Microbiological assay of vitamins B series

Here in this presentation
It cantains detailed information about microbiological assay of
vitamin B1
Vitamin B2
Vitamin B3

1. PRINCIPLES OF
MICROBIOLOGICAL ASSAY OF
VITAMIN B-SERIES
PRESENTED BY:
K. MOUNIKA
20031S0411

2. CONTENTS:
❑ Introduction
❑ Microbiological assay of vitamin B1
❑ Microbiological assay of vitamin B2
❑ Microbiological assay of vitamin B3

3. INTRODUCTION
❖VITAMINS:
Vitaminsareorganiccompoundsrequiredinthedietinsmall
amountstoperformspecificbiologicalfunctionsfornormal
maintenance  of optimum growth and health of the organism.
Vitamins arerelatively smallmolecules thatfunction most
often as co-enzyme.
Humansmustconsumeatleast12vitaminsintheirdiet,
becausewelacktheabilitytosynthesizethem.They
are classifiedasfat solubleandwater-solublevitamins
which are excreted through urine.

4. ❖MICROBIOLOGICAL  ASSAY OF VITAMINS:
Microbiologicalassayofvitaminsisatypeofbiological
assay performed with the aid of microorganisms.
Many therapeuticagents,whicheither inhibitthegrowth
of microorganismsorare essentialforthegrowthofthem
are standardized by microbial assay.

5. Principle:
Thebasisofthisassayistomeasuretheabilityoftestorganism
tosynthesisthefactorby utilizingthesubstancebeingassayed
under a proper nutritional condition.
Theresponse(thegrowthofthetestorganism)isproportionalto
the dose (amount of factor) added to  medium.
The Indian pharmacopoeia gives two methods:
1.The cylinder plate method or cup plate method
2.The tube assay method or titrimetric method.

7. Vitamin B1:
It is also known as thiamine; it is a water-soluble vitamin.
Thiamine hasspecificcoenzyme,thiaminepyrophosphate(TPP) whichis
mainly associated with carbohydrate metabolism. 
Sources:
Thiamineisfoundinawidevarietyoffoodsatlowconcentrations.Yeast,
yeast extract,legumes,fishandporkarethemosthighly
concentrated sourcesofthiamine.Ingeneral,cerealgrains arethemost
important dietary sources of thiamine.
Of these, whole grains contain more thiamine than refined grains.
Recommended dietary allowance:
▪Adults – 1-1.5mg/day (men- 1.2mg/day and women- 1.1mg/day)
▪Children – 0.7 to 1.0 mg/day

8. Functions of vitamin B1:
Thiamine is important in producing energy from carbohydrates.
Itisnecessary forproperfunctioning ofthenervoussystem,the heart
andthemusculaturesystemofthebody.Itisalsousedforstabilizingthe
appetite.
TPPwasconsideredasspecificneuroactive formof thiamine and
important in ATP production.
Vitamin B1 deficiency symptoms:
The deficiency of vitamin B1 results in a condition called beri-beri.
The early symptoms of thiamine deficiency includes -
■Anorexia, insomnia, Mental depression and Peripheral neuropathy.

9. ASSAY OF VITAMIN B1 (Thiamine)
Prerequisites:
üSTREPTOCOCCUS SALIVARIUS is the test organism used for this
assay
üBasal medium 
üStock solutions
üInoculum
üAssay procedure
§Precautions should be taken while doing the experiment

10. Test organism: 
Thetestorganismusedisastrainofstreptococcussalivarius.Members
ofthisspeciesarecharacterizedbytheirabilitytosynthesizelarge
amounts of a polysaccharide when grown on sucrose or raffinose agar.
Theseorganismscanbeseriallyculturedinamediumcontainingonly
chemicallydefinedconstituents,consistingoffiveBvitaminsandseven
aminoacids.Allstrainstestedrequiredtheadditionofthiaminetothe
medium before growth would occur.
Stock solution: 
Thestockculturesofthetestorganismaremaintainedintheformofstab
culturesinameatinfusionagarwith1%tryptone,0.1%glucose,0.2%
K2HPO4,1.5%agarandexcessCaCO3.Aftergrowth,thestabculturesare
kept in refrigerator. Stock cultures should be transferred after 6 weeks.

