Chapter 18 microbiological methods for the determination of the b group vitamins

Aliquots of a standard solution of the vitamin to be determined, or aliquots of the sample extract containing the vitamin, are added to an initiallytranslucent basal nutrient medium, com

Microbiological Methods for

the Determination of the B-Group Vitamins

18.1 Introduction

All the B-group vitamins can be assayed microbiologically The assay isms are particular strains obtained from various culture collections such

organ-as the American Type Culture Collection (ATCC) The current and previous

and measure either the lactic acid produced after a 72-h incubation period(titrimetric method) or the growth of the organism (turbidimetric method).The latter is nowadays generally preferred, as it is simpler and requiresshorter incubation times Methods based on the measurement of metaboliccarbon dioxide have also been developed

18.2 General Principles

18.2.1 Turbidimetric Methods

Turbidimetric methods are based on the absolute requirement of certainmicroorganisms for the vitamin in question; that is, the organisms canmultiply only when the vitamin is present in the surrounding medium.Conventional procedures, such as those adopted by the AOAC, use testtubes, but 48-well or 96-well microtiter plates can also be used Aliquots

of a standard solution of the vitamin to be determined, or aliquots

of the sample extract containing the vitamin, are added to an initiallytranslucent basal nutrient medium, complete in all respects except forthe vitamin in question Following inoculation with the assay organism,the organism multiplies in proportion to the concentration of limitingvitamin in the standard or sample solution, and the extent of thegrowth is ascertained by measuring the turbidity produced Over adefined concentration range, the measured response will be directlyproportional to the amount of limiting vitamin present and, within this

range, the sample solution and standard vitamin solution can be pared The response is highly dependent upon whether or not boundforms of the vitamin are released during the extraction stage of the assay.Cultures of assay organisms are despatched in lyophilized (freeze-dried)form sealed under vacuum in glass ampoules Media can be obtained in adehydrated form from Difco Laboratories, Michigan, U.S They are recon-stituted for use simply by suspending the required weighed amount indistilled water, heating to effect solution, and autoclaving to sterilizethem The use of dehydrated media allows a fresh batch of medium to bemade up in the amount required with the minimum of time and effort.Lactic acid bacteria are ideally suited as assay organisms for determin-ing all but one of the B-vitamins turbidimetrically, the one exception

specific and complex, they grow readily in synthetic and semisyntheticmedia, and they are nonpathogenic They are not prone to mutationand have maintained their characteristics unimpaired after many years

of subculture in the laboratory The rod forms (genus, Lactobacillus) aremicroaerophilic and the coccus forms (genus, Enterococcus, Leuconostoc)are facultative aerobes [1] This ability to grow well in limited amounts

of air means that test tubes of liquid medium can conveniently be usedfor assay purposes

certain species of yeasts are used instead for the determination of this

TABLE 18.1

Assay Organisms Used for Determining B-Group Vitamins

Weissella viridescens Lactobacillus viridescens 12706

Saccharomyces uvarum

9080

Poterioochromonas malhamensis Ochromonas malhamensis 11532

Lactobacillus leichmannii

Source: http://www.atcc.org/SearchCatalogs/longview.cfm

vitamin Because yeasts grow aerobically, such assays have the ence of requiring constant and uniform shaking during incubation.Protozoa have more highly developed ingestive and digestive systemsthan do bacteria and yeasts, and therefore exhibit a more mammalian-likeresponse to the various naturally occurring forms of the vitamins.However, the test growth period for protozoa is longer than that forlactic acid bacteria (3 –5 days versus 24 –48 h), conditions of growth aremore demanding, and growth response is more difficult to measure.

inconveni-18.2.2 Methods Based on the Measurement of

Metabolic Carbon Dioxide

The radiometric microbiological assay (RMA) is based upon the

TABLE 18.2

Official (AOACa) Microbiological Methods of Analysis of B-Group Vitamins

Assay Organism

Vitamin B 2 940.33 L rhamnosus (7469) All food products

Niacin 944.13 L plantarum (8014) All food products; cereal

products would require alkaline hydrolysis to completely extract bound forms of nicotinic acid Niacin 985.34 L plantarum (8014) Milk-based infant formula Vitamin B 6 961.15 S cerevisiae (9080) All food products

