assigned to each sequence using the RDP classifier (Ribosomal

Database Project) To estimate alpha diversity the OTU table was rarified and four metrics

were calculated Chao 1 to estimate the richness Observed OTUs as a measure of unique

OTUs found in the sample and the Shannon and Simpson indices All taxa abundances were

8

used to generate principal component analysis (PCA) The heatmap of important gut

microbiome families was constructed using Mev 4-9-0

28 Statistics

All data are shown as the mean plusmn SD The data were analyzed by variance and Duncanrsquos

test for multiple comparisons using SPSS ver 170 A p value lt 005 was significant

3 Results and Discussion

31 Effects of alpha-lactalbumin and beta-lactoglobulin on the photostability of cobalamin

Whey proteins were associated with slow light-induced ligand decomposition In the

absence of alpha-lactalbumin and beta-lactoglobulin the most rapid phase of CNCBL and

ADCBL decomposition spanned the first 60 and 50 min respectively (Fig 1A and 1B) After

irradiation treatment the stabilities of CNCBL and ADCBL in the presence of 025 05 and

075 μM alpha-lactalbumin were extended significantly compared to those in the absence of

alpha-lactalbumin In the presence of alpha-lactalbumin the decomposition rates of CNCBL

and ADCBL were lower than that of cobalamin alone The extent of decomposition in the

presence of alpha-lactalbumin was similar to that of cobalamin alone but was not reached

until 120 min after UV irradiation Compared with others CNCBL with 05 μM

alpha-lactalbumin had the greatest stability following irradiation In contrast to other

alpha-lactalbumin-ADCBL complexes ADCBL plus 075 μM alpha-lactalbumin had the

slowest decomposition rate

In the case of beta-lactoglobulin (Fig 1C and 1D) the stabilities of CNCBL and

ADCBL in the presence of 025 05 and 075 μM beta-lactoglobulin were also significantly

enhanced compared with that of cobalamin alone The decomposition rate of CNCBL and

ADCBL plus beta-lactoglobulin was lower than that of cobalamin alone The extent of

decomposition in the presence of beta-lactoglobulin was less than that of cobalamin alone up

to 120 min after UV irradiation Compared with others CNCBL plus 05 μM

beta-lactoglobulin had the slowest decomposition rate following irradiation In contrast to

other beta-lactoglobulin concentrations ADCBL plus 075 μM beta-lactoglobulin had the

highest rapid decomposition rate

CNCBL in the presence of beta-lactoglobulin also had a slower photodecomposition rate

than both CNCBL plus alpha-lactalbumin and cobalamin alone after 120 min irradiation

indicating that whey proteins can suppress the photodecomposition of CNCBL In regards to

CNCBL the effectiveness of beta-lactoglobulin is greater than that of alpha-lactalbumin

Conversely ADCBL plus alpha-lactalbumin was more stable than ADCBL plus

beta-lactoglobulin

9

UV-light easily induces the decomposition of cobalamin in multiple steps starting with

cleavage of the C-Co bond to form either hydroxocobalamin or aquocobalamin depending on

the pH These intermediates decompose further to deoxyadensoine to form cobamides neither

of which are bioactive in humans (Schneider amp Stroinski 1987)

Although interaction of whey proteins such as alpha-lactalbumin and beta-lactoglobulin

with vitamin B12 can delay photodecomposition of cobalamin (Gizis Kim Brunner amp

Schweigert 1965) the differences in the protective effects of the various proteins can be

attributed to their interactions with cobalamin and its analogues Dalsgaard Otzen Nielsen amp

