Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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importance in the production of fermented beverages.

Commercial purified pectinolytic enzymes have been

applied to improve clarification processes and aroma

and color compound extraction, in beverage indus-

tries. The beverage industry has used genetic engin-

eering techniques mainly for the selection of yeasts.

Biotechnology in Cereal Industries

0057 The most important field has been fermentation. Isol-

ation and characterization of both desirable and

undesirable microorganisms (pathogens) have been

carried out. The role of cereal as a substrate to obtain

antimutagenic or anticarcinogenic fungic compounds

has been evaluated.

0058 The cereal breeding industry is also investigating

the application of genetic techniques to improve

major grain parameters (e.g., resistance to herbicides,

viruses, fungal disease, temperature variations).

Biotechnology in Dairy Industries

0059 A great deal of research has been carried out on

selection, industrial production conditions, immobil-

izing processes, and applications of different dairy

starters. The most studied microorganisms group

has been the lactic acid bacteria (LAB), which have

been applied to improve and increase industrial

productions of traditional food, without any loss of

popular acceptance; to increase the nutritional and

health value of dairy foods; to increase selected

flavor generation; and to improve the texture of

dairy products.

0060 Potential health benefits of bifidobacteria have

been studied, and their application in yogurt and

cheese production has been described. Some LAB

have been genetically modified to utilize the lactose,

and have been applied to obtain new yogurts with a

sweet alanine flavor, longer shelf-life, or improved

clinical probiotic value.

0061 Other important studies have been concerned with

the isolation of microbial enzymes, developments in

GM enzymes, and their application in dairy indus-

tries. Clotting enzymes (chymosin), proteases, and

lipases are the most studied and applied. Immobilized

or free lipases have been used in many different

aspects, such as for the production of food additives

(e.g., antioxidants by esterification of phenols) and

desirable flavors (e.g., in cheese, buttermilk), for syn-

thesis of flavor thioesters, or as emulsifiers or food

spoilage agents. Immobilized esterases have been

employed in butter oil to obtain dairy flavor products.

Thermally stable microbial b-glycosidase has been

used to avoid problems of human lactose intolerance.

0062 Some microorganisms are selected to improve

the safety of dairy products, which are able to

biosynthesize biopreservative compounds (e.g., reute-

nin). Different biotechnological practices (selected

microorganisms or their metabolites) can be applied

to control and predict bacteriophage contamination

in dairy products, and bacteriocins isolated from dif-

ferent cultures have been applied to render inactive

milk pathogens.

0063Many papers on probiotic dairy food have been

published in relation to mechanisms, underlying

their effects on human health, and some microorgan-

isms have been applied to reduce the level of choles-

terol and increase the availability of provitamin D

0064Recent works have studied the use of genetic engin-

eering to modify the composition of milk indirectly by

using transgenic animals. These studies have been

carried out to obtain low-lactose milk or to improve

the quality of milk (increasing the fat level) to make

special cheeses such as mozzarella.

0065Different papers have shown biotechnological ap-

plications on dairy effluents such as cheese whey.

Microbial fermentation of whey for the production

of biosurfactant sophorolipids or some enzymes with

important industrial and environmental applications

has been described.

Biotechnology in the Meat-product Industries

0066Fermentation processes in order to increase microbial

activities and the production of bacteriocins have

been extensively studied with satisfactory results,

and new starter cultures (LAB) have been developed.

0067In addition, different kinds of proteinases alone or

accompanied by lipases and esterases have been stud-

ied and applied usefully for the tenderization and

ripening of meats and their products. Proteinases

have been also used to hydrolyze peptides isolated

from different animal meats to obtain new flavors.

0068In meat production, amylases, cellulases, and pec-

tinases have been mixed and applied to obtain new

animal feed, which increases the body weight and the

percentage of protein and ash of the meat.

0069The main genetic technique used is the polymerase

chain reaction, which uses a DNA-based identifica-

tion system for testing the species origin of meat

samples and is suitable for authenticating processed

meat products.

Biotechnology in the Fish-product Industries

0070The development of new fermented products (fish

and sauces) with special attention on desirable sens-

ory properties has been one of the most important

fields of study in these industries.

0071The second field has been the production of o-3

polyunsaturated fatty acids, using immobilized lipase,

or looking for marine organisms (bacteria, microalga,

BIOTECHNOLOGY IN FOOD PRODUCTION 505

fungi, and zooplankton) that can biosynthesize these

acids.

0072 Other applications have been the use of different

enzymes for the tenderization and ripening of fish and

to obtain new flavors. Fish enzymes and proteins have

been isolated and applied in the food industry. LAB

and their bacteriocins isolated from fish intestines

have been genetically characterized, and their poten-

tial use in food preservation evaluated.

See also: European Union: European Food Law

Harmonization; Food and Drug Administration; Food

and Agriculture Organization of the United Nations;

Genetically Modified Foods; Legislation: International

Standards; Codex; World Health Organization

Further Reading

Altman A (1998) Agricultural Biotechnology. New York:

Marcel Dekker.