Vitamin B 6 985.32 S cerevisiae (9080) Milk-based infant formula Pantothenic acid

(free form only)

945.74 L plantarum (8014) Vitamin preparations; double

enzyme treatment would

be required to liberate pantothenic acid from coenzyme A in food samples Pantothenic acid 992.07 L plantarum (8014) Milk-based infant formula Folate

(free form only)

944.12 E hirae (8043) Vitamin preparations;

enzymatic deconjugation would be required to determine total folate Folate (total) 992.05 L rhamnosus (7469) Milk-based infant formula Vitamin B 12 952.20 L delbrueckii

subsp lactis (7830)

All food products Vitamin B 12 986.23 L delbrueckii

subsp lactis (7830)

Milk-based infant formula

a Official Methods of Analysis of AOAC International, 17th ed., revision 2 (2003).

substrate by the test organism in the presence of the specific vitamin to

be analyzed This technique has been applied to the determination

foods using the yeast Kloeckera apiculata as the test organism and

Lactobacillus delbrueckii subsp lactis [4] In these applications, the activity is measured automatically by means of a commercially availablegas flow system incorporating an ionization chamber [2] The RMAcombines the biological specificity of measuring a vitamin-dependentmicrobiological metabolic reaction with the sensitivity and accuracy ofradioactive decay measurement Extraction methods suitable for the

RMA have been discussed by Guilarte [5] Sample preparation issimplified because colored, turbid, or precipitated debris do not interfere

of glassware, so important for conventional tube assays, is unnecessary

18.3 Conventional Turbidimetric Method Using Test Tubes18.3.1 Summary

In the standard procedure, the appropriate assay medium, free of thevitamin to be determined, is prepared at twice its final concentration(i.e., double strength) Multiple aliquots of a standard solution of thepure vitamin and of suitably prepared extracts of the test food areadded to a series of uniform test tubes in amounts suitable to producegradations in growth between no growth and maximum growth The con-tents of all tubes are diluted with water to the same volume, and an equalvolume of the translucent assay medium is added The tubes are thencovered and sterilized by autoclaving After cooling to a uniform temp-erature, the tubes are aseptically inoculated with an actively growingculture of the test organism The tubes are then incubated for 22– 24 h

at a constant temperature near the optimum for the test organism untilgrowth has reached the maximum permitted by the limiting vitaminpresent The growth response to standard and test extract is determined

by measuring the turbidity produced in the tubes The data obtainedfrom the standards are used to construct a standard curve from whichthe vitamin concentrations of the various sample aliquots are derived

The use of multiple aliquots allows a validity check to be carried out: thevitamin concentration found should be directly proportional to thevolume of aliquot taken The amount of vitamin present in the originalsample is then calculated at the different test levels, and the results areaveraged to obtain the final result.

18.3.2 Laboratory Facilities and Cleaning of Glassware

Microbiological manipulations should be performed well away fromthe sample preparation area, preferably in a separate room of struc-tural simplicity to facilitate cleaning An essential requirement is areadily available supply of fresh glass-distilled water; deionized

assays [7] All glassware and other items should be kept exclusivelyfor microbiological assays and segregated A meticulous washing-uproutine is essential to ensure that the glassware is scrupulously

involves the following steps:

1 Boiling in a water bath containing a special type of detergent for 3 h

2 Machine-washing and rinsing with deionized water

3 Soaking in dilute acid

4 Rinsing with deionized water

5 Baking in an oven at 1008C overnight [8]

The use of disposable glassware eliminates this meticulous washing,but items that are not disposable require special attention [9]

Essential equipment includes:

1 An autoclave large enough to admit all racks of tubes for agiven assay and capable of accurate adjustment to a constant

an electrically heated autoclave, because it allows steaming at1008C and has a more rapid overall operation sequence fromheating up to cooling down

2 Incubators of the forced draught or circulating water bathtype, capable of maintaining a constant accurate temperature(+0.58C) in the range of 27–378C A shaking incubator or waterbath is required for assays with yeasts; assays with protozoarequire incubation with both shaking and illumination

3 A nephelometer equipped to accept the assay tubes for directmeasurement of turbidity

18.3.3 Media

Three types of media are used:

1 Maintenance media to preserve the viability and sensitivity of theassay organism Such media contain agar and all the nutritionalfactors essential for the organism’s normal growth and metabolism

A buffer salt is included to prevent the pH from rapidly dropping

to levels which would inhibit growth, although growth willproceed until a pH of at least 4 is reached The medium is formulated

to give an initial pH (typically 6.8) that is somewhat higher than theoptimum pH of 5.5–6.5 for most lactic acid bacteria [10] This is toallow for the production of acid that occurs when the medium is ster-ilized by autoclaving

2 Inoculum media (broths) to condition the test culture for immediateuse Difco supply dehydrated inoculum media, whose formulationsare the same as those of the corresponding maintenance media,except that the agar component is omitted The vitamin to beassayed is present in an amount that barely supports the growth

of the assay organism This limits the accumulation of the vitamin

in the bacterial cell and thus maintains the sensitivity of the assay

3 Assay (basal) media to permit quantitation of the vitamin under test.The assay medium contains all the nutritional factors necessary forthe normal growth and metabolism of the assay organism, exceptthe vitamin to be determined It is formulated from highly purifiednatural products, synthetic vitamins, and other reagent-grade com-pounds An assay medium used for lactic acid bacteria mustcontain a fermentable carbohydrate, a variable assortment of essen-tial amino acids, various vitamins and mineral salts, certain purineand pyrimidine bases, and an appropriate buffer system Glucose isuniversally used as a source of carbon and energy; the amino acidsare provided in the form of acid-hydrolyzed casein plus tryptophan

or a mixture of the specific amino acids; and the buffer salt is usuallysodium acetate, which also has a stimulatory effect on growth.18.3.4 General Assay Procedure

The standard assay procedure using lactic acid bacteria can be brokendown into a number of steps:

1 Maintenance of stock cultures

2 Preparation of the inoculum culture

3 Preparation of the assay medium

4 Extraction of the vitamin from the test material

5 Setting up the assay

6 Quantification

A scheme of steps 1 and 2 is depicted in Figure 18.1

18.3.4.1 Maintenance of Stock Cultures

The assay organism purchased in lyophilized culture is regenerated byincubation for 24 h at 378C in sterile inoculum broth The cells are thenconcentrated by centrifugation and transferred by stab to tubes contain-ing agar-based maintenance medium These tubes are the original stockcultures Stock cultures of the microaerophilic lactobacilli can be main-tained over a period of several years by employing a regular schedule

of subculturing into maintenance medium At regular weekly ormonthly intervals (depending on the medium employed), three freshstab cultures are prepared from a refrigerated stock culture One ofthese cultures is reserved as the new stock culture until the next timefor transfer, when it will be used to prepare three more agar stabs Theother two cultures are used to prepare inocula for the assay

The usual procedure for subculturing lactobacilli is to distribute 10-mlquantities of molten maintenance medium into lipless 16– 20-mm dia-meter test tubes and autoclave the plugged tubes for 20 min at 15 lbpressure (1218C) After cooling, stab inoculations are made into thesolidified agar The tubes are incubated under the appropriate conditionsfor each vitamin that produce distinctly visible growth along the line

of the stab Since the growth rate of an organism is a function oftemperature, the incubation must be precisely controlled to within+0.58C of the selected temperature The stab cultures thus prepared arestored in the refrigerator under aseptic conditions

Original stock culture

Stab transfer into maintenance medium (agar) and incubate

Culture for preparing

Inoculum

Transfer into inoculum

medium broth and incubate

Inoculum culture

Centrifuge, resuspend cells

in sterile 0.9% NaCl, then dilute

Use to inoculate assay tubes

1st stock culture (store in refrigerator)

Cultures for preparing inoculum

For the determination of vitamin B6, Bell [11] maintained the yeastSaccharomyces cerevisiae under aerobic conditions on an agar slope using

10 ml of sterilized maintenance medium in a 1-oz McCartney bottle.The yeast was subcultured weekly by taking a loopful of yeast from thecurrent culture onto a fresh agar slope, incubating at 328C overnight,and storing in a refrigerator