Larsen (2007) demonstrated that photooxidation can lead to changes in the primary structures

of alpha-lactalbumin and beta-lactoglobulin in the presence of the photosensitizer riboflavin

as well as folic acid (Liang Zhang Zhou amp Subirade 2013) During photooxidation the

carbonyl content was increased due to oxidation of tryptophan histidine and methionine in

all proteins (Gizis et al 1965) In particular the tryptophan residues in alpha-lactalbumin

were more stable than those in beta-lactoglobulin and were located in the hydrophobic interior

of both molecules Thus whey proteins can offer protection to cobalamin against

photodecomposition However a strange phenomenon was observed alpha-lactalbumin

which contains one methionine residue provided better protection to ADCBL during the

irradiation than beta-lactoglobulin which contains four methionine residues This

phenomenon is most likely because exposed methionine residues and thiol groups of proteins

have a strong affinity for the deoxyadenosyl radical from ADCBL produced after irradiation

(Padmanabhan Jost Drennan amp Eliacuteas-Arnanz 2017) forming S-adenosyl-methionine

(Bridwell-Rabb amp Drennan 2017) Furthermore as the concentration of whey protein

increases the decomposition rate of CNCBL also increases Additionally during irradiation

treatment higher concentrations of whey protein can lead to more substances with thiol

groups By way of an explanation a recent study stated that cysteine and other reducing

substances such as methional and dimethyl disulfide from methionine can rapidly destroy

CNCBL under irradiation (Mukherjee amp Sen 1957)

32 Effects of alpha-lactalbumin and beta-lactoglobulin on the thermal stability of

cobalamin

Thermal treatment is an essential element in food processing and the stability of

nutrients during thermal treatment is directly related to food quality Cobalamin is not only

photosensitive but also heat-sensitive In the absence of whey proteins cobalamin was

reduced by ca 60 after 30 min at 80 degC The stability of cobalamin was enhanced

significantly in the presence of 075 μM alpha-lactalbumin or beta-lactoglobulin compared

with that of cobalamin alone or other supplements (Fig 2) Beta-lactoglobulin could protect

10

CNCBL from thermal decomposition even after 120 min of heat treatment at 80 degC

Conversely the decomposition rate of ADCBL supplemented with alpha-lactalbumin was

significantly reduced in contrast to those of others In contrast to CNCBL alone whey protein

enhanced the thermal stability of CNCBL at the beginning of 30 min by ca 15 even at low

concentrations (Fig 2) There were no great differences between ADCBL alone and ADCBL

complexes at the beginning indicating that ADCBL is not as stable as CNCBL even in the

presence of whey proteins Similarly there were no differences in the CNCBL decomposition

rates in the first 45 min suggesting that even low concentrations of whey protein afford

sufficient protection Similar results were observed for ADCBL-whey protein complexes in

the first 30 min These results indicate that low concentrations of whey protein are an efficient

protective agent for cobalamin during food processing

In solution all forms of cobalamin are liable in the presence of vitamin C thiamine

nicotinamide etc especially during heating treatment Watanabe et al reported that ca 40

of vitamin B12 in food was appreciably degraded during microwave heating (Watanabe et al

1998) Vitamin B12 is sensitive to heat treatment and decomposes in multiple steps starting

from hydrolysis of propionamide side chains in the corrin ring to form deamidated

cobalamins which are nonactive in humans (Schneider amp Stroinski 1987) Researchers also

stated that hydrolysis of vitamin B12 in vials and glass containers occurred during heat

sterilization The native structures of beta-lactoglobulin and alpha-lactalbumin are destroyed

during thermal denaturing albeit this does affect their binding affinity for cobalamin (Gizis

Kim Brunner amp Schweigert 1965) A previous study has shown that both alpha-lactalbumin

and beta-lactoglobulin can repeatedly undergo a similar thermal transition under heat

treatment (Hong amp Creamer 2002) The transition temperature of alpha-lactalbumin is lower

than that of beta-lactoglobulin Extensive heat treatment gives rise to alpha-lactalbumin

dimers and trimers via disulfide bond formation Meanwhile the thiol group in

beta-lactoglobulin becomes solvent-accessible during heat treatment The dimers and trimers

of beta-lactoglobulin some of which are hydrophobically associated products are also

formed via intermolecular thiol-catalyzed disulfide bond interchanges The stable

improvement of cobalamin is due to an increase in the number of exposed hydrophilic

binding sites and fewer unexposed thiol groups during heating unlike the changes caused by

irradiation treatment

Many studies demonstrated that more attention has been paid to the stability of

cobalamin under different storage conditions than to the light and thermal sensitivities of

cobalamin (US pat 9089582B2 2015) The simple solution to the problem is to isolate

vitamin B12 from substances by encapsulation lyophilization and addition of iron salts