European Federation of Biotechnology (EFB) Task Group

on Public Perceptions of Biotechnology (1997). Biotech-

nology in Foods and Drinks Inform. London, UK: EFB

IFT (2000) Genetically modified organisms. Food Technol-

ogy 54: 42–45.

Kaufman PB, Cseke LJ, Warber S, Duke JA and Brielmann

HL (1999) Natural Products from Plants. Boca Raton,

FL: CRC Press.

Lappe M and Bailey B (1999) Against the Grain. The

Genetic Transformation of Global Agriculture. London:

Earthscan Publications.

Lee BH (1996) Fundamentals of Food Biotechnology. New

York: VCH Publishers.

Menrad K, Agrafiotis D, Enzing CM, Lemkow L and Ter-

ragni F (1999) Future Impacts of Biotechnology on

Agriculture, Food Production and Food Processing.

London: Springer.

Nelson GC (2001) Genetically Modified Organisms in

Agriculture, Economics and Politics. London: Academic

Press.

Pandey A, Benjamin S, Soccol CR et al. (1999) The realm of

microbial lipases in biotechnology. Biotechnology and

Applied Biochemistry 29: 119–131.

Ratledge C and Kristiansen B (2001) Basic Biotechnology.

Part I and II. New York: Cambridge University Press.

Roller S and Harlander S (1998) Genetic Modification in

the Food Industry: A Strategy for Food Quality Im-

provement. London: Blackie Academic & Professional.

Ryan-Harshman M (1998) Food biotechnology: food

industry, nutrition and public health. Proceedings of

the Nutrition Society 56: 845–854.

Shahidi F, Jones Y and Kitts DD (1997) Seafood Safety,

Processing and Biotechnology. Basle: Technomic Pub-

lishing.

Thompson PB (1997) Food Biotechnology in Ethical Per-

spective. London: Blackie Academic & Professional.

Tzotzos GT and Skryabin KG (2000) Biotechnology in the

Developing World and Countries in Economic Transi-

tion. Wallingford, UK: CABI Publishing.

BIOTIN

Contents

Properties and Determination

Physiology

Properties and Determination

R Bitsch, Friedrich-Schiller-University, Jena, Germany

Biotin

0001 The molecular structure of biotin is characterized by

three chiralic C atoms leading theoretically to eight

stereoisomers. Only the d(þ)-isomer is biologically

active. The most commonly used microbiological

assays (bioassays) for quantitation are more recently

replaced by protein binding assays, with avidin

showing higher precision and enhanced detection

limits. Besides these methods, biotin can also be ana-

lyzed mainly in pharmaceuticals using photometric

and fluorometric procedures or chromatographic

techniques (TLC and HPLC).

Properties

0002The molecular structure of biotin (CAS No. 58–85–5)

is a bicyclic ring system comprising an imidazolidone

ring fused with a tetrahydrothiophene ring that is

substituted with a valeric acid side chain. The stereo-

chemistry reveals that both ring systems are fused in a

506 BIOTIN/Properties and Determination

boat-like configuration at an angle of 62



(Figure 1).

The chemical nomenclature of this compound is like a

cis-hexahydro-2-oxo-1H-thieno[3,4-d] imidazole-4-

pentanoic acid as was elucidated by Ko

gl 1937 and

Du Vigneaud in 1941. The molecule contains three

chiralic centers (*), and so eight stereoisomers are

possible. Only the d-(þ) isomer with a cis-configur-

ation at the chiralic centers possesses vitamin activity

and is found in natural products. The empiric formula

is C

S (molecular weight 244.3 Da). The

molecular structure was confirmed in 1943 by Harris

et al., who were the first to achieve total biotin syn-

thesis. In the meantime, a stereospecific synthesis for

commercial production was developed by Goldberg

and Sternbach in 1949.

Physicochemical Characteristics

0003 d-Biotin as a free acid with a pK

value of 4.51

crystallizes in colorless needles with a melting point

of 230–232



C (decomposition). The substance is

slightly soluble in water (0.82 mM ¼0.2 g l

1



C) and ethanol 95% (3.27 mM ¼0.8 g l

1

readily soluble in dilute alkali, and almost insoluble

in most organic solvents. The specific optical rotation

[a]

in 0.1 N NaOH is þ 92



C(c ¼0.3) (Table 1).

0004Crystalline d-biotin is fairly stable on exposure to

air, daylight, and heat. Gradual destruction is ob-

served on exposure to ultraviolet radiation. In weakly

acid or alkaline solutions between pH 4.0 and 9.0, it

is relatively stable, too. The molecule is also resistant

to autoclaving in 2 N or 6 N sulfuric acid for 2 h (the

commonly used extraction procedure).

