Microbiological assay of Vitamins- B series Simplified

1. VITAMIN ANALYSIS
Dr Gana Manjusha K
Associate Professor
Vignan Institute of Pharmaceutical Technology

2. Definition and Importance
• Vitamins are defined as relatively low-molecular
weight compounds which humans, and for that
matter, any living organism that depends on organic
matter as a source of nutrients, require small
quantities for normal metabolism.
• With few exceptions, humans cannot synthesize
most vitamins and therefore need to obtain them
from food and supplements.
• Insufficient levels of vitamins result in deficiency
diseases [e.g., scurvy and pellagra, which are due to
the lack of ascorbic acid (vitamin C) and niacin,
respectively].

3. Importance of Analysis
• Vitamin analysis of food and other biological samples has
played a critical role in determining animal and human
nutritional requirements.
• Furthermore, accurate food composition information is required
to determine dietary intakes to assess diet adequacy and improve
human nutrition worldwide.
• From the consumer and industry points of view, reliable assay
methods are required to ensure accuracy of food labeling.
• This chapter provides an overview of techniques for analysis of
the vitamin content of food and some of the problems associated
with these techniques.
• Please note that the sections below on bioassay,
microbiological, and chemical methods are not comprehensive,
but rather just give examples of each type of analysis.

4. METHODS
Vitamin assays can be classified as follows:
Bioassays
involving humans
and animals.
Microbiological
assays
use of protozoan
organisms, bacteria,
and yeast.
Physicochemical
assays
that include
spectrophotometric,
fluorometric,
chromatographic,
enzymatic,
immunological, and
radiometric methods.

5. Extraction Methods
• Vitamin assays in most instances involve
the extraction of a vitamin from its
biological matrix prior to analysis.
• This generally includes one or several of the
following treatments: heat, acid, alkali,
solvents, and enzymes.

6. Extraction Methods
• In general, extraction procedures are
specific for each vitamin and designed to
stabilize the vitamin.
• In some instances, some procedures are
applicable to the combined extraction of
more than one vitamin, for example, for
thiamine and riboflavin as well as some of
the fat-soluble vitamins

7. Typical extraction procedures are as follows:
• • Ascorbic acid: Cold extraction with metaphosphoric
acid/acetic acid.
• • Vitamin B1 and B2: Boiling or autoclaving in acid
plus enzyme treatment.
• • Niacin: Autoclaving in acid (noncereal products) or
alkali (cereal products).
• • Folate: Enzyme extraction with α-amylase, protease
and γ-glutamyl hydrolase(conjugase)
• • Vitamins A, E, or D: Organic solvent extraction,
saponification, and re-extraction with organic solvents.
For unstable vitamins such as these, antioxidants are
routinely added to inhibit oxidation.

8. • Analysis of fat-soluble vitamins may require
saponification, generally either overnight at room
temperature or by refluxing at 70◦C. In the latter case,
an air-cooled reflux vessel as depicted in Fig. 11-1
provides excellent control of conditions conducive to
oxidation.

9. Microbiological assays
• Microbiological assays for the determination of
vitamins are dependent upon the specific growth
requirements of selected microorganisms, usually
lactic acid bacteria.
• The first MBA for riboflavin was performed by Snell
in 1939 using lactic acid bacteria because their
growth requirements had been studied by dairy
bacteriologists, and as a group their requirements
were complex but specific.
• Lactobacilli have the additional advantage that their
growth can be easily followed by turbidometric or
optical density measurement or by titration of the
lactic acid produced during growth.

10. • Their choice as assay organisms is amply justified as
they are still used today for the determination of B-group
vitamins.
• The basis of a MBA is addition of a dilution series of the
sample extract to a basal medium which contains all the
growth requirements for the test organism except the
vitamin to be determined; the mixture is then inoculated
with the test organism and incubated.
• The test organism will grow in proportion to the vitamin
content of the sample extract and quantitation is
achieved by inclusion of a range of vitamin standards in
the MBA.
• The growth of the test organism is measured as
mentioned above.