18.3.4.2 Preparation of the Inoculum Culture

The inoculum is prepared 1 day before the assay day by making a transferfrom the stab culture into a tube containing sterile inoculum medium.Following an overnight incubation at 378C, the cells of the resultantculture are washed three times with sterile saline (0.9% NaCl solution)

to reduce carry-over of vitamin into the assay tubes during the sequent inoculation This washing operation is performed by centrifugingthe tube contents, discarding the supernant, and resuspending the cells in

sub-10 ml of saline After the third wash, the cells are resuspended in sterileassay medium and incubated at 378C until used The final inoculum isprepared by diluting the vitamin-depleted cell suspension with assaymedium

Bell [11] found that procedures using centrifugal washing gave aninoculum in the lag phase of growth, thus necessitating unduly longassay incubation periods Problems of airborne bacterial contaminationwere also encountered Bell prepared inocula for the determination of

using Bacto-Micro Inoculum Broth (Difco Code 0320) as the inoculummedium The common procedure for all these determinations was tosubculture the assay organism from the most recent agar stab into 5 ml

of sterile inoculum broth and incubate overnight at 378C The followingmorning, one drop of this subculture was added to 5 ml of single-strengthbasal medium (basal medium diluted with an equal volume of water)containing a controlled amount of the vitamin being assayed Theseamounts were 25 ng nicotinic acid/ml, 20 ng pantothenic acid/ml,0.05 ng biotin/ml, 2 ng riboflavin/ml, 1.0 ng folic acid/ml, and 0.04 ngcyanocobalamin/ml Both the inoculum broth and the basal mediumwere held at 378C during transfer of the organism After a further 6-hincubation at 378C, two drops of the resultant culture were transferred

to 10 ml of single-strength basal medium (without added vitamin) Thisfinal suspension was used as the inoculum and contained cells in theexponential (acceleration) growth phase

similar manner as described earlier, except that the inoculum mediumwas Bacto-APT Broth (Difco Code 0655) and the incubation temperature

a loop of S cerevisiae from the agar slope was added to 5 ml of strength medium containing a glass bead, and this subculture wasincubated for 20 h at 278C with constant shaking One milliliter of the sus-pension obtained was pipetted into a tube containing 5 ml of sterilesingle-strength basal medium and mixed to give the inoculum culture.Variations of the inoculum culture can be eliminated by the use ofglycerol-cryoprotected lactobacilli This entails preparing a largevolume of inoculated basal medium, adding glycerol, and dispensing

bac-teria stored in this manner remain viable for several months A thawedvial is then used as the inoculum for each assay This technique has thepractical advantages over serial agar-stab culturing in diminishing theneed for microbiological expertise and saving time The washing ofcells by centrifugation with saline is unnecessary, thus saving furthertime and effort Standard curves made from a single batch of cryopro-tected inoculum are superimposable Because blank values are lower, aheavier inoculum can be used, which results in a greater growth rateand hence a reduction in incubation time Thus, assays using cryopro-tected inocula are more sensitive and reproducible, besides beingcheaper and quicker [12]

Horne [13] prepared glycerol-cryoprotected L rhamnosus as inoculumfor a microtiter plate assay of folate as follows Dissolve 9.4 g of dehy-drated basal medium (Bacto-Folic Acid Casei Medium; Difco Code No.0822) and 50 mg of ascorbic acid in 200 ml of distilled water To thissingle-strength basal medium add 120 ng of (6RS)-5-formyl-THF,calcium salt pentahydrate, and then sterilize by filtration (0.22-mm poresize) Suspend the lyophilized L rhamnosus, in its shipping vial, in 1 ml

of the prepared medium Transfer 0.25 ml of this suspension to theremaining 199 ml of medium and incubate at 35– 378C for about 18 h.Cool in an ice bath and add a equal volume of cold, sterile glycerol(80%, v/v in water) Store 4-ml aliquots in sterile tubes at about 2708C