0005Biotin is, however, susceptible to oxidation at the

sulfur atom of the thiophene ring with the sequential

formation of biotin sulfoxide and biotin sulfone. This

oxidation, which is negligible in concentrated solu-

tions, provides a primary step for vitamin activity

losses in processed foods producing a mixture of d-

and l-sulfoxides. While d-biotin sulfoxide exhibits a

similar growth promoting activity for some micro-

organisms to that of biotin (e.g. Lactobacillus and

Saccharomyces sp.), subsequent oxidation to biotin

sulfone leads to complete loss of the biological activ-

ity. Biotin sulfoxide also seems to be a product of the

biotin metabolism and can be detected as a minor

excretion component in urine. In addition, microbial

growth can drastically lower the concentration of

nonsterile dilute solutions of this vitamin.

0006Besides the sulfoxide and sulfone, further metabol-

ites and analogs have been identified as intermediate

products of the biosynthesis (e.g., dethiobiotin) or

of the metabolism (e.g., bis- and tetranorbiotin) in

bacteria, yeasts, and fungi or as byproducts of the

chemical synthesis (e.g., oxybiotin) (Figure 2). The

growth response of microorganisms to these analogs

is variable and thus may lead to conflicting results in

terms of vitamin activity in biological samples when

measured by means of microbial assays (see Table 2).

Bisnorbiotin, tetranorbiotin, and biotin sulfone, as

well as other derivatives, are potent antagonists of

biotin and do not support microbial growth. Apart

from the sulfoxide and sulfone, these analogs are of

little practical importance in foods. Nevertheless,

more recent results indicate that in human urine

samples, besides unchanged biotin, about 11%

d-biotin-sulfoxide, 29% bisnorbiotin, and 4% biotin

sulfone plus bisnorbiotinmethyl-ketone and tetra-

norbiotin-l-sulfoxide could be identified, but no

62˚

fig0001 Figure 1 D(þ)-Biotin. *, chiralic centers.

tbl0001 Table 1 Physicochemical properties of biotin

Brutto formula Molecular weight Melting point Specific opticalrotation

S 244.3 Da 230–232



C (decomposition) [a]

¼þ92



(0.1 N NaOH, c ¼ 3.0)

Solubility

Water Ethanol (95%) Lipophilic solvent Dilute alkali

0.82 mM ¼ 0.2 g l

1

(25



C) 3.27 mM ¼ 0.8 g l

1

Insoluble Readily soluble

BIOTIN/Properties and Determination 507

degradation products of the cyclic urea ring were

found to be excreted.

0007 In natural products, biotin exists either in free form

or protein-bound. Generally, the free form predomin-

ates in plant food, whereas a greater part in animal

food is protein-bound. Therefore, less drastic hy-

drolysis with 2 N sulfuric acid is required to liberate

biotin from plant material than from products

of animal origin, which need 6N sulfuric acid.

The biotin content in selected foods is shown in

Table 3.

0008If protein-bound, the vitamin is linked through an

amide bond between the carboxyl group of the valeric

acid side chain and an e-amino group of terminating

lysine. This e-N-biotinyl-l-lysine (biocytin) is natur-

ally occurring, too, and is partly formed by proteo-

lysis of biotin enzymes. Occasionally, biocytin is

indicated as the real prosthetic group of biotin-

dependent enzymes.

0009The most prominent antagonist of biotin is avidin.

This is a glycoprotein from egg white with a MW of

68 000, which is formed within the oviduct of avian

(CH

)

(CH

)

Oxybiotin Biotinsulfoxide

Dethiobiotin

Biocytin (ε-N-Biotinyl-

L-lysine)

Biotinsulfone

(CH

)

(CH

)

(CH

)

(CH

)

enzyme

n = 2, Bisnorbiotin

n = 0, Tetranorbiotin

fig0002 Figure 2 Biotin derivatives and analogs.

508 BIOTIN/Properties and Determination

species and reptiles. It has a tetrameric structure with

four identical subunits (MW 17 000), each containing

a binding site for biotin, so 4 moles of biotin are

bound by 1 mole of avidin.

0010 The interaction of avidin with biotin is the

strongest noncovalent binding ever found in living

organisms with a dissociation constant of the binding

sites, K

¼ 10

15

M. Because avidin is a basic protein

with an isoelectric point of pH 10.5, by oral intake,

not only the dietary but also the enterally synthesized

and enzymatically bound biotin can be irreversibly

bound. The avidin–biotin complex is not cleaved

within the gastrointestinal tract.

0011 Raw egg white contains 2 mg of avidin per 100 g,

so about 15 mg of biotin are fixed by one egg (c.50 g)

and made unavailable for man and animal. Not only

free or enzymatically bound biotin but the structural

isomers with an intact ureido ring in the cis configur-

ation compete for complexing, too, as is shown in

Table 4. The very stable avidin–biotin complex is

only split by autoclaving with strong acid. Avidin,

however, is only in raw egg white active as an inhibi-

tor of the biotin availability and is denaturated by

heat, thereafter losing its binding capacity.

0012The exact physiological role of avidin is not well

understood, but it is generally thought to be effective

as a bacteriostat. With respect to the stoichiometric

binding, avidin has served as a useful diagnostic tool

in the identification and study of biotin-dependent

enzymes.

