Determination of Water-soluble Vitamin by Microbiological Assay
BY MICROBIOLOGICAL ASSAY METHOD General information of microbiological assay is presented in section
“Determination of Water-soluble Vitamin by Microbiological Assay”
1. PURPOSE/SCOPE
A method is described for the determination of niacin (nicotinic acid) in foods by microbiological assay. Results are expressed as milligram per 100g of food.
This method can apply for all foods. In general, the vitamin is present in foods of animal origin.
2. SAFETY -
3. REFERENCES
1. AOAC (2000) Microbiological Methods, 17th Ed., Ch 50.1.19. pp. 21 2. Difco Manual of Dehydrated Culture Media and Reagent for
Microbiology (1984) Media for the Microbiological Assay of Vitamins and Amino Acids, 10th Ed., Difco Laboratories, Detroit, Michigan, USA, pp1055-1114.
4. DEFINITION -
5. PRINCIPLE
See section 2 in “Determination of Water-soluble Vitamin by Microbiological Assay”
6. REAGENTS
6.1 Culture media
6.1.1 Micro Assay Culture Agar (Difco Cat. No. 0319-01-5) 6.1.2 Micro Inoculum Broth (Difco Cat. No. 0320-02) 6.1.3 Niacin Assay Medium (Difco Cat. No. 2003-03-31) 6.2 Assay medium
See section 8.2 in “Determination of Water-soluble Vitamin by Microbiological Assay”.
6.3 Stock culture and test organism
6.3.1 Stock culture of Lactobacillus plantarum ATCC 8014
- Stab culture in 3 tubes of 10 ml micro assay culture agar (6.1.1).
- Incubate at 35-37oC for 16 – 24 hours.
- Store the tubes at 4oC and subculture every two weeks in triplicate.
6.3.2 Inoculum
- Subculture Lactobacillus plantarum from a stock culture to a tube containing 5 ml of micro inoculum broth (6.1.2).
- Incubate at 35-37oC for 16 - 24 hours under aseptic condition.
- Centrifuge the culture at 2000 rpm for 10 minutes.
- Discard the supernatant and wash cells three times with 10 ml steriled normal saline solution (NSS).
- Centrifuge the culture at 2000 rpm for 10 minutes.
- Discard the last supernatant and dilute cell to an appropriate inoculum with steriled NSS. (Mc Farland No 1.0)
- Mix thoroughly and use 1 drop to inoculate the vitamin assay tubes.
6.4 Reagents
For general reagents, see section 6.4 in “Determination of Water-soluble Vitamin by Microbiological Assay”.
6.4.1 NaOH, 1 N: Dissolve 40 g sodium hydroxide pellets (Merck Cat.
No. 1.06498.1000) in 1 L deionised water.
6.4.2 H2SO4, 1 N: Dilute 28 ml Sulfulric acid to 1 L with deionesed water.
7. APPARATUS
See section 7 in “Determination of Water-soluble Vitamin by Microbiological Assay”
8. PROCEDURE
8.1 Preparation of sample
See section 8.1 in “Determination of Water-soluble Vitamin by Microbiological Assay”.
8.2 Extraction
8.2.1 Weigh accurately about 1-2 g dry sample (3 significant figures) or pipette 10 ml of liquid sample into a 250 ml Erlenmeyer flask.
8.2.2 For %recovery study, spike the recovery sample (based on the in-house control sample) with 3 ml of 8 μg/ml standard solution.
8.2.3 Add about 100 ml 1 N Sulfuric acid (6.4.2) to all flasks, cover with aluminum foil and mix.
8.2.4 Autoclave at 121-123oC for 30 minutes.
8.2.5 Cool to room temperature.
8.2.6 Adjust pH to pH 4.5 with 1 N NaOH (6.4.1) and quantitatively transfer into a 200 ml volumetric flask.
8.2.7 Dilute to the volume with deionised water.
8.2.8 Filter through filter paper (7.8) and collect the filtrate in a 125 ml Erlenmeyer flask. Adjust pH of a portion of the clear filtrate to pH 6.8 and dilute to concentration of about 20 ng niacin/ml.
8.3 Preparation of standard and sample set
See section 8.3 in “Determination of Water-soluble Vitamin by Microbiological Assay”.
8.3.1 Stock niacin Ι, 100μg/ml. (for standard curves)
Accurately weigh 0.05g nicotinamide (BDH Cat. No. 44068) into a 500 ml volumetric flask. Dissolve and dilute to the volume with 25% ethanol.
