Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
24.1 Introduction
Biotin is an essential micronutrient for all living mammals because of its role as
a cofactor of carboxylation and decarboxylation enzymes. Human cells have
four biotin-dependent carboxylases that catalyse key reactions in gluconeo-
genesis, amino acid catabolism and fatty acid synthesis. Mammalian cells
cannot biosynthesize biotin, and thus it becomes an essential component of the
diet and is therefore classied as a vitamin. Biotin is widely distributed in the
human diet, either mostly free in the case of milk, vegetables and fruit, or partly
bound in animal tissues and plant seeds. Protein-bound biotin is generally
formed via covalent linkage to lysine residues, and gastrointestinal (GI) tract
proteases can release biotin in the form of biocytin (e-N-biotinyl-L-lysine).
The biotin content of cows milk is considered to be modest at approximately
2 mg/100 g. In the context of this discussion it is also relevant to note that biotin
is central to several metabolic pathways involved with mammalian milk
biosynthesis.
The structure of biotin has three asymmetric carbons and can therefore exist
as eight potential stereoisomers, but only the d-biotin form is both biologically
377
378 Chapter 24
Table 24.1 Daily intake of biotin to meet nutritional needs.a
Life stage and gender Amount (mg)
Babies: birth to 6 months 5
Infants: 712 months 6
Children: 13 years 8
Children: 48 years 12
Children: 913 years 20
Teens: 1418 years 25
Adults 30
Pregnant women 30
Breast-feeding women 35
a
Estimated recommended human daily intake required as a function of age
(source: Institute of Medicine).
active and found in nature, and is the only form of the parent vitamin that
requires consideration. Biotin rarely exists in the free form in animal tissues, the
majority being bound as biocytin within apoenzyme protein structures via an
amide link between its terminal carboxyl group and protein lysine residues.
Clinical deciencies of biotin in humans are not common in view of the low
dietary intake levels required (see Table 24.1), but have been reported when
prolonged and excessive amounts of raw egg white are consumed. However, in
infants, biotin deciency can occur as a consequence of insucient production of
biotinidase, the enzyme required to release bioavailable biotin from biocytin.
Rare genetic deciencies of biotinidase are also known. Obvious cutaneous
symptoms include hair loss and skin rashes, but many neurological problems can
develop. Deciency during pregnancy is considered to be tetragenic in the new-
born. Biotin-producing microorganisms within the intestine can contribute to the
human biotin pool and complicate the evaluation of nutritional need.
As with all nutritional studies, reliable analytical methods are a pre-requisite
for population-based policies related to Recommended Daily Allowances
(RDA), total diet surveys and label claims, as well as supporting clinical and
food compliance testing of biotin and its biomarkers. In general, for an esti-
mation of total biotin in food, a proteolytic step is required to release the
bioactive biocytin, which may then be included with free biotin in the analysis.
The specic extraction procedures employed for the release of matrix-bound
biotin remain a major analytical challenge and in fact may inuence results
even more than the end-point analytical measurement technique. Several
extraction strategies developed for the analysis of bound biotin have, depending
on sample type, most commonly employed high temperature mineral acid
digestion and/or ambient temperature enzymatic hydrolysis techniques.
The rst biotin tests for foods relied on microbiological principles, although
some non-specic colorimetric tests were developed for biotin in pharma-
ceutical preparations. This chapter summarizes the important analytical
techniques that have been employed, or are in use today, with emphasis
on dairy products. The analytical techniques reported for the determination of
biotin content can be categorized under (i) microbiological, (ii) biological,
Biotin Analysis in Dairy Products 379
Table 24.2 Overview of the attributes and limitations of the principal analy-
tical strategies available for the analysis of biotin in foods.
Principle Key steps Advantages Limitations
Microbiological Extraction of biotin Low equipment Test microorganism
assay (MBA). and biocytin from costs. Versatile, vulnerable to
Growth of sample. Incubate sensitive, reference growth inhibition
biotin-dependent with microorgan- Infant Formula or stimulation
microorganism ism and measure Council method. from matrix com-
proportional to turbidity. ponents. Highly
biotin in sample manipulative,
extract. time-consuming.
High performance Extraction of High specicity for High equipment
liquid chromato- biotin forms, biotin. Proven costs
graphy (HPLC). purication, and commonly
Chromatographic chromatographic available
separation of bio- separation, technology.
tin species with post-column
quantitation based derivatisation -
on detector uorescence, or
response. MS detection.