EDTA alkali metal chelating agents polyvalent alcohols and butanol but this process leads

11

to its degradation However all of the aforementioned methods were associated with an

enhanced stability of cobalamin in pharmaceutics products all of which contain a high

amount of cobalamin In US pat 6500472B2 (2002) Toshiaki amp Toshiaki reported that

lactoferrin provided adequate protection to cobalamin against light-induced decomposition

during food processing By contrast in this study the addition of beta-lactoglobulin or

alpha-lactalbumin offered a more concise and efficient method to avoid the loss of low

concentrations of cobalamin due to light and heat treatments during food processing

33 Effects of cobalaminwhey protein complexes on cobalamin stability during in vitro

stomach digestion

Due to the low pH ADCBL was reduced by approximately 25 after 120 min of

treatment in an in vitro stomach digestion system (IVSDS) (Fig 3) However CNCBL was

reduced by only 10 after 120 min treatment in IVSDS With supplementation of

alpha-lactalbumin or beta-lactoglobulin the stabilities of CNCBL and ADCBL were

significantly enhanced after 60 min treatment in IVSDS Both whey proteins ceased the

decrease in cobalamin during stomach digestion Even after 120 min of treatment in IVSDS

almost 95 of CNCBL and ADCBL were retained with the protection provided by

beta-lactoglobulin Meanwhile alpha-lactalbumin has also improved the stabilities of CNCBL

(75) and ADCBL (90) Whey proteins show a high capability to protect cobalamin

following the same trend as that observed in heat treatment

The bioavailability of vitamin B12 in humans and animals is low as a substantial

amount of vitamin B12 is destructed during stomach digestion (Artegoitia de Veth Harte

Ouellet amp Girard 2015) Vitamin B12 is a polyacidic base with a pKa of 33 which can be

easily ionized even under neutral conditions (Schneider amp Stroinski 1987) At a pH of 3-4

vitamins such as thiamin and niacinamide contribute to the destruction of vitamin B12 (Blitz

Eigen amp Gunsberg 1954) Eighty percent of cobalamin was decomposed in the rumen of

dairy cows and only 25 of cobalamin that escaped from rumen digestion was absorbed in

the small intestine (Artegoitia de Veth Harte Ouellet amp Girard 2015) Furthermore some

studies have reported that cobalamin from milk is more efficiently absorbed than a single

supplementation Researchers also reported that the stability of cobalamin at pH 3 was

significantly increased by 22 by the protection of lactoferri (US pat 6500472B2 2002) In

the in vitro stomach digestion model used in this study both beta-lactoglobulin and

alpha-lactalbumin enhanced the stability of cobalamin in the stomach environment

Particularly with the protection conferred by whey protein the stability of ADCBL was

significantly improved in contrast to that of single ADCBL The addition of

beta-lactoglobulin or alpha-lactalbumin enhanced the acid tolerance of cobalamin which

12

indicates that this method can certainly increase cobalamin bioactivity for food industrial

applications

34 Effects of cobalaminwhey protein complexes on microecology

Most microbiome studies on the human intestine have relied on data obtained from stool

samples (Clarke et al 2014) and some studies were unable to find significant influences of

prebiotics (Rajkumar et al 2014) on microecology This lack of influence is likely the case

because some influences of those substance may function in the upper portion of the intestinal

tract Vitamin B12 is normally absorbed at the end of small intestine Excess vitamin B12 that

has escaped absorption can mostly influence the microbiome of the proximal colon and we

herein simulated the microbial community using an in vitro intestinal digestion model to