8.3.2 Intermediate standard Ι, 1 μg/ml (for standard curves)
Pipette 5 ml of 8.3.1 into a 500 ml volumetric flask. Dilute to the volume with deionised water.
8.3.3 Working standard 40 ng/ml. (for standard curves)
Pipette 4 ml of 8.3.2 into a 100 ml volumetric flask. Dilute to the volume with deionised water
8.3.4 Stock niacin ΙΙ, 100 μg/ml (for %recovery test)
Accurately weigh 0.05 g nicotinamide (BDH Cat. No. 44068) into a 500 ml volumetric flask. Dissolve and dilute to the volume with 25% ethanol.
8.3.5 Intermediate standard ΙΙ, 8 μg/ml (for %recovery test)
Pipette 4 ml of 8.3.4 into a 50 ml volumetric flask. Dilute to the volume with deionised water.
Note : Stock standards kept at 4oC and protected from light in amber glass bottles.
: Prepare fresh stock standards every three months.
8.4 Inoculation
See section 8.5 in “Determination of Water-soluble Vitamin by Microbiological Assay”.
8.5 Growth measurement
Incubate the whole set of tubes at 35-37 oC for 16-18 hours. Check the turbidity regularly after 16 h incubation.
8.6 Preparation of standard growth curve
See section 8.7 in “Determination of Water-soluble Vitamin by Microbiological Assay”.
9. CALCULATION, UNIT OF EXPRESSION AND TEST REPORT 9.1 Vitamin concentration in unknown sample
Vitamin (mg/100g) = Sample conc. (ng/ml) X the appropriate dilution factor X 100 sample wt. (g)
The niacin concentration in the sample is taken from the standard growth curve.
9.2 Recovery Calculation
% Recovery = the recovery sample conc. (ng/ml) - control sample conc. (ng/ml) X 100 added standard conc. (ng/ml)
The concentration of niacin in the recovery sample and control sample are taken from the standard growth curve.
9.3 Report
Results are reported to the nearest 0.1 mg/100g, or 1 significant figure.
10. ACCEPTANCE OF RESULTS
See section 10 in “Determination of Water-soluble Vitamin by Microbiological Assay”.
11. METHOD VALIDATION -
12. APPENDIX
See section 12 in “Determination of Water-soluble Vitamin by Microbiological Assay”.
DETERMINATION OF NIACIN BY COLORIMETRIC METHOD
1. PURPOSE/SCOPEC
This method is described for the determination of niacin (nicotinic acid) in foods by colorimetric method. Results are expressed as milligram per 100g of food.
2. SAFETY
Cyanogen bromide is extremely poisonous. All operations involving this reagent should be carried out in an efficient fume cupboard. Do not breathe any vapour, and if solution comes in contact with skin, wash immediately with water.
3. REFERENCE
3.1 Deutsch MJ (1984). Vitamins and other nutrients. In Official Methods of Analysis of the Association of Official Analytical Chemists. 14th Edition (Williams S, ed.), AOAC, Virginia; pp. 841-842.
3.2 Association of Vitamin Chemists, Inc. (1966). Methods of Vitamin Assay.
3rd Edition, Interscience Publishers, New York;pp. 169-172.
4. DEFINITION
Niacin, and the amide of niacin, niacinamide, is widely distributed in foods.
5. PRINCIPLE
Niacin, and the amide of niacin, niacinamide, are usually bound to other chemical compounds and must therefore be freed by hydrolysis with strong acid or alkali or by enzymatic treatment prior to analysis by chemical methods.
Hence niacin derivatives such as coenzymes and niacinamide are converted into free niacin, which is being estimated by this procedure. To eliminate the interference of biologically inactive materials that may be present, purification procedures have to be carried out and blank determinations are necessary during colour development. Niacin in the extract is reacted with cyanogen bromide to give a pyridinium compound. The latter undergoes rearrangement yielding derivatives that couple with aromatic amines to produce coloured compounds. Under proper conditions the density of the colour produced is proportional to the niacin present and may be measured in a colorimeter.
It is to be noted that niacin and niacinamide are both stable in the dry form and in aqueous solutions and are unaffected by light and pH.
6. REAGENTS
6.1 Dilute ammonium hydroxide solution Dilute 5 ml ammonia to 250 ml with water 6.2 1N sodium hydroxide
6.3 10 N sodium hydroxide