11. Standard solution of thiamine:
Weigh10mgofthiamineisdissolvedin100mlof0.1Macetatebuffer,
pH4.5.Thissolutionissterilizedbyautoclavefor15minandstored
intheicebox. Eachmlofthissolutioncontains 0.1µgofthiamine.
It is prepared only on the day of use.
Inoculum preparation:
Itcanbepreparedbytransferringthestockculturefromtheagar
stabdirectlyintothebasalmedium,towhich10millimicrograms
of thiamine per 10ml have been added.
Thenthiscultureisincubatedat37°cfor24hrs(or)untilthe
growth occurs, before being used to inoculate the assay tubes.

12. INGREDIENTS QUANTITY 
Casein hydrolysate 0.5gm
Thiamine-free yeast extract 0.3gm
Glucose 1.0gm
Phosphate buffer 10ml
Salt solution 1ml
Sodium thioglycolate 10mg
Uracil  0.5mg
Nicotinic acid 50µg
Riboflavin  50µg
Calcium pantothenate 50µg
Biotin 0.1µg
Distilled water to 100.0ml
  BASAL MEDIUM STOCK SOLUTION (THIA MINE FREE):

13. Assay procedure:
Take 7steriletesttubesand fillthemwith0,0.5,1.0,1.5,2.0,2.5and
3.0 ml of standard thiamine solution, respectively.
Make up the volume in each test tube up to 5ml using distilled water.
Add5mlofthiaminefreebasalmediumineachofthetesttubes
and sterilize.
Takeanother7steriletesttubesandaddincreasingquantitiesof
dilutedsolutionswith0,0.1,0.2, 0.4,0.7,1.0and2.0milli
micrograms of the test samples.
Make up the volume in each test tube to 5ml using distilled water.
Add 5ml of thiamine free basal medium to each test tube and sterilize.

14. Ø0.5ml of inoculumismixed with 10mlmlofsalinesolution,from
which 1drop is used to inoculate each test tube of the medium.
ØThetubes arethenrotatedinordertoobtaina uniformmixture of
thiamine and inoculum throughout the medium.
ØThetesttubesareincubatedat37°Cfor24hrs,thenthegrowth
response is measured either by turbidimetric or titrimetric method.
Measurement of growth response:
Turbidimetricmeasurementswithaphotoelectriccalorimeterhave
beenfoundtobemoresatisfactoryformeasuringgrowthresponsethan
titratingthedevelopedacid,sincethemaximumquantityofacid
producedisnotsolargeasthatwhichcanbeattainedwithLactobacilli.
Goodresultshavebeenobtained,however,bytitratingwith0.05N
NaOH, with bromothymol blue as an indicator. 

15. Results:
Atypicalcurveisobtainedresultingfromresponsetothiamine.The
thiaminecontentofthetestsubstancemaybedeterminedby
comparingthegrowthresponsetothatproducedbythiamineonthe
standard curve. 

17. Vitamin B2:
Itisalsoknownasriboflavin.Itisaneasilyabsorbedmicronutrientwitha
key role of maintaining health in humans and animals.
Itisthecentral component ofthetwocofactors FAD (Flavinadenine
nucleotide)andFMN (Flavin mononucleotide)andisthereforerequired by
all flavoproteins.
Sources:
Rich sources - Milk, cheese, eggs, meat, liver, kidney, legumes  
Moderate sources - Green leafy vegetables and cereals
✔When these sources are exposed to light destroys riboflavin.
Recommended dietary allowance:
Daily intake of vitamin B2 is recommended for-
▪Men - 1.3mg/day
▪Women - 1.1mg/day

18. Functions of B2:
It is used in the energy production and cell respiration.
Flavoproteinsplaysanimportantroleincarbohydrate,fatandprotein
metabolism.
It is necessary for the maintenance of good vision, skin, nails and hair.
It is used for the formation of antibodies and red blood cells.
Vitamin B2 deficiency symptoms:
▪Blurred vision
▪Reddening of cornea
▪Dermatitis
▪Anaemia
▪Oily skin