investigate the effects of cobalamin

After quality-filtering 16S rDNA sequencing results produced 710640 reads giving a

mean sample depth of 19740 reads with a standard deviation of 7932 reads Alpha diversity

analysis demonstrated a significant contrast among various supplementation combinations of

cobalamin and whey protein (Fig 4A) Differences in the alpha-diversity indexes between

alpha-lactalbumin alone and ADCBL alone were not significant The alpha diversity indexes

of the other combinations were similar However a gradual increase in the alpha diversities of

beta-lactoglobulin alone and CNCBL alone compared to that of the combinations was

observed Supplementation of alpha-lactalbumin or ADCBL can be especially beneficial for

recovering from gastrointestinal problems unlike supplementation of beta-lactoglobulin and

CNCBL due to an increase in the alpha diversity of the gut microbiome

The bacterial community composition was different between single and combination

supplementation of cobalamin These differences were shown by PCA plots which explained

79 of the total variation in the three primary principal axes and showed clear grouping

based on single or combination supplementation (Fig 5A) The microbial ecology with

different supplementation was diverse at the phylum level However the most abundant

populations were Firmicutes Bacteroidetes and Verrucomicrobia which composed a

majority of the communities in all samples (Fig 6) The combination of CNCBL and whey

protein significantly improved the relative abundance of Verrucomicrobia by 34 After

adding cobalamin the ratios of BacteroidetesFirmicutes increased up to 2 fold indicating

that cobalamin can enhance the growth of Bacteroidetes meanwhile whey protein improved

the growth of Firmicutes (Fig 4B)

Among various supplementations significant differences in heatmaps were obvious at

the family level (Fig 5B) Twenty-four families of phylogenetic groups were compared

between all samples by the mean relative abundances to identify the bacterial composition

13

influenced by various cobalamin supplementations The hierarchical clustering (Fig 5B) of

microbiota profiles represents eight cobalamin supplementation diets The samples taken from

whey protein supplementation alone and the CNCBL-beta lactoglobulin combination were

distinguished from the others Differences in the stabilities of the microbiota profiles under

cobalamin and combination supplementation did not reach significance which was in contrast

to the variation in whey protein supplementation alone The CNCBL supplementation

stimulated microorganisms related to Christensenellaceae Prevotellaceae Cariobacteriacese

Clostridiaceae Chloroplast Lactobacillaceae Enterococcaceae and Lachnospiraceae (Fig

5B) while those in Bifidobacteriaceae were starkly reduced ADCBL strongly increased

Bifidobacteriaceae Beta-lactoglobulin stimulated Bacteroidaceae Eubacteriaceae

Rikenellaceae Prophyromonadaceae and Verrucomicrobiaceae Moreover

alpha-lactalbumin was able to stimulate Peptostreptococcaceae Erysipelotrichaceae

Christensenellaceae Prevotellaceae Cariobacteriacese and Clostridiaceae

Combined supplementation increased the proportions of Firmicutes and Bacteroidetes

and reduced the proportions of Proteobacteria which includes several genera of pathogens

such as EscherichiaShigella spp and Pseudomonas spp Some studies have reported that

Bacteroidetes spp can outcompete opportunistic pathogens such as Enterohemorrhagic

Escherichia coli by taking up the limited amount of vitamin B12 available (Cordonnier et al

2016) Some researchers demonstrated that ADCBL directly binds the riboswitch region

leading to conformational changes in the secondary structure of mRNA which masks the

ribosome-binding site (RBS) and thus inhibits expression (Nahvi et al 2002) Vitreschak

Rodionov Mironov amp Gelfand (2003) summarized almost 200 B12 elements from 66

bacterial genomes using a multiple alignment program Riboswitch is a 5rsquo-untranslated leader

sequence of a corresponding mRNA that regulates translation initiation and gene expression

by binding a special molecule (Winkler amp Breaker 2005) Expression of both the cobalamin

biosynthetic cob operon and the transporter btuB gene was repressed by the addition of

vitamin B12 particularly ADCBL Depending on which B12 transporter is present on the

bacterial membrane surface and B12-dependent riboswitches (Degnan Barry Mok Taga amp