0013Besides avidin, streptavidin (MW 60 000) has been

isolated from Streptomyces avidinii, which is also

composed of four subunits with a biotin-binding

affinity similar to that of avidin. On the basis of

the stoichiometric binding of biotin by avidin and

streptavidin in a stable complex, current analytical

methods to quantitate biotin and its metabolites in

biological materials have been developed.

Determination

Microbiological Assays

0014As with other B vitamins, biotin has been exclusively

assayed for longer times by microbiological methods.

Biotin is a growth factor for a variety of bacteria,

fungi, yeasts, and flagellatae. For assay purposes, in

foods and other biological materials such as blood,

plasma/serum, urine, and various tissues (liver, brain),

Lactobacillus plantarum, L. casei, Saccharomyces

cerevisiae, Neurospora crassa,andOchromonas

danica have been used. Besides the variable response

to biotin analogs, as shown in Table 2, the lack of

specifity is also due to certain amino acids, fatty acids

and their metabolites, which are able to stimulate the

growth and replace biotin, thus affecting the validity

of the assay. Results achieved with different microbial

assays are often therefore hardly comparable,

particularly if the relationship of biotin to these

interfering factors is poor.

tbl0002 Table 2 Growth response of biotin derivatives to some microorganisms

Microorganism Biotin-

D -sulfoxide Oxybiotin D-Dethiobiotin Biocytin

Lactobacillus plantarum (ATCC 8014) þþ þ þ 

Lactobacillus casei (ATCC 7469) þþþ

Ochromonas danica þþ

Saccharomyces cerevisiae (ATCC 7754) þþ (þ) þþ þþ

Neurospora crassa þþþþ

Kloeckeria brevis (ATCC 9774) þþ  þþ

þþ, identical activity to equimolar biotin; þ, about half of the activity of equimolar biotin; , no growth response.

tbl0003 Table 3 Biotin content in selected foods

Food Biotin content

(mgper100g)

Nutrient

density (mg

biotin per

10 0 0 kcal or

4.2 MJ)

Fruits

Apples, apricots, bananas,

grapefruits, pears, strawberries

0.4–5.5 8–125

Vegetables

Asparagus, cauliflower, peas,

potatoes, spinach, tomatoes

1–4 51–314

Eggs (whole) 25 162

Nuts

Almonds, peanuts, walnuts 10–34 18–61

Milk and cheese

Whole milk, brie, gouda 1.5–6 4–54

Meat

Pork and beef, lean 4–5 27–28

Liver

Pork and beef 23–92 194–536

Fish

Cod, herring, redfish, trout 2–10 22–80

Cereals

Wheat flour, oats, rice polished,

rice (wholegrain), rye

(wholemeal)

1.5–17 4.5–34

Modified, according to the databases of Bundeslebensmittelschl

ssel ll.2,

1996, of the Bundesinstitut f

r gesundheitlichen Verbraucherschutz und

Veterina

rmedizin, Berlin, with permission.

BIOTIN/Properties and Determination 509

0015 Minor growth activity for L. plantarum exhibits

oleic acid, elaidinic acid, linoleic and linolenic acid, as

well as a few other lipids, which have to be removed

either by filtration or by ether extraction. Aspartate

and several other amino acid metabolites, pimelic

acid, and oleic acid interfere with the Saccharomyces

assay. Less frequently used microorganisms are N.

crassa and O. danica, although O. danica is rather

specific and responds, besides biotin, only to biocytin

and protein-bound vitamin. The O. danica assay was

applied for biotin assessments in blood, serum, urine,

liver, brain, and muscle. The validity of the method

established by recovery experiments was demon-

strated to 96–103% biotin recovery.

0016 In order to extract the vitamin, in most assays, the

biological material is subjected to acid (sulfuric acid)

or enzymatic hydrolysis. Protein-bound biotin is also

cleaved by papain, a proteolytic enzyme hydrolyzing

specifically lysine-bound substances.

0017 Principle of the Lactobacillus plantarum (ATCC

8014, formerly L. arabinosus) assay To liberate

protein-bound biotin, foods, feeds and other bio-

logical samples are extracted with sulfuric acid for

2 h at 121



C. Plant products as well as whole blood,

plasma, and urine samples are autoclaved with 2 N

sulfuric acid, and animal samples otherwise with

6 N sulfuric acid. The ratio of sample to acid should

be 1:20, whole blood, plasma or urine at a ratio of

3 ml of sample to 8 ml of acid. For pharmaceuticals

and premixes, dilution with 0.1 N sodium hydroxide

at a concentration of about 0.2 ng ml

1

and, if needed,

centrifugation or filtration to clarify is the preferred

extraction mode. Stronger acid concentrations for

sample extraction should be avoided, because a high

sodium sulfate concentration in the assay is inhibitory

to the test organism. Furthermore, using strong sul-

furic acid, followed by autoclaving, biotin may be

partly oxidized to inactive biotin sulfone.

0018 Hydrochloric acid should not be used in place of

sulfuric acid, as the hydrochloric acid may inactivate

biotin probably because of the presence of chlorine.