11. The basic procedure for a MBA is the same for all the
vitamins and can be broken down into a series of stages as
follows:
• 1. Preparation of media for maintaining stock cultures
of the test organisms.
• 2. Preparation of a basal medium deficient in the
vitamin to be determined.
• 3. Preparation of the inoculum medium and inoculum
culture.
• 4. Extraction of the vitamin from the samples.
• 5. Setting up the assay.

12. • 6. Sterilisation of the assay tubes and media.
• 7. Inoculation of assay tubes with the test organism.
• 8. Incubation (18-24 h).
• 9. Measurement of the growth response of the test
organism and calculation of results.

13. • Stages 1-3 require media which are capable of
supporting the growth of the test organism to
be used and for lactic acid bacteria they must
contain amino acids, vitamins, purine and
pyrimidine bases, fermentable carbohydrate,
mineral salts and buffers.
• These media are available commercially which
eliminates the possible variation in
composition experienced when media are
prepared from basic ingredients in the
laboratory.

14. • Test organisms must be obtained from a
recognised national culture collection and
stock cultures have traditionally been
maintained as agar stabs.

15. • Preparation of the basal assay medium is
conveniently performed by rehydrating
commercially available dried media
according to suppliers instructions.
• The preparation of the inoculum has shown
variation over time, and to some extent is
dependent upon the method to be used at
the end of the incubation period to measure
bacterial growth.

16. • One classic procedure involves taking a
stab-culture from the stock culture into a
sterile lactobacilli broth and incubating
overnight before required.
• The broth is centrifuged, the supernatant
discarded, the bacteria washed several times
with sterile saline and then suspended in
sterile saline and used for inoculation of the
assay tubes.

19. Extraction of the Vitamin from the
Test Material (Stage 4)
• The vitamins are extracted from the food
matrix in a form that can be utilized by the
particular assay organism being used.
• This generally involves autoclaving the
food sample in the presence of acid or, for
acid labile vitamins, digesting the sample
with suitable enzymes.

20. • After precipitating the proteins at their
isoelectric point, the pH of the 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 not altered by the addition of
different amounts of the extract.

21. • The extract is then diluted to bring the
concentration of the vitamin to be assayed within
the range of the standard curve.
• Hopefully, the dilution factor is sufficiently high
to dilute out any interfering substances that would
cause drift and invalidate the assay.
• The minimum dilutions of foods necessary to
avoid the inhibitory effects of food preservatives
and neutralization salts.
• Finally, the extracts are filtered to remove the
precipitated protein and lipoidal material, and to
obtain a clear solution for assay.

23. •Setting up the assay (stage 5) is almost the same for
all vitamins.
0-5 ml of sample extracts and standards are pipetted
into the tubes, the volume made up to 5 ml as
necessary and 5 ml of assay medium added to each
tube.

24. • Inoculation (stage 7) involves adding the same
amount of inoculum to each assay tube then the
whole rack of tubes is placed in a water bath at the
required temperature for incubation.
• After incubation for 18 to 24 h bacterial growth in
standards and sample assay tubes should be visible
and the most convenient way to measure growth is
by the use of a nephelometer or by measurement of
optical density.

25. • It is essential that the measurement of growth
is determined after carefully defined times if
results are to be valid, and these times must be
determined within the laboratory using a
defined assay protocol.
• Growth measurements from standards are
plotted against vitamin concentration and
results for samples obtained by interpolation
from this calibration line.