18.3.4.3 Preparation of the Assay (Basal) Medium

In traditional assay procedures, the assay media are prepared at doublestrength, either from commercial dehydrated formulations (if available)

or from individual ingredients

In addition to factors essential for bacterial growth, other substanceswhich stimulate growth must be considered Ideally, the medium shouldcontain sufficient amount of all stimulatory substances so that theeffects of these nutrients added with the hydrolyzed food extract beingassayed will be eliminated In practice, the ideal is seldom achievedand the adequacy of a basal medium depends upon the experimental con-ditions [10] A basal medium which, when supplied with the missing

vitamin, contains all of the nutrients essential for growth, but is lacking inone or more substances that markedly stimulate growth, may givesatisfactory assays if the period of incubation is long enough to eliminatethe effects of the nonessential growth stimulants Such a medium will also

be satisfactory for assaying samples which are rich in the vitamin to bedetermined, as the high dilution of sample will result in a negligibleaddition of stimulatory substances Conversely, such a medium willtend to give erroneous results if used with a short incubation period,and with samples of low vitamin potency

Fatty acids are notorious growth stimulants for a number of lacticacid bacteria [14,15], but it is not customary to add these acids to thebasal medium; rather they are removed from the food sample after theextraction step

18.3.4.4 Extraction of the Vitamin from the Test Material

The vitamins are extracted from the food matrix in a form that can beutilized by the particular assay organism being used This generallyinvolves autoclaving the food sample in the presence of acid or, foracid-labile vitamins, digesting the sample with suitable enzymes Afterprecipitating the proteins at their isoelectric point (ca pH 4), the pH ofthe extract is adjusted to that of the basal medium (typically pH 6.8).This step is necessary to ensure that the pH of the medium is notaltered by the addition of different amounts of the extract The extract isthen diluted to bring the concentration of the vitamin to be assayedwithin the range of the standard curve Hopefully, the dilution factorwill be sufficiently high to dilute out any interfering substances thatwould cause drift and invalidate the assay The minimum dilutions offoods necessary to avoid the inhibitory effects of food preservatives andneutralization salts have been calculated [16] Finally, the extracts arefiltered to remove the precipitated protein and lipoidal material, and toobtain a clear solution for assay

18.3.4.5 Setting Up the Assay

At all stages during the analytical procedure, the solutions must be tected from daylight Aliquots of the working standard vitamin solutionare added in increasing volumes up to 5 ml to a duplicate series oftubes for the construction of a standard curve; duplicate blanks contain-ing no vitamin are included Similar volumes of the neutralized testextracts are added to a single series of tubes A range of concentrationlevels of each test extract is assayed in the expectation that at least threewill fall on the standard curve Fresh glass-distilled water is added toall tubes to bring the volume in each tube to 5.0 ml, after which 5.0 ml

pro-of double-strength basal medium is added so that the total volume is

10 ml The addition of basal medium to the extract and water givesbetter mixing of the two solutions than adding the extract and water tothe medium The entire rack of filled tubes is covered with a sheet ofaluminum foil and sterilized in an autoclave The tubes are removedimmediately from the autoclave when atmospheric pressure is reached,and the tubes are cooled to below the subsequent incubation temperature

It is imperative that all tubes are cooled to the same temperature, becauseturbidity is a measure of the rate of growth rather than the extent ofgrowth, and small differences in the temperature between tubes at thestart of the incubation influence the growth rate

All tubes are inoculated with one drop of freshly prepared inoculumculture The inclusion of a control run, which is not inoculated butotherwise treated identically, is advisable to check the sterility of thebasal medium For turbidimetric assays using lactic acid bacteria oryeasts, the tubes are incubated for 22– 24 h at a constant temperaturenear the optimum for these organisms Suitable temperatures are 378Cfor L rhamnosus, L plantarum, and L delbrueckii subsp lactis; 308C forWeissella viridescens; and 288C (with constant shaking) for S cerevisiaeand K apiculata

The setting-up procedure described by Bell [11] involves less pulations than the conventional procedure, owing to the volumes of stan-dard or sample assay solutions being reduced from milliliter aliquots tomicroliter aliquots ranging from 0 to 250 ml The addition of such smallvolumes to 10 ml of assay medium has two advantages First, no signifi-cant volume change occurs so that single-strength medium can be used.Second, the need for adjustment of the pH value of samples extractedwith acids is greatly reduced For example, no pH adjustment is requiredwhen acids less than 0.1 N are used, and for strong acid hydrolysatesthe solution need only be adjusted to pH 1 –2

mani-18.3.4.6 Quantification

At the end of the incubation period, the cells are uniformly suspended byshaking the tubes, and time is allowed for the air bubbles to dispersebefore measurement The turbidities of all tubes are measured in anephelometer using a neutral filter, colorimeter with a filter in theregion of 640 nm, or spectrophotometer at 540 –660-nm wavelength Theturbidity may be expressed as an absorption (extinction), as a transmit-