19. ASSAY OF VITAMIN B2 (riboflavin)
Prerequisites:
✔LACTOBACILLUS CASEI is the test organism used for this assay
✔Basal medium 
✔Stock solutions
✔Inoculum
✔Assay procedure
▪Precautions should be taken while doing the experiment

20. BASAL MEDIUM STOCK SOLUTION (RIBOFLA VIN FREE):
INGREDIENTS QUANTITY 
Casein, tryptic digest 1000mg
Casein, acid hydrolysate 1000mg
Glycine, 1-cysteine, MgSO4.7H2O Each 40mg
1-aspargine, dl-tryptophane Each 20mg
Adenine, guanine, uracil, MnSO4.4H20 Each 2mg
Dextrose 4000mg
Sodium acetate, anhydrous 1200mg
NaCl 1000mg
KH2PO4, K2HPO4 Each 100mg
Thiamine HCL, P-amino benzoic acid 40µg
Niacin, calcium pantothenate 80µg
Pyridoxine hydrochloride 160µg
Folic acid 2µg
Biotin 0.4µg
Dissolve
in
 100ml
of 
  water

21. Reagents:
 Stock solution A of riboflavin
 A 50mg of B2 is weighed & suspended
in350ml of distilled water containing
0.6ml of GAA
                        (warmed to 80°c until
                         complete dissolution)
           cool the solution 
Transfer to 500ml volumetric flask &
make up with distilled water
          
Stored under toluene in a dark bottle
in the refrigerator   
   Stock solution B of riboflavin
  10ml of stock solution A were
accurately diluted to 100ml with
distilled water 
Store under toluene in a dark
bottle in the refrigerator
Solution can be for a week
containing 10mg/ml

22. Standard solution of riboflavin:
1mlofstocksolutionBofriboflavinwasaccuratelydilutedto100mlwith
Distilled water. This solution containing 0.1µg of riboflavin per ml.
Preparation of inoculum:
Take  10 ml of centrifugal tube
To  this add 5ml of basal medium and 
5ml of vit B2 solution (0.2micro gram/ml) and sterilize
Then add Lactobacillus casaei to it and incubate at 37C for 2hrs
Centrifuge it & decant off the supernatant liquid and 
suspend the bacterial cells in 10ml sterile saline
Centrifuge again and suspend the bacterial cells
in 10 ml sterile solution and inoculum is ready.

23. Assay procedure:
Take 7steriletesttubesand fillthemwith0,0.5,1.0,1.5,2.0,2.5and3.0mlof
standard riboflavin solution, respectively.
Make up the volume in each test tube up to 5ml using distilled water. 
Add 5ml of riboflavin free basal medium in each of the test tubes and sterilize.
Takeanother5steriletesttubesandfillthemwith0.5,1.0,1.5,2.0and2.5ml
of the test samples.
Make up the volume in each test tube up to 5ml using distilled water.
Add 5ml of riboflavin free basal medium in each of the test tubes and sterilize.
Coolallthetesttubestoroomtemperatureandinoculateeachtesttubewith
a drop (0.6ml) of inoculum.
Incubate at 37°C for 72hrs, then the growth response is measured.

24. Interpretation of results for titrimetry:
Determinetheaverageoftitrationvaluesofeachlevelofbothstandard&
test solutions .
Plotagraphconsideringaveragetitrationvalues(inml)of0.1NNaOH
against concentration of standard riboflavin solution. 
A linear graph is obtained .
Byinterpolatingthestandardcurvedeterminetheconcentrationasactivity
per ml of vitamin B2 .
Fromthegraphtheconcentrationoftestsolutionofriboflavinisfoundand
reported.
Titrimetric method:
Afterincubationperiodtitratethecontentsofeachtesttubeseparatelywith
0.1NNaOHusingbromothymolblueasindicatoratPH6.8untilgreencolouris
produced. Record all the titre readings clearly.