26. QUANTIFICATION
• At the end of the incubation period, the cells are
uniformly suspended by shaking the tubes, and
time is allowed for the air bubbles to disperse
before measurement.
• The turbidities of all tubes are measured in a
nephelometer using a neutral filter, colorimeter
with a filter in the region of 640 nm or
spectrophotometer at 540-660-nm wavelength.
• The turbidity may be expressed as an
extinction, as a transmittance (in % T), as a
difference 100 - T (in %) or simply as a
galvanometer reading

27. • The arithmetic means of the replicates are calculated,
and the means for the standard solutions are plotted on
semi logarithmic graph paper with the turbidity values
as ordinates (linear scale) and concentrations in ng/ml
as abscissae (logarithmic scale). The calibration curve is
drawn through these points (Figure 7.2).

29. • The vitamin contents of the sample tubes are read
off from the calibration curve and the values for
the original samples are calculated from simple
dilution factors.
• Values for the vitamin content of a given sample
calculated from at least three dilutions should
check within the limits of error of the assay, which
is usually considered to be ±10-15%; that is, they
should not differ by more than 15% from their
common mean.
• If this condition is not fulfilled, the determination
must be repeated.

30. • The reliability of a determination can be assessed by
testing for the presence or absence of drift.
• This simply entails plotting on the calibration curve the
mean turbidity values for the sample dilutions against
the corresponding dilution factors.
• In the example given in Figure 7.2, three dilutions, 1 :
200, 1: 500 and 1 : 1000, give mean percentage
turbidities of 43%, 25% and 14%, respectively.
• Joining the points together gives a check curve which is
roughly parallel to the calibration curve, signifying the
absence of drift.

31. • Drift is manifested by a check curve that deviates
widely from the calibration curve, either increasing or
decreasing with concentration of the sample as shown
in Figure 7.3.
• The occurrence of drift in assay values is evidence for
the presence of interfering materials in the test solution
presented for assay.

33. • The occurrence of drift in assay values is evidence for
the presence of interfering materials in the test solution
presented for assay.
• Snell (1948) recognized three general causes of drift:
(1) a substance chemically unrelated to the vitamin may
stimulate or inhibit the response of the assay organism
to suboptimal levels of the vitamin (e.g. free fatty acids
affect the response of L. casei to riboflavin and L.
plantarum to pantothenic acid);

34. (2) substances chemically related to the vitamin may
replace the vitamin in the nutrition of the test organism,
but the dose-response curve to the vitamin and the
related compound may be completely different (e.g.
polyglutamyl folates with 4-7 glutamate residues cause
positive drift in folate assays using L. casei); and
(3) substances physiologically related to the vitamin may
also replace it for growth (e.g. D-alanine may replace
vitamin B6)'
• If a drift has been established, the determination is
invalid and the assay must be repeated.

35. Thiamine - vitamin B1
• Introduction: Vitamin B1exists in tissue as thiamine, thiamine
monophosphate, thiamine diphosphate (pyrophosphate,
cocarboxylase), thiamine triphosphate and in protein bound
forms.
• The most common way to determine the vitamin B1 content of
food stuffs has been to release and extract thiamine and its
phosphate esters using acid hydrolysis followed by enzymatic
dephosphorylation of the esters and subsequent determination of
thiamine.
• The determination of thiamine has traditionally been achieved
by fluorimetry after oxidation of thiamine to the fluorescent
compound thiochrome, by using microbiological assay (MBA)
and more recently by high performance liquid chromatography
(HPLC).

36. Assay organisms
• Two Lactobacilli have been widely used as
assay organisms for the determination of
thiamin, namely L. fermentum (ATCC No.
9338) and L. viridescens (ATCC No.
12706). Of the two, the latter is preferred as
it is less susceptible to inhibitory or
stimulatory substances

37. • Extraction
• In order to ensure the complete utilization of total
thiamine by L. viridescens, the extraction procedure
involves hot acid digestion, followed by enzymatic
hydrolysis as a means of liberating free thiaminee from
all bound forms.
• The enzyme treatment is omitted for the analysis of
grain products and milk, and for the determination of
the added thiamine hydrochloride in fortified foods.