Goodman 2014) vitamin B12 and complexes could mediate the microecological structure

especially promoting Firmicutes abundance An increase in the ratios of

ClostridiaEubateriaceae and RuminococceaeEubateriaceae was observed upon CNCBL

supplementation which was partially related to IBD These results also indicate that excess

CNCBL would likely lead to an inflammatory environment

The effect of a 10-day high-dose whey protein and cobalamin supplementation on the

microbiome in the proximal colon was demonstrated in our study Large taxonomic shifts

have also been observed between carnivores and vegans who normally have deficient vitamin

14

B12 intake (Allen amp Stabler 2008) Here we reported that the microbial turnover during

cobalamin supplementation was as dramatic as that of vitamin D intake (Bashir et al 2015)

Those changes included a reduction in potential pathogens and an increase in bacterial

diversity richness Thus far no systematic research on the effect of cobalamin on the

microbiome has been reported although many people exhibit excess vitamin B12 upon

supplementation Recently researchers have paid a substantial amount of attention on the

effect of cobalamin on the microbial community However most research has focused on the

inhibition and mediation capabilities of various structures of cobalamin (Mok amp Taga 2013)

Degnan Barry Mok Taga amp Goodman (2014) have researched multiple transporters in

human gut microbes to distinguish differences in their interactions with cobalamin Our

results demonstrate that supplementation of cobalamin and whey can enhance the proportions

of the phyla Firmicutes and Bacteroidetes and reduce that of the phylum Proteobacteria

4 Conclusion

Vitamin B12 is a light- and thermosensitive substance while alpha-lactalbumin and

beta-lactoglobulin can be useful protectors for cobalamin against physical destruction during

food processing With the protection provided by beta-lactoglobulin or alpha-lactalbumin the

thermal and photostabilities of ADCBL and CNCBL were remarkably increased by 20

Meanwhile the pH stability of the cobalamin-complex is of paramount importance for

bioavailability during stomach digestion Under the protection of whey proteins the stabilities

of ADCBL and CNCBL in gastric juice for 2 h were increased by 197 and 22

respectively compared with those of the others Following absorption of cobalamin in the

small intestine the residual cobalamin or complexes worked as a modular unit on the gut

microbiome Cobalamin supplementation in an in vitro colonic stimulation enhanced the

ratios of the phyla Bacteroidetes and Firmicutes and reduced the abundance of the phylum

Proteobacteria which led to a better and healthier colonic environment Despite the relatively

small sample size we observed a significant modulatory effect of vitamin B12 on the gut

microbiome Whey protein-ligand complexes with cobalamin significantly improved the

stability and bioavailability of cobalamins and mediated the gut microbiome thus influencing

human nutrition

Acknowledgements

This work was supported by National Natural Science Foundation of China (No

31501452 and 81302781) the Scientific Research Foundation of Education Department of

Zhejiang Province (No Y201432277) Foundation of Food Science and Engineering the

most important Discipline of Zhejiang Province (No JYTsp20141082) and Project of

15

international communication for construction of the first ranked discipline of Zhejiang

Gongshang Univerisity (No 2017SICR106)

Conflict of interest statement

The authors declare that they have no conflict of interest

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solution Journal of Pharmaceutical Analysis 10 9-15

Allen L H (2010) Bioavailability of vitamin B12 International Journal for Vitamin and

Nutrition Research 80 330-335 Allen R H amp Stabler S P (2008) Identification and quantitation of cobalamin and

cobalamin analogues in human feces American Journal of Clinical Nutrition 87(5)

1324-1335 Artegoitia V M de Veth M J Harte F Ouellet D R amp Girard C L (2015) Short

communication Casein hydrolysate and whey proteins as excipients for

cyanocobalamin to increase intestinal absorption in the lactating dairy cow Journal of Dairy Science 98(11) 8128-8132