25. OVERVIEW
Turbidimetric method:
Afterincubationperiod,thevitaminBcanbeassayedbyUV/visible
spectrophotometerat440nm.Calculatetheconcentrationoftestsolutionusing
calibration curve graph.

27. Vitamin B3:
Itisalsoknownasniacin(or)nicotinicacid.NiacinisaprecursorforNAD+
andNADP+ neededforglycolysis,NADHgetsoxidizedinelectrontransport
chain.
Two types -
▪Niacinamide (nicotinamide) - does not regulate cholesterol
▪Niacin (nicotinic acid) - highly toxic in large doses
Sources:
Dietary sources are milk, eggs, meat, nuts & all protein containing foods.
Recommended dietary allowance:
ØDaily intake of vitamin B3 is recommended for-
§Men - 16mg/day
§Women - 14mg/day
§Children – 15mg/day

28. Functions of vitamin B3:
Vitamin B3 helps in releasing energy from carbohydrates, fats and proteins.
It is very essential for DNA synthesis
It is necessary for healthy skin, nerves and digestive system
Highdosesofniacinmedicationsareusedtopreventdevelopmentof
atherosclerosis and improves circulation.
Vitamin B2 deficiency symptoms:
Deficiency of vitamin B3 may lead to pellagra disease.
The early symptoms of vitamin B3 deficiency lead to-
▪Gastrointestinal disturbance, loss of appetite 
▪Headache, insomnia
▪Nervousness and irritability

29. ASSAY OF VITAMIN B3 (niacin)
Prerequisites:
üLACTOBACILLUSPLANTARUM isthetestorganismusedforthis
assay
üBasal medium 
üStock solutions
üInoculum
üAssay procedure
▪Precautions should be taken while doing the experiment

30. Reagents:
Standard niacin stock solution –1?
50mgofniacinistakenin500mlofvolumetricflaskanddissolvein
alcoholanddiluteitwithalcohol uptothemarkandmix.Storeitina
refrigerator. Each ml of this solution contains 100µg of niacin.
Standard niacin stock solution –2?
100mlof standardniacinstock solution-1wereaccuratelydilutedto
1000mlwithwaterandstoredundertolueneinarefrigerator.Each
ml of this solution contains 10µg of niacin.
Standard niacin solution:
1Mlofstandardniacinstocksolution-2istransferredinto100ml
Volumetricflaskanddilutedto100mlwithdistilledwater.Eachmlof
thissolutioncontains0.1µgofniacin.It ispreparedonlyontheday
of use.

31. BASAL MEDIUM STOCK SOLUTION:

32. Culture medium:
Take basal medium stock solution of 5ml   +  5ml of water containing 1.0µg of
niacin 
      plug the tubes with cotton
          sterilize for 15 mins in an autoclave at 121°C and cool
Preparation of inoculum:
Transfer Lactobacillus plantarum into a sterile test tube containing 10ml of
culture medium
            Incubate this culture for 16 to 24 hrs at 30-37°C
Inoculum is ready

33. Assay procedure:
Take tensteriletesttubes&add0,0.5,1.0,1.5,2.0,2.5,3.0,3.5,4.0,4.5and5
ml of standard niacin solution, respectively.
Toeachtesttubeadd5mlofbasalmediumstocksolutionandmakeupthe
volume to 10ml with distilled water.
Takeanother4steriletesttubes & add1,2,3and4mloftestsolutionof
material to be assayed. 
Toeach tubeadd5mlofbasalmediumstocksolution&adjustvolumeto10ml
with distilled water. 
Aftermixing,plugthetubeswithcottonandsterilizeallthetesttubesinan
autoclave at 121°C  for 15 mins & cool to the room temperature.
Inoculateeachtesttubewithadropofinoculumpreparedandincubate for
16-24 hrs at 30-37°C.  

34. OVERVIEW
Turbidimetric method:
Afterincubationperiod,thevitaminB3canbeassayedby
visible spectrophotometer ataspecific wavelength between540nmand660nm
and then record all the readings.