Ligand-binding Extraction of biotin Rapid, specic High equipment
assays, relying on species. Direct or inexpensive equip- costs if non-
biospecic mole- indirect detection ment costs if labelled platform
cular recognition by biotin-specic labelled technique. utilised.
of biotin. Labelled antibody or bind- Proprietary
or non-labelled ing protein in kit-based assays
platforms either a labelled or available.
available. non- labelled het-
erogenous format.
800
3.181
600
mAU
400
200
0
0 1 2 3 4 5
min
B
800
3.241
600
mAU
400
200
0
0 1 2 3 4 5
min
Figure 24.1 HPLC chromatogram of biotin standard with post-column avidin reac-
tion. Note the fast chromatography permitted with this sensitive and
specic reaction. Detection is using uorescence. A: biotin standard,
0.04 ug/mL. B: supplemented milk powder extract with 12 ug/100 g.
12000
R2 = 0.9999
8000
6000
4000
2000
0
0.00 0.02 0.04 0.06 0.08 0.10
mg/L Biotin
Figure 24.2 Non-linear calibration curve for biotin by post-column avidin reaction.
The curve ts very well to a quadratic equation. It is important to have
many data points to describe the curvature unless a narrow concentra-
tion range is selected and sample extracts diluted into this range.
ionization eciency. These issues can be resolved using isotope dilution tech-
niques when deuterated internal standards become available. Nevertheless, the
use of positive ESI and [2H2]-biotin has successfully allowed biotin levels to be
characterized in NIST 3280 multivitamin tablets (Nelson et al. 2006). Although
for food samples, sample preparation restrictions will limit LC-MS ability to
quantitatively analyse all total B vitamins in a single run, gains are currently
being made to detect free (non-bound) vitamins in supplemented infant formula.
4000
4
3000
3
2000
5
Response (RU)
a
b
1000 c
d
e
1 7
0
2
1000
6
2000
3000
100 0 100 200 300
Time (s)
In view of the low molecular mass of the vitamins, biosensor assays employ
the principle of competitive inhibition. Prior to the analysis for biotin, a biotin
derivative is covalently tethered to the sensor surface. Sample extract or biotin
standard is mixed under equilibrium conditions with an excess of a biotin-
specic antibody, and unbound antibody binds to the surface generating an
SPR binding response in real-time, as illustrated in Figure 24.3.
The relative binding response is interpolated from a calibration curve in
order to compute concentration. As an inhibition assay, the response is
inversely related to biotin concentration and exhibits a sigmoidal dose
response relationship that is typical of most ligand-binding assays. With respect
to specicity, the routine compliance assay is targeted to the quantitation of
free biotin only in nutritional dairy products, and therefore does not include
biocytin (Indyk et al. 2000). However, in milk and supplemented infant for-
mulas, the overwhelming majority of biotin is present in the free form.
It remains speculative as to the potential impact on biotin analysis that may
result from current innovations in the eld of biosensor technologies. New
alternatives to classical biorecognition elements will include articial receptors
such as molecularly imprinted polymers and aptamers, which promise
increased stability. Although they are currently not widely utilized in com-
mercial biosensors, such biosensor innovations are likely to oer future
advantages.
Biotin Analysis in Dairy Products 389
24.6 Biotin Forms and Concentration in Milk
and Dairy Products
A large number of clinical studies, spearheaded by Donald Mock and his
associates (Mock et al. 1992, 1997; Staggs et al. 2004), have been conducted
concerning the distribution of biotin in tissues and biological uids. These have
in turn attempted to assess the extent to which human nutritional requirements
vary with age and gender. Biotin is required in such low quantity that average
diets, whether of animal or vegetable origin, supply sucient vitamin to prevent
widespread deciency states, making it problematical to determine recom-
mended dietary intake (RDI) in humans without deliberate dietary interven-
tion. It is also suggested that microbiological fauna in the intestinal tract
contribute to the host biotin pool. Other measures, such as comparison of
consumed versus excreted biotin, homeostasis of circulatory biotin and use of
radiolabelled (tritiated) biotin have all helped to determine the current RDI for
adults at 2030 mg/day, increasing during pregnancy and lactation to 3035 mg/
day, while other estimates are 1.5 mg/kg body weight across all age groups.