Bashir M Prietl B Tauschmann M Mautner S I Kump P K Treiber G Pieber T R

(2015) Effects of high doses of vitamin D3 on mucosa-associated gut microbiome vary

between regions of the human gastrointestinal tract European Journal of Nutrition 55(4) 1479-148

Blitz M Eigen E amp Gunsberg E (1954) Vitamin B12 studies the instability of vitamin

B12 in the presence of thiamine and niacinamide Journal of the American Pharmaceutical Association 43(11) 651-653

Bridwell-Rabb J amp Drennan C L (2017) Vitamin B12 in the spotlight again Current

Opinion in Chemical Biology 37 63-70 Cawthern K M Narayan M Chaudhuri D Permyakov E a amp Berliner L J (1997)

Interactions of alpha-lactalbumin with fatty acids and spin label analogs The Journal of

Biological Chemistry 272(49) 30812-30816

Cinquin C Le Blay G Fliss I amp Lacroix C (2006) New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota FEMS Microbiology

Ecology 57(2) 324336

Clarke S F Murphy E F OrsquoSullivan O Lucey A J Humphreys M Hogan A Hayes P OReilly M Jeffery IB Wood-Martin R Kerins DM Quigley E Ross RP OToole

PW Molloy MG Falvey E Shanahan F Cotter PD (2014) Exercise and associated

dietary extremes impact on gut microbial diversity Gut 63(12) 1913-1920

Cordonnier C Le Bihan G Emond-Rheault J-G Garrivier A Harel J amp Jubelin G (2016) Vitamin B12 Uptake by the Gut Commensal Bacteria Bacteroides

thetaiotaomicron Limits the Production of Shiga Toxin by Enterohemorrhagic

Escherichia coli Toxins 8(1) 11-19 Dalsgaard T K Otzen D Nielsen J H amp Larsen L B (2007) Changes in structures of

milk proteins upon photo-oxidation Journal of Agricultural and Food Chemistry 55

10968-10973 de Wolf F a amp Brett G M (2000) Ligand-binding proteins their potential for application

in systems for controlled delivery and uptake of ligands Pharmacological Reviews

52(2) 207-236

Degnan P H Barry N a Mok K C Taga M E amp Goodman A L (2014) Human gut microbes use multiple transporters to distinguish vitamin B 12 analogs and compete in

the gut Cell Host and Microbe 15(1) 47-57

16

Demerre L J amp Wilson C (1956) Photolysis of Vitamin B12 Journal of the American

Pharmacists Association 45(3) 129-134 Gizis E Kim Y P Brunner J R amp Schweigert B S (1965) Vitamin B12 content and

binding capacity of cowrsquos milk proteins Journal of Nutrition 87(3) 349-352

Gu Q Zhang C Song D Li P amp Zhu X (2015) Enhancing vitamin B12 content in

soy-yogurt by Lactobacillus reuteri International Journal of Food Microbiology 206 56-59

Heep I amp Odenthal H (2015) US Pat 9089582B2

Hong Y H amp Creamer L K (2002) Changed protein structures of bovine β-lactoglobulin B and α-lactalbumin as a consequence of heat treatment International Dairy Journal

12(4) 345-359

Johns P W Das A Kuil E M Jacobs W A Schimpf K J amp Schmitz D J (2015) Cocoa polyphenols accelerate vitamin B12 degradation in heated chocolate milk

International Journal of Food Science and Technology 50 421-430

Liang L Zhang J Zhou P amp Subirade M (2013) Protective effect of ligand-binding

proteins against folic acid loss due to photodecomposition Food Chemistry 141(2) 754-761

Macfarlane G T Macfarlane S amp Gibson G R (1998) Validation of a three-stage

compound continuous culture system for investigating the effect of retention time on the ecology and metabolism of bacteria in the human colon Microbial Ecology 32(2)

180-187

Martens J H Barg H Warren M amp Jahn D (2002) Microbial production of vitamin B12 Applied Microbiology and Biotechnology 58(3) 275-285