38. • In a procedure recommended by Pearson
(1967b), a suitable weight of the finely ground
or homogenized material is suspended or
dissolved in at least 15 times its weight of 0.1 N
HCl. The mixture is autoclaved for 15 min at
121°C or steamed in an autoclave for 30 min.
• A lower autoclaving temperature of 108-109 °C
is required for the digestion of grain products,
which contain mostly non phosphorylated
thiamine. Distinctly acid conditions (pH 1.0-
1.5) must be maintained during the digestion.

39. • The mixture is then cooled to room temperature
and adjusted to pH 4.0-4.5 with 2.5 M sodium
acetate solution.
• A 5-ml quantity of a freshly prepared 6%
solution of Takadiastase (or other diastatic
enzyme preparation) in 2.5 M sodium acetate is
added, and the mixture is incubated for 3 h at
45-50 °C or overnight at 37°C.

40. • If overnight incubation is used, the mixture
must be protected from bacterial action with a
layer of toluene.
• After incubation, the extract is steamed in an
autoclave for 20 min to deactivate the enzyme,
cooled, adjusted to pH 6.5-6.6, and diluted to
give an appropriate thiamine concentration for
the assay.
• If the extract is cloudy at this point, it may be
filtered through Whatman No. 1 filter paper. A
reagent blank is taken through the same
procedure.

41. Riboflavin - vitamin B2
• Introduction: Natural riboflavin occurs in foods as
free riboflavin or as the protein bound riboflavin-5'-
phosphate (FMN, flavin mononucleotide) and flavin
adenine dinucleotide (FAD).
• Extraction of these bound forms of the vitamin is
most commonly achieved by hydrolysis with a dilute
mineral acid (e.g. 0.1 M HCl); this stage in the
extraction also hydrolyses most FAD to FMN.
• If we are to determine 'free' riboflavin, the FMN has
to be further hydrolysed enzymatically using a
commercial enzyme preparation such as Takadiastase
or Clarase.

42. • As stated previously, these enzyme preparations
contain phosphates (as 'impurities') in addition to
amylases, therefore, FMN can be dephosphorylated
to yield riboflavin; the amylase has the advantage of
hydrolysing starch which aids sample digestion
when carbohydrate rich foods are being examined.

43. Assay organisms
• The organism traditionally used for determining riboflavin is
Lactobacillus casei subsp. rhamnosus (ATCC No. 7469).
• Lactic acid bacteria cannot utilize FAD, and the growth
response of L. casei, measured turbidimetrically, differs
significantly between riboflavin and FMN .
• As most of the vitamin B2 activity is present in food sources as
FMN after acid extraction, it would be more accurate to use
FMN as the standard in the microbiological assay instead of
riboflavin.
• Kornberg, Langdon and Cheldelin (1948) proposed the use of
Enterococcus faecalis (ATCC No. 10100) which, with a
sensitivity to 0.1 ng riboflavin/ml, is 50 times more sensitive
compared with L. casei.

44. Extraction
• The flavins are released from their intimate association
with proteins by autoclaving the sample at 121°C for
15 min in the presence of 0,1 N HCL.
• For cooked wheaten products, such as bread, the
autoclaving time must be increased to 30 min .
• As a result of the acid digestion, FAD, which cannot
be utilized by lactic acid bacteria, is completely
degraded to FMN and riboflavin, and some of the
FMN is also degraded to riboflavin.
• The autoclaving of food samples with 0.1 N HCl
hydrolyses the starch, but it also liberates sufficient
amounts of free fatty acids to cause an interference in
the L. casei assay. The use of Takadiastase has been
suggested as a means of hydrolysing starch .

45. Niacin
• Introduction: Niacin is the collective name for
nicotinic acid and nicotinamide. Nicotinic acid is
readily converted to the physiologically active form,
nicotinamide which functions as a component of the
coenzymes nicotinamide adenine dinucleotide (NAD)
and nicotinamide adenine dinucleotide phosphate
(NADP).
• Nicotinic acid, NAD and NADP occur in almost all
foodstuffs and provide sources of the vitamin which
are available to man, but the vitamin does occur
bound as nicotinyl esters to polysaccharides, peptides
and glycopeptides which are not metabolised by the
human gut.