Human milk is the primary agent for infant nutriture and thereby guides the
composition of manufactured infant formula and milk substitutes. The
reported concentration of biotin in human milk is variable with lactation (and
unfortunately between analytical methods), but is more than sucient to
supply the newborn infant with the RDI of 56 mg/day, as evidenced by the
absence of reported deciency syndromes in breast-fed babies. Interestingly,
most biotin in milk is present in a free form and therefore unbound with any
macromolecules. As expected, when milk is separated into its fat and aqueous
fractions, the water-soluble biotin is found predominantly in the skim-milk
phase. Biotin has some lipophilicity and so a small percentage is carried into the
cream as part of the fat-globule membrane. The total concentration of human
milk is not large and somewhat similar to bovine milk. With respect to breast
milk substitutes, it is necessary to ensure the biotin status remains comparable,
thus international guidelines recommend 0.42.4 mg/100 kJ of reconstituted or
ready-to-feed infant formula.
Table 24.3 shows biotin levels in selected consumer dairy products and
compares the milk of the cow with that of the goat and human. When com-
mercial milk is converted by spray-drying into dehydrated powders, the biotin
contents increase proportionately. Skim-milk powder thus becomes a reason-
able source of the vitamin at 1315 mg/100 g. Puried casein contains essentially
Table 24.3 Typical biotin content of species milks and cows milk consumer
products (mg/100 g).a
Cow
Goat Human
Milk Yoghurt Cheese Butter Ice-cream Milk Milk
2.04.3 0.94.0 0.85.9 0.5 1.1 3.23.9 0.40.8
a
Data from cited references and Food Standards Australia New Zealand (2010).
390
60
20089 Dairy Season 200910 Dairy Season 201011 Dairy Season
50
40
30
Biotin (ug/100 g)
20
10
0
08 08 08 08 08 08 09 09 09 09 09 09 09 09 09 09 09 09 10 10 10 10 10 10 10 10 10 10 10 11 11 11 11 11 11 11 11 11
0 7. 08. 09. 10. 11. 12. 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. 01. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. 01. 02. 03. 04. 05. 06. 07. 08. 09.
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
21 21 21 22 22 23 23 23 26 26 27 27 28 28 28 29 29 30 30 2 2 3 3 4 4 4 5 5 6 6 6 9 9 10 10 11 11 11
Figure 24.4 Seasonal trend in biotin levels in skim-milk powder (mg/100 g) over three consecutive production seasons in New Zealand
(20082011) resulting from an extensive pasture grazing dairy practice. Biotin concentration was estimated using a biosensor-
based immunoassay.
Chapter 24
Biotin Analysis in Dairy Products 391
no biotin, as it is removed during the precipitation and washing processing
stages. Although the whey fraction of processed milk contains the majority of
the endogenous milk biotin, puried whey proteins lose most of their associated
biotin during ultraltration, since in milk, biotin is not covalently bound to this
fraction.
Countries with high biotin intake, as shown in total diet surveys, generally
consume more meat, eggs and sh, which represent the predominant sources of
the vitamin (Murakami et al. 2008). However, cheese contains measurable
biotin across a range of concentrations (approximately 16 mg/100 g) and can
contribute signicant dietary biotin in some European societies where cheese
consumption is high. On a solids basis, cheese has less biotin than its raw
material (milk), so speculation that starter microorganisms can contribute
additional biotin during production is probably false and, in fact, may consume
biotin. However, hard cheeses such as Cheddar and Gruyere contain lesser
amounts of biotin than soft cheeses (e.g. Brie). Similarly, yoghurt and other
cultured dairy products have diminished biotin levels as compared to the parent
milk on a solids-non-fat basis.
Recent work in New Zealand has shown a seasonality of biotin levels in
bovine milk, as illustrated in Figure 24.4. Extensive pasture grazing and animal
husbandry practices based on herd calving scheduled to maximize lactating
cows at peak grass growth result in a minor but consistent seasonal trend.
Summary Points
This chapter focuses on the analysis of biotin in dairy products.
Biotin is a member of the B group of water-soluble vitamins.
As a vitamin, biotin is an essential component of the human diet.
Levels of biotin in dairy products formulated for infant nutrition are
rigorously regulated.
Reliable analytical methods are essential to support the formulation and
compliance of manufactured dairy products.
The attributes and limitations of dierent analytical platforms for the
measurement of biotin are reviewed.
Key Facts
Biotin is a vitamin required in very small (microgram) quantities as a
cofactor to essential enzyme reactions.
Biotin is hard to detect in dairy and food samples, dierent methods often
giving dierent answers.
Detection using microbiological organisms has traditionally been used,
but is currently being replaced by modern chromatographic and immu-
nochemical techniques.