Mok K C amp Taga M E (2013) Growth inhibition of Sporomusa ovata by incorporation of

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Mukherjee S L amp Sen S P (1957) THE STABILITY OF VITAMIN B12 Protection by Iron Salts Against Destruction by Aneurine and Nicotinamide Journal of Pharmacy and

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1043-1049

Padmanabhan S Jost M Drennan C L amp Eliacuteas-Arnanz M (2017) A New Facet of

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Rucker B R Suttie J W McCormick B D amp Machilin L J (2001) Handbook of vitamin New York Marcel Dekker Inc

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Schneider Z amp Stroinski A (1987) Comprehensive B12 chemistry biochemistry nutrition ecology medicine Berlin Verlag WAlter de Gruyter

Tokle T Lesmes U Decker E A amp McClements D J (2012) Impact of dietary fiber

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Toshiaki U amp Toshiaki S amp H (2002) US Pat 6500472B2

Vitreschak A G Rodionov D A Mironov A A amp Gelfand M S (2003) Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural

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Watanabe F Abe K Fujita T Goto M Hiemori M amp Nakano Y (1998) Effects of

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17

Winkler W C amp Breaker R R (2005) Regulation of Bacterial Gene Expression by

Riboswitches Annual Review of Microbiology 59 487-517

18

Figure legends

Fig 1 Stability of cobalamin after irradiation treatment in the presence or absence of

alpha-lactalbumin or beta-lactoglobulin

A) The triangle block diamond and circle markers represent CNCBL (025 μM) in the

presence of 075 05 025 μM alpha-lactalbumin (alpha-LA) and alone respectively B) The

triangle block diamond and round markers represent ADCBL (025 μM) in the presence of

075 05 025 μM alpha-LA and alone respectively C) The triangle block diamond and

round markers represent CNCBL (025 μM) in the presence of 075 05 025 μM

beta-lactoglobulin (beta-LG) and alone respectively D) The triangle block diamond and

round markers represent ADCBL (025 μM) in the presence of 075 05 025 μM beta-LG

and alone respectively

Fig 2 Stability of cobalamin after heat treatment in the presence or absence of


A) The black white dot pattern and gray bars represent CNCBL (025 μM) in the presence

of 075 05 025 μM alpha-LA and alone respectively B) The black white dot pattern and

gray bars represent ADCBL (025 μM) in the presence of 075 05 025 μM alpha-LA and

alone respectively C) The black white dot pattern and gray bars represent CNCBL (025

μM) in the presence of 075 05 025 μM beta-LG and alone respectively D) The black

white dot pattern and gray bars represent ADCBL (025 μM) in the presence of 075 05

025 μM beta-LG and alone respectively

Fig 3 Stability of cobalamin during stomach digestion in the presence of

alpha-lactalbumin (gray bar with a gray frame) beta-lactoglobulin (black bar) or alone

(gray bar)

A) ADCBL B) CNCBL

Fig 4 Effect of alpha-lactalbuminbeta-lactoglobulin and cobalamins on microbiota

from an in vitro intestinal simulator

A) Alpha diversity of bacterial populations from an in vitro intestinal simulator (n=3) a b c

d e f g h ndash each letter indicates that a significant difference (plt005) exists B) Comparison

of Bacteroidetes and Firmicutes from an in vitro intestinal simulator (n=3) a b c d ndash each

letter indicates that a significant difference (plt005) exists

19

Fig 5 Data analysis of microbiota from an in vitro intestinal simulator

A) Principal component analysis plots of microbiota from in vitro intestinal simulations by

complexes (blue) or single supplementation (red) B) Heatmap of changes in bacterial

abundance from in vitro intestinal simulations Each row represents family-like phylogenetic

groups of bacteria whose mean abundance differed significantly between complexes

Hierarchical clustering of the bacterial fingerprints and various supplementations collected

from samples of in vitro intestinal simulations

Fig 6 Stacked bar plots showing the average percentage of bacterial populations from

an in vitro intestinal simulator in the presence of cobalamin and complexes

20

21

22

23

24

25

26

Highlights

Whey protein enhanced thermalphoto stability of cobalamin during food processing and

storage

Whey protein improved the stability and bioavailability of cobalamin during digestion