High-fat samples must be extracted, prior to acid

cleavage, with hexane or ethyl ether to avoid fatty

acid stimulation.

0019After cooling, the samples are adjusted to pH 6.75

with concentrated NaOH, and biotin in the extract is

determined by means of its growth response to an

inoculum suspension prepared from a stock culture

of L. plantarum and diluted with culture medium.

After incubation at 37



C, the absorbance (nephelo-

metric) is measured at 660 nm, and the biotin content

is calculated by comparing with a standard curve

using d(þ)-biotin concentrations of up to 0.80 ng

per 5 ml. The detection limit of this microbiological

assay is 122 pg biotin ml

1

(0.5 pmol ml

1

). The co-

efficient of variation (CV) for whole blood analysis

amounts to about 19%.

0020A modification of the standard biotin assay

with L. plantarum has been developed by a radiomet-

ric-microbiological assay (RMA) based on the

measurement of

from the metabolism of l-1-

[

C]-methionine by Kloeckeria brevis (ATCC 9774).

The amount of

is measured with the aid of a gas-

flow ionization chamber system and is proportional to

the present biotin amount. The assay sensitivity

has been found to be 10 pg of biotin (41 fmol)/vial

(¼5.2 ml). This test has been employed for plasma,

as well as food, analysis. Biocytin and biotin-d-sulf-

oxide have the same biological activity as biotin in

equimolar concentrations, but dethiobiotin and other

analogs as well as fatty acids and aspartate did not

interfere with the biotin measurement (see Table 2).

The RMA allows some simplification of the sample

preparation and assay procedure (no filtration of the

sample extract and no turbidimetric measurement).

Its application for biotin analyses, however, is limited

by the need for special laboratory devices.

0021The application and detection limits of the most

commonly used microbial assays are summarized in

Table 5.

Isotope Dilution Assays (Radioligand Binding

Assays)

0022The strong and specific affinity of the glycoprotein

avidin to biotin was taken very early on as the basis

tbl0004 Table 4 Quantitation (%) of biotin analogs and derivatives by various binding proteins and microbial assay (comparing equimolar

amounts), according to Baur, Helbich-Endermann, and Salz (unpublished results)

Derivative Avidin Streptavidin Neutravidin Lactobacillusplantarum

Biotin 99 88 100 98

Biocytin 103 98 83 0.1

Biotin-

D-sulfoxide 97 87 35 83

Biotin-

L-sulfoxide 96 36 24.5 14

Biotinsulfone 96 54 28 0.2

Bisnorbiotin 49 0.4 5.5 0.2

Dethiobiotin 10 – – 0

510 BIOTIN/Properties and Determination

for methods to quantitate biotin and certain metabol-

ites in biological materials. The competition between

radioactive and nonradioactive (cold) biotin for avi-

din binding is the principle of isotope dilution assays.

The radioactivity of the avidin–biotin complex

depends on the dilution of the added radioactively

labeled biotin by the unknown (nonradioactive) vita-

min content in the sample.

C-Biotin,

125

I-biotin, or

H-biotin can be used as a competing tracer. Tech-

niques to separate protein bound from unbound

biotin differ in adsorption and separation of either

the avidin–biotin complex (bentonite or nitrocellu-

lose) or the exceeding free biotin (charcoal) in the

sample.

0023 Radiochemical assays previously using

C-labeled

material (biotin) have often lacked the sensitivity ne-

cessary to measure the physiological concentrations

of biotin because of the low specific activity of the

available [

C]-carboxybiotin (45 mCi per mmol of

biotin). The detection limit of 1 ng of biotin per

assay (4.0 pmol) or 5 mg of biotin kg

1

(20.5 mmol)

was only capable of detecting the biotin content in

tissue samples and animal feed.

0024 By using a

125

I-labeled conjugated of biotin, a

biotin detection limit in the femtomolar range has

been made possible, thus being sensitive enough to

determine the biotin content in the small volumes of

blood available in pediatric studies. The drawback of

this assay, however, is the extensive and cumbersome

synthesis of the

125

I-biotin beforehand.

0025 The lack of sensitivity has been overcome when

using

H-labeled biotin as a tracer, which is commer-

cially available with a considerably enhanced specific

radioactivity (30–46 Ci per mmol of biotin). Assays

based on

H-biotin are sensitive enough to quantify

biotin in the picomolar, or even femtomolar, range, as

required for biological material (tissue, blood, and

urine).

0026 Non-bound biotin, as well as free

H-biotin, is

separated by means of adsorption of the avidin–biotin

complex on bentonite or filtration through nitro-

cellulose filters. Alternatively, nonbound biotin can

be adsorbed on dextran-coated charcoal. In principle,

biotin analogs or metabolites with an intact ureido

ring as well as protein-bound biotin are also com-

plexed by avidin and, if present in the sample, should

be assayed by the radioisotope dilution methods (see

Table 4).

0027A problem may arise in terms of the extraction

procedure of bound biotin from the sample material.