reading The arithmetic means of the replicates are calculated, and themeans for the standard solutions are plotted on semilogarithmic graphpaper with the turbidity values as ordinates (linear scale) and concen-trations in ng/ml as abscissae (logarithmic scale) The calibration curve

is drawn through these points The vitamin concentrations of the sampletubes are read off from the calibration curve and the concentration valuesfor the original samples are calculated from simple dilution factors Con-centration values for a given sample calculated from at least three

mean [17]

The reliability of a determination can be assessed by testing for the sence or absence of drift This simply entails plotting the mean turbidityvalues on the calibration curve for the sample dilutions against the corre-sponding dilution factors A check curve that is roughly parallel to thecalibration curve signifies the absence of drift Drift is manifested by acheck curve that deviates widely from the calibration curve, eitherincreasing or decreasing with concentration of the sample The occurrence

pre-of drift in assay solutions is evidence for the presence pre-of interferingmaterials in the test solution presented for assay If a drift has been esta-blished, the determination is invalid and the assay must be repeated Inthis event, measures must be taken to remove the interfering substances

by improving the efficiency of the extraction procedure If these measuresfail, the assay conditions must be changed or a different assay organismemployed [17]

18.3.5 Partial Automation of the Assay Procedure

Haggett et al [18] described an inter-connected modular system whichprovided automated dispensing of sample, buffer, and assay mediuminto the tubes; automatic measurement of growth response by turbidime-try after incubation; and computer software to control the equipmentand to process the analytical data The automation process facilitateddetection of errors and halved the operator time per assay

18.4 Turbidimetric Method Using Microtiter Plates

A microbiological assay for folate adapted for the use of disposable96-well microtiter plates and an automatic plate reader was introduced

by Newman and Tsai [19] The organism used in this assay was afolate-depleted culture of L rhamnosus prepared by serial agar-stabculturing and centrifugal washing Horne and Patterson [20] simplifiedthis method by using the glycerol-cryoprotected L rhamnosus, whichrequires only dilution before use as the inoculum An inherent problem

of overestimated results in the two perimeter rows of clear-wall 96-wellplates was overcome by the use of plates with opaque, black walls [13]

The use of microtiter plates allows automated determination andcomputer analysis of data The saving in time is substantial owing tothe speed of multiwell plate readers, which can measure the light trans-mission in 96 wells in less than 2 min Other advantages over the standardtube assay are minimized reagent costs and a 10-fold increase insensitivity.

Molloy and Scott [21] provided details of a microtiter plate method fordetermining folate in blood, which can be applied to suitably preparedfood extracts For each diluted sample extract in duplicate (replicates aand b), duplicate aliquots of 100 and 50 ml are transferred to four separatewells of a 96-well microtiter plate The working folate standard is dis-pensed into wells of a separate microtiter plate at volumes of 0 (blanks),

5, 10, 15, 20, 25, 30, 40, 60, 80, and 100 ml Eight replicates of eachvolume from 5 to 100 ml and 16 blank replicates are dispensed Volumes

of all wells are made up to 100 ml with 0.5% (w/v) sodium ascorbateand to each well is added 200 ml of inoculum (a cryopreserved, chloram-phenicol-resistant strain of L rhamnosus in assay medium) Each plate iscovered with a plastic plate sealer and a roller is used to ensure that allwells are sealed and air bubbles removed Plates are incubated at 378Cfor about 42 h After this time, each plate is inverted and mixed to produce

an even cell suspension and the plate sealer is removed The opticaldensities at 590 nm are measured in a microtiter plate reader linked to acomputer A commercial enzyme-linked immunosorbent assay (ELISA)program is used to collect data, draw a standard curve, and calculatethe concentration of folate in each well Appropriate dilution factors areused to calculate folate concentrations in the sample extracts Forsample replicate a, the concentration to be reported is calculated by aver-aging the final results (ng/ml) from the duplicated 100-ml and 50-mlaliquots The 50-ml average is then checked against the 100-ml averageand the result is accepted if there is no more than a 10% difference betweenthe two The mean of these two averages is then taken as the result forreplicate a This procedure is repeated for replicate b Finally, the resultsfor replicates a and b are checked against each other If there is agreement