Whey proteins reduced relative abundances of Proteobacteria induced by cyanocobalamin

1

Stability of Vitamin B12 with the Protection of Whey Proteins and Their Effects on the

Gut Microbiome

Running title Stability and Effect of Vitamin B12Whey Complexes

HuanhuanWangadagger huanvalhznueducn

Yikai Shouadagger 403278497qqcom

Xuan Zhub zhuxuanmailzjgsueducn

Yuanyuan Xub 362120957qqcom

Lihua Shib 624872970qqcom

Shasha Xiangb xsscherishqqcom

Xiao Fengb 249259608qqcom

Jianzhong Hanb jzhan99zjgsueducn

aSchool of Medicine Hangzhou Normal University Hangzhou 310018 China

bSchool of Food Science and Bioengineering Zhejiang Gongshang University Hangzhou

310018China

daggerThese authors contributed equally to this work

Corresponding author Dr Xuan Zhu Email zhuxuanmailzjgsueducn Phone

+8657128008902 School of Food Science and Bioengineering Zhejiang Gongshang

University Xuezheng Str 18 Hangzhou 310018 China

Abstract

Cobalamin degrades in the presence of light and heat which causes spectral changes and loss

of coenzyme activity In the presence of beta-lactoglobulin or alpha-lactalbumin the thermal-

and photostabilities of adenosylcobalamin (ADCBL) and cyanocobalamin (CNCBL) are

increased by 10-30 Similarly the stabilities of ADCBL and CNCBL are increased in the

presence of whey proteins by 197 and 22 respectively when tested in gastric juice for 2

h Due to the limited absorption of cobalamin during digestion excess cobalamin can enter

the colon and modulate the gut microbiome In a colonic model in vitro supplementation with

cobalamin and whey enhanced the proportions of Firmicutes and Bacteroidetes spp and

reduced those of Proteobacteria spp which includes pathogens such as Escherichia and

Shigella spp and Pseudomonas spp Thus while complex formation could improve the

stability and bioavailability of cobalamin these complexes might also mediate gut

microecology to influence human nutrition and health

2


3

1 Introduction





























and metabolites







4


































5



































6



































7




































8



28 Statistics































beta-lactoglobulin

9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



2


3

1 Introduction





























and metabolites







4


































5



































6



































7




































8



28 Statistics































beta-lactoglobulin

9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



3

1 Introduction





























and metabolites







4


































5



































6



































7




































8



28 Statistics































beta-lactoglobulin

9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



4


































5



































6



































7




































8



28 Statistics































beta-lactoglobulin

9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



5



































6



































7




































8



28 Statistics































beta-lactoglobulin

9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



6



































7




































8



28 Statistics































beta-lactoglobulin

9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



7




































8



28 Statistics































beta-lactoglobulin

9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



8



28 Statistics































beta-lactoglobulin

9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



9





























cobalamin







10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



10





































11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



11









stomach digestion



























12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



12


applications


































13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



13





































14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



14













4 Conclusion
















human nutrition

Acknowledgements





15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



15





References



















Ecology 57(2) 324336










52(2) 207-236



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



16








12(4) 345-359







180-187







1043-1049

















17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



17



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



18

Figure legends






















(gray bar)

A) ADCBL B) CNCBL







19










20

21

22

23

24

25

26

Highlights


storage



19










20

21

22

23

24

25

26

Highlights


storage



20

21

22

23

24

25

26

Highlights


storage



21

22

23

24

25

26

Highlights


storage



22

23

24

25

26

Highlights


storage



23

24

25

26

Highlights


storage



24

25

26

Highlights


storage



25

26

Highlights


storage



26

Highlights


storage



stability of vitamin b12 with the protection of whey

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