With acid hydrolysis, there is the problem of achiev-

ing maximal liberation of bound forms without any

detectable vitamin losses or oxidative damage. Fur-

thermore, large amounts of salt are produced on neu-

tralization of the acid extracts, which may interfere

with the following adsorption of biotin on bentonite

or charcoal. Dilution is necessary, therefore, to lower

the high salt concentration, but this may create prob-

lems in bringing the biotin content to within an

appropriate sensitivity range. Such difficulties can be

avoided by means of enzymatic cleavage by papain

digestion, instead of acid hydrolysis. A typical cali-

bration curve using

H-biotin as a tracer and avidin as

a binder is shown in Figure 3.

0028This modification of the radioligand test has been

applied to biotin determinations in plasma, urine,

and animal and plant food samples. Food samples

are pretreated with liquid nitrogen followed by

homogenization and papain digestion. Comparing

analyses by use of the microbiological assay with

L. plantarum correlated well with this radioligand

test. The detection limit is 0.4 pmol of biotin per

tube (¼ 0.1 ng of biotin).

tbl0005 Table 5 Microbiological assays

Microorganism Principle of assay Biotinrelease Application Detectionlimit

Lactobacillus plantarum

ATCC 8014

Growth-promoting

acticity

2–6 N H

(121



C, 2 h)

Biological samples,

food analysis

122 pg ml

1

(0.5 pmol ml

1

)

Ochromonas danica Growth-promoting

activity

Papain proteolysis Biological samples 3 pg ml

1

(12 fmol ml

1

)

Kloeckeria brevis ATCC

9774

detection from

[

C]-methionine

2NH

(foods)

papain proteolysis

(plasma)

Plasma, food analysis 10 pg per vial (5.2 ml)

(41 fmol)

0.1

100

0.2 0.4 0.6

Biotin (ng)

B/MB

100

0.8 1.0 1.2

fig0003Figure 3 Calibration curve. B, bound

H-biotin; MB, maximal

binding capacity. B=bound

H-biotin, MB=maximal binding cap-

acity. From Deutsche Lebensmittel-Rundschau 82 (1986) 80–83,

with permission.

BIOTIN/Properties and Determination 511

0029 Another modification makes use of a sequential

solid-phase assay based on

125

I-labeled avidin. A

fixed amount of

125

I-labeled avidin is incubated with

either standards of biotin or sample biotin in serum or

urine. An aliquot of this incubate is then transferred to

microtiter plates previously coated with biotin linked

to bovine serum albumin. Depending on the amount

of biotin bound to

125

I-biotin in the first incubation

step, the counts bound in the second incubation step

vary and can be compared with the counts for the

standards. The assay sensitivity is 10 fmol per assay

well (100 fmol ml

1

0030 Table 6 summarizes details of the aforementioned

isotope dilution assays.

Assessment by Competitive Enzyme Protein

Binding Assay (EPBA) or Sequential Solid-Phase

Assay

0031 Very recently, highly improved and sensitive protein

binding assays have been developed by means of

either enzyme-linked avidin or streptavidin, or bio-

tinylated chromophores or fluorophores, respectively.

0032 The principle of the EPBA is similar to isotope

dilution assays, to prevent enzyme-coupled avidin

from binding to a biotinylated protein adsorbed to

plastic by the unknown sample’s vitamin content.

These tests have been improved by the introduction

of microtiter plates in solid-phase sequential assays.

In most cases, the detector molecule is avidin linked

to horse-radish peroxidase (HRP), and the microtiter

plates are coated with bovine serum albumin (BSA)

covalently linked to biotin.

0033The typical sequence of a sequential solid phase

assay is as follows:

0034A constant amount of avidin linked to horse-radish

peroxidase (HRP-avidin) is incubated in a micro-

titer plate with known amounts of biotin and with

dilutions of the sample to produce the standard

curve and the unknown response (incubation 1).

0035An aliquot of the first incubation is then trans-

ferred to corresponding microtiter plates previ-

ously coated with biotin covalently linked to

bovine serum albumin (biotinyl-BSA) and incu-

bated, too (incubation 2). During this incubation

step, HRP-avidin molecules in surplus with un-

occupied biotin-binding sites will bind to the bio-

tinyl-BSA. Unbound HRP-avidin molecules with

no available biotin-binding sites are washed away

thereafter.

0036The amount of HRP-avidin bound to the biotinyl-

BSA is quantitated by measuring the rate of

tbl0006 Table 6 Isotope dilution techniques of biotin determination

Tracer Binder Application Separation of free and bound

tracer

Detectionlimit Author

125

l-Biotin

conjugate

Avidin Physiological

fluids

Adsorption of unbound

biotin on dextran-coated

charcoal

<1.25 pmol ml

1

T Horsburgh/D Gompertz:

Clinica Chimica Acta 82

(1978) 215

C-Biotin Avidin Biological

tissue, animal

feed

Precipitation of avidin–

biotin

20 pmol ml

1

R L Hood: Methods in

Enzymology 62 (1979) 279

H-Biotin Avidin Blood serum Precipitation of avidin–

biotin

1.0 pmol ml

1

H-Biotin Avidin Tissues, serum Adsorption of avidin–biotin

on bentonite

0.5 pmol ml

1

K Dakshinamurti/L Allan:

Methods in Enzymology 62

(1979) 284

H-Biotin Avidin Biotin analogs Adsorption of avidin–biotin

on bentonite

4 fmol ml

1

A L Landman: International

Journal for Vitamin and

Nutrition Research 46

(1976) 310

H-Biotin Avidin Plasma, urine Adsorption of avidin–biotin

on nitrocellulose

1.0 pmol ml

1

R S Sanghvi/R M Lemons/

H Baker/J G Thoene:

Clinica Chimica Acta 124

(1982) 85

H-Biotin Avidin Plasma, urine,

animal and

plant food

Adsorption of unbound

biotin on dextran-coated

charcoal

0.2 pmol ml

1

R Bitsch/l Salz/D Hoetzel:

International Journal for

Vitamin and Nutrition

Research 59 (1989) 59

125

l-Avidin Biotin–bovine

serum

albumin

Serum, urine Sequential solid-phase

assay (molecular sieve

chromatography on

Sephadex)

0.1 pmol ml

1

D M Mock: Methods in

Enzymology 184 (1990)

224

512 BIOTIN/Properties and Determination

peroxidation of o-phenylenediamine (OPD) used as

an indicator dye. Peroxidation is stopped by the

addition of acid (e.g., H

), which also intensi-

fies the absorbance of the peroxidized dye (OPD).

The absorbance maximum at 490 nm is finally

quantitated in a plate-reader spectrophotometer.

Analogous to RIA, a biotin increase in the standard

additions or unknown samples decreases the number

of free biotin-binding sites on HRP-avidin during the

initial incubation. Thus, fewer molecules of free

HRP-avidin are bound to the plates coated with

biotinyl-BSA, and the final color development is

progressively less.

0037 This sequential solid-phase test was applied for

biotin measurement in serum, urine, and milk. The

detection limit is reported to about 5 fmol of biotin

and about 10 parts per trillion (p.p.t.). Separate quan-

titation of biotin and analogs such as biotinsulfoxide

and bisnorbiotin is necessary, because the biotin-

binding affinity of HRP-avidin varies among biotin

and its metabolites. These derivatives thus have to be

separated beforehand by HPLC on RP18 columns,

and the binding affinity of each metabolite has

to be determined separately. The measured total

avidin-binding substances therefore do not equal the

stoichiometric sum of the vitamin and its metabolites.

0038 Variations of this sequential solid phase test are

described using streptavidin as the binder and bioti-

nylated enzyme, e.g., alkaline phosphatase, and free

sample vitamin as the competitors for binding. The

activity of alkaline phosphatase as an indicator is

determined by measuring the absorbance of p-nitro-

phenylphosphate as a chromogenic enzyme substrate

at 405 nm. The biotin-binding affinity of streptavidin

is slightly lower than that of avidin, but biotin metab-

olites are bound much more weakly by streptavidin

(see Table 4).

0039 Assays utilizing enzyme-linked biotin as an indica-

tor are applicable to quantify either biotin or avidin/

streptavidin. The lower limits of those assays are

0.2–20 pg of biotin per well (0.8 fmol to 0.08 pmol).

0040 A superior replacement for avidin or streptavidin

appears to be the introduction of neutravidin, a modi-

fied avidin with no carbohydrate components. The

deglycosylation procedure is carried out under mild

conditions, so as not to affect the stability or the

biotin-binding affinity. The suitability for biotin as-

sessments seems to be more advantageous in so far as

the cross-reactivity for structure analogs is weaker

compared with the other binding proteins (Table 4).

An ELISA test has been developed, based on neutra-

vidin-coated microtiter plates and biotinylated HRP

competing with sample biotin. The absorbance of the

chromogen tetramethylbenzidine after oxidation by

the enzyme conjugate and urea peroxide is detected at

450 nm in a plate-reader photometer. The detection

limit of this commercial test is 1.1 pg of biotin

(4.5 fmol) or 11 ng of biotin per liter of sample

(45 pmol). Using this assay, the biotin content of bio-

logical materials (plasma, urine) as well as in several

plant and animal food samples was analyzed after

release of the protein bound vitamin by papain diges-

tion. The biotin content correlated well with micro-

biologically determined vitamin using L. plantarum

(r ¼0.973). A typical biotin calibration curve

obtained when using the RIDASCREEN

assay

with neutravidin as a solid-phase binder is shown in

Figure 4. The principles and detection limits of typical

competitive EPBAs are summarized in Table 7.

Enzymatic Determination

0041An older enzymatic assay is based on the formation of

holopyruvate carboxylase from apopyruvate carbox-

ylase and biotin, followed by determination of the

pyruvate carboxylase activity in the presence of holo-

enzyme synthetase, measuring the incorporation of

H-



into oxaloacetate, which is reduced to

acid-stable malate. The apoenzyme and the holoen-

zyme synthetase have been obtained from the biotin-

free cultivated yeast, S. cerevisiae.