46. • If we are to determine 'available' niacin then
care has to be exercised over choice
ofextraction conditions used.
• Acid hydrolysis will release niacin from NAD
and NADP but will not hydrolyse nicotinyl
esters; alkali hydrolysis is required to achieve
this.
• Thus if we wish to determine 'available' niacin,
acid hydrolysis should be used, whereas alkali
hydrolysis will yield 'total' niacin.
• Bound forms of niacin occur mainly in cereal
products.

47. Assay Organisms
• Lactobacillus plantarum ATCCTM 8014 is the test
organism.
• A stock culture needs to be prepared and maintained
by inoculating the freeze dried culture on Bacto
Lactobacilli agar followed by incubation at 37◦C for
24 h prior to sample and standard inoculation.
• A second transfer may be advisable in the case of poor
growth of the inoculum culture.
• In general, growth is measured by turbidity.
• If lactobacilli are employed as the test organism,
acidimetric measurements can be used as well.

49. Folates
• Folate is the general term including folic acid
(pteroylglutamate, PteGln) and poly-γ-glutamyl
conjugates with the biological activity of folic
acid.
• Folates present a diverse array of compounds
that vary by oxidation state of the pteridine ring
structure.

50. • Folates are labile to oxidation, light, thermal
losses, and leaching when foods are processed.
• Because of the presence of multiple forms in
food products and its instability, folate presents a
rather difficult analytical problem.

51. Assay organisms
• Three organisms, Lactobacillus casei subsp.
rhamnosus (ATCC No. 7469), Enterococcus
hirae (ATCC No. 8043) and Pediococcus
acidilactici (ATCC No. 8081), have been
routinely employed in folate assays because
they respond specifically to certain metabolic
forms of folate, and therefore can be used to
distinguish between the different forms present
in the assay material.

53. Vitamin B12
• Vitamin B12 is a member of a group of
compounds known as cobalamins and exists in
foodstuffs mainly in coenzyme forms which are
wholly or partly bound to cellular protein
constituents.
• Good sources of the vitamin are offal meats
followed by muscle meats, dairy produce and
fish.
• VitaminB12 is present in food stuffs at very
low levels and MBAs are the only way to
estimate the B12 content of food satisfactorily.

54. • Assay Organisms
• The choice of test organism for the
determination of vitamin B12 has been a
topic of great debate over the years.
• The two most commonly used are
Ochromonas malhamensis and
Lactobacillus leichmannii. 0. malhamensis
has a greater specificity for cobalamins than
L. leichmannii and is claimed to provide a
truer estimate of B12 biological activity.

55. Extraction
– The extraction procedure employed in the AOAC
microbiological method for determining vitamin
B12 activity in vitamin preparations is also
applicable to foods, having been found satisfactory
by inter laboratory collaborative analysis of a crude
liver paste, condensed fish solubles and a crude
vitamin B12 fermentation product.

56. • The procedure entails homogenizing the sample
with a 0.1 M phosphate-citrate buffer at pH 4.5
containing freshly prepared sodium
metabisulphite (Na2S20S), and then
autoclaving the mixture for 10 min at 121°C.
• The metabisulphite converts the various
cobalamins to the more stable
sulphitocobalamin as soon as they are released
from their association with proteins.

57. • For the determination of vitamin B12 activity in
milk-based infant formula, protein is removed
by filtration after adjustment of the autoclaved
extract to the point of maximum precipitation (c.
pH 4.5).
• Methods in which the sample is heated on a
boiling water bath, rather than autoclaved, may
not completely extract all of the bound vitamin.
• Prior treatment with Takadiastase may be
employed for starchy samples that yield turbid
extracts after filtration or centrifugation.

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