Biotin levels in dairy products are only moderate, and insucient to
supply human daily needs. However, since it is ubiquitous in many foods,
deciency states are uncommon.
Infants fed on dairy-based formulae need additional biotin added to their food.
392 Chapter 24
Denitions of Words and Terms
Affinity chromatography. A separation technique that exploits the high biological
specicity and anity of specic naturally occurring proteins for the analyte.
Apoenzyme. An enzyme devoid of its cofactor.
Avidin, streptavidin. Avidin is a protein expressed in the white of the reptilian,
amphibian and avian egg with very high binding anity for biotin. Strep-
tavidin is a structurally related protein expressed in certain bacteria that
exhibits comparable anity for biotin.
Biosensor. An analytical device for the detection of an analyte that combines a
biospecic detection element with a physicochemical detector that may be
based on a number of principles.
Chromatography. A group of analytical techniques whereby complex mixtures
of compounds are both separated into individual forms and then sequentially
detected by a range of techniques. The techniques described in this chapter
are predominantly based on a high-pressure liquid chromatographic (HPLC)
platform.
Cofactor. A non-protein compound that is essential for facilitating the biolo-
gical activity of an enzyme.
Enzyme. A protein that catalyses the conversion of one biologically active
compound to another.
Ligand-binding. The biospecic binding interaction between an analyte and a
biological macromolecule, usually a protein. Such an interaction is non-
covalent, usually reversible and either crucial for biological activity, or can be
exploited for analytical purposes.
Mass spectrometry. An analytical detection technique that measures the mass-
to-charge ratio of charged particles, and provides conrmatory identication
of target analytes separated by chromatography.
Stereoisomers. Compounds that have the same molecular formula and
sequence of bonded atoms, but dier in the three-dimensional orientations of
those atoms.
Vitamin. An organic compound that the body cannot biosynthesize yet is
essential for normal growth and development and therefore must be derived
from the diet.
List of Abbreviations
ADAM 9-anthryldiazomethane
EPBA enzyme protein binding assay
ESI electrospray ionisation interface
FID ame ionization detection
FITC uorescein isothiocyanate
GC gas chromatography
GC-MS gas chromatographymass spectrometry
GI gastrointestinal
HABA p-hydroxy- azobenzene-2 0 -carboxylic acid
Biotin Analysis in Dairy Products 393
HPLC high-performance liquid chromatography
IC ion chromatography
MBA microbiological assay
QC quality control
RDA Recommended Daily Allowance
RDI recommended dietary intake
SPE solid-phase extraction
SPR surface plasmon resonance
TLC thin-layer chromatography
TMB 3,3 0 ,5,5 0 -tetramethyl benzidine
UV ultraviolet
References
Angyal, G. (ed.), 1996. Biotin. In: US Food and Drug Administration. Methods
for the Microbiological Analysis of Selected Nutrients. Association of Ocial
Analytical Chemists, Arlington, VA, USA, pp. 910.
Bell, J.S., 1974. Microbiological assay of vitamins of the B-group in foodstus.
Laboratory Practice. 23: 235241.
Bitsch, R., Salz, I., and Hotzel, D., 1989. Biotin assessment in foods and body
uids by a protein binding assay (PBA). International Journal of Vitamins and
Nutritional Research. 59: 5964.
Blake, C.J., 2007. Analytical procedures for water-soluble vitamins in foods
and dietary supplements: a review. Analytical and Bioanalytical Chemistry.
389: 6376.
Campos-Gimenez, E.C., Trisconi, M-J., Kilinc, T., and Andrieux, P., 2010.
Optimization and validation of an LC-FLD method for biotin in infant
formula, infant cereals, cocoa-malt beverages and clinical nutrition products.
Journal of AOAC International. 93: 14941502.
Eitenmiller R.R., Landen Jr., W.O., and Ye, L., 2007. Biotin. In: Vitamin
Analysis for the Health and Food Sciences, 2nd ed. CRC Press, Boca Raton,
FL, USA, pp. 535560.
European Committee for Standardization, 2009. EN 15607: Foodstus.
Determination of d-biotin by HPLC. CEN, Brussels, Belgium. 14 pages.
Finglas, P.M., Faulks, R.M., and Morgan, M.R.A., 1986. The analysis of
biotin in liver using a protein-binding assay. Journal of Micronutrient Ana-
lysis. 2: 247257.
Finglas, P.M., and Morgan, M.R.A., 1994. Application of biospecic methods
to the determination of B-group vitamins in food: a review. Food Chemistry.
49: 191201.