18.5 Assays of Individual B-Group Vitamins

Two Lactobacilli species have been widely used as assay organisms for the

Of the two, the latter is generally preferred as it is less susceptible to

inhibitory or stimulatory substances [22] W viridescens requires the intactthiamin molecule for growth Thiamin pyrophosphate is approximately60% as active as thiamin when equimolar concentrations are compared[23] The conventional tube assay using W viridescens has been comparedwith manual and semiautomated fluorometric methods in terms of itsability to recover added thiamin hydrochloride from commercially

thiaminase [24] The microbiological method gave the best results for

Bui [25] used L fermentum maintained as stab cultures

the extraction procedure involves both acid and enzymatic hydrolysis as

treat-ment can be omitted for the analysis of grain products and milk, and forthe determination of the added thiamin hydrochloride in fortified foods

Lactic acid bacteria cannot utilize flavin adenine dinucleotide (FAD), andthe growth response of L rhamnosus, measured turbidimetrically, differssignificantly between riboflavin and flavin mononucleotide (FMN) [26]

FMN after acid extraction, it would be more accurate to use FMN as thestandard in the microbiological assay instead of riboflavin

L rhamnosus is stimulated by starch [27], and either stimulated orinhibited by long-chain free fatty acids (e.g., palmitic, stearic, oleic, andlinoleic acids) and other lipids, including lecithin [28 – 30] Kornberg

et al [31] proposed the use of Enterococcus faecalis which, with a sensitivity

to 0.1 ng riboflavin/ml [32], is 50 times more sensitive than L rhamnosus.Lipids also stimulate the growth of E faecalis, but its higher sensitivity toriboflavin allows samples to be prepared for assay at a higherdilution, resulting in negligible amounts of interfering lipids and otherextraneous matter in the assay tubes Barton-Wright [33] used a basalmedium that was more synthetic in composition than the originalmedium used by Kornberg et al [31] Comparative assay values usingmany of the foods tested The higher value of 0.33 mg/g for white flourusing L rhamnosus is apparently due to the stimulatory effect of lipids,

as a prior ether extraction lowered this value to 0.20 mg/g, making itcomparable to the value of 0.21 mg/g using E faecalis The ether extractionproduced no lowering of the E faecalis assay value On the basis of thesecomparisons, E faecalis is preferred to L rhamnosus as an assay organism

The extraction procedure for the L rhamnosus assay necessitates claving the food sample with 0.1 N HCl at 1218C for 30 min Duringacid hydrolysis, FAD, which cannot be utilized by lactic acid bacteria, iscompletely degraded to FMN and riboflavin, and some of the FMN

auto-is also degraded to riboflavin The complete conversion of FMN to flavin is not necessary, because FMN, riboflavin, and the variousisomeric riboflavin monophosphates are all nutrients for the growth of

ribo-L rhamnosus The acid digestion eliminates the troublesome starch, but

it liberates interfering free fatty acids

The general extraction procedure used in L rhamnosus assays for lyzing foods of very low fat content (such foods include many cereals)involves acid hydrolysis, followed by precipitation of the denaturedproteins at pH 4.5 The precipitated proteins, together with the smallamount of lipoidal material and any nonhydrolyzed starch, are removed

ana-by simply filtering through paper For samples of negligible fat content,the filtration step results in an assay free from drift Omission of the

whole wheat flour, which was typical of cereal products in general [29].High-fat foods, such as wheat germ, maize, oats, soya beans, meat,cheese, and mixed diets, require a preliminary extraction with petroleumether (bp 40– 608C) before acid hydrolysis to remove neutral fats Thisinvolves extracting the dried, finely ground sample with petroleumether for 16– 18 h in a Soxhlet apparatus The defatted material is auto-claved, adjusted to pH 4.5, diluted and filtered A 50-ml aliquot of thefiltrate is shaken with two or three 30-ml portions of diethyl ether in a sep-arating funnel The combined ether extracts are washed two or three times

Source: From Kornberg, H.A., Langdon, R.S., and Cheldelin, V.H., Anal Chem., 20, 81, 1948 With permission from American Chemical Society.