0042The reaction of the test is as follows:

+ biotin

Holoenzyme

nthetase

Pyruvate

carboxylase

Apopyruvate

carboxylase

ATP, Mg

, K

C-oxaloacetate.

Pyruvate carboxylase

Pyruvate

H-

−

Absorbance (%)

100

37 111

Biotin (ng l

−1

)

333 1000

fig0004Figure 4 Calibration curve of RIDASCREEN

with biotin.

BIOTIN/Properties and Determination 513

The enzyme activity is proportional to the amount of

available biotin. The method is somewhat difficult,

and the absolute minimum amount of biotin detect-

able is 5 pg. Of the biotin analogs and precursors,

only biocytin has been found to interfere in this test.

Photometric and Fluorimetric Determinations

0043 Based on the high affinity of biotin, earlier methods

took advantage of the spectral shift, the fluorescence

change, or the chemiluminescence induction after

displacement of an avidin-bound dye by biotin.

After addition of the azodye 2-(4

-hydroxyazo-

benzene) benzoic acid (HABA) to avidin, a complex

is formed with an absorbance maximum at 500 nm.

Titration with biotin quantitatively displaces the

dyefrom avidin, leading to a spectral shift, which

can be monitored spectrophotometrically. This test

can be employed for the quantitation of biotin as

well as avidin. The sensitivity is no higher than 2 mg

of biotin per ml (8 nmol ml

1

). Biotin analogs with an

intact ureido ring react in a similar way. Depending

on their binding constants, these analogs may reduce

the sharpness of the endpoint but do not affect the

titer.

0044 Another spectrophotometric assay for biotin and

avidin has been proposed, which is based on the

inactivation of biotinylated glucose-6-phosphate

dehydrogenase when complexed with avidin. The

degree of inactivation of the biotinylated enzyme in

relation to the avidin concentration is detected in a

coupled reaction with NADH at 340 nm. The detec-

tion limits have been reported to be 100 ng of biotin

per ml (0.4 nmol ml

1

) and 2.5 ng of avidin per ml.

0045 Fluorescence and chemiluminescence determina-

tions The competition between biotin and a biotin

luminol derivative for avidin was utilized for a

chemolumimetric detection. Biotin is covalently

linked to the amine function of isoluminol to yield

the conjugate. This conjugate emits light when

oxidized by either a hydrogenperoxidase–lactoperox-

idase or a superoxide. The peak light intensity in-

creases about 10-fold when the conjugate is

incubated with avidin prior to oxidation. When the

isoluminol-biotin conjugate and free biotin compete

for the avidin binding sites, the peak light intensity

produced in subsequent chemiluminescent reactions

decreases with increasing biotin levels. In this way,

biotin is measured quantitatively at levels as low as

50 nM.

0046A greater sensitivity is achieved by measuring the

quenching of the chemiluminescence of aminobutyl-

ethylisoluminol (ABEI)-labeled biotin when bound to

fluorescein-labeled avidin. The chemiluminescence

decrease in the range of 460–525 nm compared with

unbound ABEI-biotin is capable of detecting biotin in

human sera in the range of 1.2–4.3 nM. However, the

disadvantage with this is the laborious preparation

of the ABEI-biotin and its purification by TLC and

HPLC.

0047Another modification of fluorescence detection in a

competitive binding assay includes HPLC separation

of biotin and biocytin coupled with a postcolumn

reaction detection system. In one analytical approach,

biotin or biocytin displacement of 2-anilinonaphtha-

lene-6-sulfonic acid from its binding site to avidin

yields a decrease in the fluorescence intensity at

438 nm. The other analytical approach utilizes the

increase in fluorescence of (strept) avidin labeled

with fluorescein isothiocyanate (FITC) at 518 nm

after binding with the analyte (biotin, biocytin) in

the postcolumn eluate. However, the detection limits

of both methods are relatively low in the range of

5 10

7

M up to 1.8 10

8

M biotin or biocytin,

tbl0007 Table 7 Enzyme immuno(sorbent) assays

Enzyme conjugate Indicator dye Solid phase

(microtiterplates)

Application Detection limit Author

HRP-Avidin OPD Biotinyl-BSA Serum, milk, urine 5 fmol per well

(10 p.p.t.)

D Mock: Methods in

Enzymology 279

(1997), 265

HRP-Streptavidin OPD Biotinyl-BSA Aqueous solution 0.1 pmol per well E Z Huang: Methods in

Enzymology 279

(1997) 304

HRP-Streptavidin [2,2-azinodi(3-ethyl-

benzthiazoline)

sulfonic acid

Biotinyl-goat

antirabbit IgG

Aqueous solution,

clinical samples

1pgml

1

(4 fmol) D Shiuan: Methods in

Enzymology 279

(1997) 321

Biotinyl-HRP Tetramethylbenzidine

þ urea peroxide

Neutravidin Plasma, urine, food

samples

4.5 fmol per well M Helbich-

Endermann:

Doctoral thesis

(1999) Jena/

Germany

514 BIOTIN/Properties and Determination