Frappier, F., 1993. Biotin: properties and determination. In: Macrae, R.,
Robinson, R.K., and Sadler M.J. (ed.) Encyclopedia of Food Science,
Food Technology and Nutrition., Academic Press, London, UK, Vol. 1, pp.
395399.
394 Chapter 24
Hayakawa, K., Katsumata, N., Abe, K., Hirano, M., Yoshikawa, K., Ogata,
T., Horikawa, R., and Nagamine, T., 2009. Wide range of biotin (vitamin H)
content in foodstus and powdered milks as assessed by high-performance
anity chromatography. Clinical Pediatric Endocrinology. 18: 4149.
Holler, U., Wachter, F., Wehrli, C., and Fizet, C., 2006. Quantication of
biotin in feed, food, tablets and premixes using HPLC-MS/MS. Journal of
Chromatography B. 831: 816.
Hoppner, K., and Lampi, B., 1992. Biotin content of cheese products. Food
Research International. 25: 4143.
Indyk, H.E., Evans, E.A., Bostrom-Caselunghe, M.C., Persson, B.S., Finglas,
P.M., and Woollard, D.C., 2000. Determination of biotin and folate in infant
formula and milk by optical biosensor-based immunoassay. Journal of
AOAC International. 83: 11411148.
Kalman, A., Caelen, I., and Svorc, J. 2006. Vitamin and psuedovitamin ana-
lysis with biosensors in food products: a review. Journal of AOAC Interna-
tional. 89: 819825.
Lahely, S., Ndaw, S., Arella, F., and Hasselmann, C., 1999. Determination of
biotin in foods by high-performance liquid chromatography with post-col-
umn derivatization and uorimetric detection. Food Chemistry. 65: 253258.
Livaniou, E., Costopoulou, D., Vassiliadou, I., Leondiadis, L., Nyalala, J.O.,
Ithakissios, D. S., and Evangelatos, G.P., 2000. Analytical techniques for
determining biotin. Journal of Chromatography A 881: 331343.
Mock, D.M., Mock, N.I., and Langbehn, S.E., 1992. Biotin in human milk:
methods, location and chemical form. Journal of Nutrition. 122: 535545.
Mock, D.M., Mock, N.I., and Stratton, S., 1997. Concentrations of biotin
metabolites in human milk. Journal of Pediatrics. 131: 456458.
Murakami, T., Yamano, T., Nakama, A., and Mori, Y., 2008. Estimation of
dietary intake of biotin and its measurement uncertainty using total diet
samples in Osaka, Japan. Journal of AOAC International. 91: 14021408.
Nelson, B.C., Sharpless, K.E., and Sander, L.C,. 2006. Improved liquid chro-
matography methods for the separation and quantitation of biotin in NIST
standard reference material 3280: multivitamin/multielement tablets. Journal
of Agricultural and Food Chemistry. 54: 87108716.
Ploux, O., 2000. Chapter 11 Biotin. In: De Leenheer, A.P., Lambert, W.E.,
and Van Bocxlaer, J.F. (ed.) Modern Chromatographic Analysis of Vitamins,
3rd ed. Marcel Dekker, New York, USA, pp. 479509.
Reyes, F.D., Romero, J.M.F., and de Castro, M.D.L., 2001. Determination of
biotin in foodstus and pharmaceutical preparations using a biosensing
system based on the streptavidin-biotin interaction. Analytica Chimica Acta.
436: 109117.
Ridascreens. Enzyme binding assay for the quantitative analysis of biotin. Cat.
No. R2201, R-Biopharm GmbH, Darmstadt, Germany.
Staggs, C.G., Sealey, W.M., McCabe, B.J., Teague, A.M., and Mock, D.M.,
2004. Determination of the biotin content of select foods using accurate and
sensitive HPLC/avidin binding. Journal of Food Composition and Analysis.
17: 767776.
Biotin Analysis in Dairy Products 395
Thompson, L.B., Schmitz, D.J., and Pan, S-J., 2006. Determination of biotin
by high-performance liquid chromatography in infant formula, medical
nutritional products and vitamin premixes. Journal of AOAC International.
89: 15151518.
Walash, M.I., Rizk, M., Sheribah, Z.A., and Salim, M.M., 2008. Kinetic
spectrophotometric determination of biotin in pharmaceutical preparations.
International Journal of Biomedical Science. 4: 238244.
VitaFasts. Quantitative determination of biotin by microbiological assay. Cat.
No. P1003. R-Biopharm GmbH, Darmstadt, Germany.