biotin 2
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Experiment 7Biotin Assay
GROUP 4Mary Ann Claudine Avena
Jerome BedañoKeesha Dimaano
Gillian Anthony MamuricJane Carol Pastrana
Introduction
Vitamin H, more commonly known as biotin, is part of the B complex group of vitamins. All B
vitamins help the body to convert food (carbohydrates) into fuel (glucose), which is "burned" to produce
energy. These B vitamins, often referred to as B complex vitamins, also help the body metabolize fats
and protein. B complex vitamins are necessary for healthy skin, hair, eyes, and liver. They also help the
nervous system function properly.
The body needs biotin to metabolize carbohydrates, fats, and amino acids (the building blocks of
protein). Biotin is often recommended for strengthening hair and nails and it's found in many cosmetic
products for hair and skin. It is a water-soluble vitamin, meaning the body does not store it; however,
bacteria in the intestine can make biotin. It is also available in small amounts a number of foods. Biotin is
also important for normal embryonic growth, making it a critical nutrient during pregnancy.
Biotin is found in small quantities in many foods. Bacteria in the large intestine also make biotin.
Unlike some vitamins, biotin is recycled and reused by the body. Daily intake does not need to be high
because only small amounts are lost in urine. Biotin is stable and little is lost when foods are exposed to
heat, light, or air. In dry form biotin is quite durable, but in highly acid and alkaline solutions it loses its
biological activity, especially at high temperatures.
D-biotin is the only naturally occurring isomer, it is often used synonymously with Coenzyme-
R. Biotin acts as a co-enzyme in four different carboxylase enzymes of the body: acetyl-CoA carboxylase,
propionyl-CoA carboxylase, b-methylcrotonyl-CoA carboxylase and pyruvate carboxylate. These enzyme
complexes play a role in the metabolism of lipids, proteins and carbohydrates.
Snell & Wright (1941) were the first to point out that their method for the assay of nicotinic acid
could be applied to the determination of biotin and pantothenic acid. Landy & Dicken (1942) published a
general procedure for the estimation of six vitamins, using Lactobacillus casei as the test organism. The
microbiological determination of biotin (using L. casei) has been investigated from the standpoint of
growth stimulants. It is reported that Lactobacillus plantarum converts excess biotin to two vitamers not
utilizable for growth. More recently, it was observed that L. plantarum can degrade oxybiotin and
desthiobiotin as well as convert biotin to vitamers. In a present study, L. casei, an organism
physiologically similar to L. plantarum, was found to metabolize all three materials to products. the
degradation of desthiobiotin is shown to impart a definite physiological advantage to L. casei.
Often abbreviated to MRS, this type of bacterial growth medium is so-named by its
inventors: de Man, Rogosa and Sharpe. Developed in 1960, this medium was designed to favour the
luxuriant growth of Lactobacilli for lab study. It contains sodium acetate, which suppresses the growth
of many competing bacteria (although some other Lactobacillales, like Leuconostoc and Pediococcus,
may grow). This medium has a clear brown colour. The yeast and meat extracts and peptone provide
sources of carbon, nitrogen and vitamins for general bacterial growth. The yeast extract also contains
vitamins and amino acids specifically required by Lactobacilli. polysorbate 80 is a surfactant which
assists in nutrient uptake by Lactobacilli. Magnesium sulfate and manganese sulfate provide cations
used in metabolism.
Lactic acid is a commercially viable product. It is used in such things as: meat and poultry
preservation, cosmetics, oral and health care products and baked goods. One way to produced lactic
acid is through the fermentation of sugar from the microorganism Lactobacillus. Under optimal
conditions of 37 degrees Celsius and with the sugar glucose present Lactobacillus will convert glucose to
lactic acid with one hundred percent yield. However, glucose is only one of many sugars found in
nature.
MATERIALS
Sterile saline solution 50% and 95% ethyl alcohol
Test tubes Bromthymol blue
0.1/10 N NaOH 6 N H2SO4
METHOD
The standard and sample solutions are
quantitatively measured and dispensed.
The tubes were plugged with cotton and was placed in the autoclave for 5mins
at 120 Degrees Celsius
Each tube was inoculated with 1
drop of the inoculum.
the inoculated assay was incubated at 30
Degrees Celsius
Assays that have been incubated were read on a spectrophotometry at an absorbance of 540nm
Tests incubated for 65-72 hrs were titrated
with 0.1 NaOH just to a definite blue color.
A standard curve values were obtained
by titration or turbidity measurement.
RESULTS
Tube # mL of biotin (0.4µg/mL)
0.1 µg biotin per tube
Distilled water
TITRATIONInitial
volumeFinal
volumeTitrationvolume
% LacticAcid
1 0 0 5 0 0.5 0 0%
2 0 0 5 0.5 0.6 0 0%
3 0.5 0.2 4.5 0.6 0.8 0.2 2.36%
4 1.0 0.4 4 0.8 1.0 0.2 2.36%
5 1.5 0.6 3.5 1.0 1.1 0.1 2.18%
6 2.0 0.8 3 1.1 1.2 0.1 2.18%
7 3.0 1.2 2 1.2 1.6 0.4 2.72%
8 5.0 2.0 0 1.6 1.7 0.1 2.18%
Yeast Extract
9 1.0mL 0.92 4 1.7 13.7 12 23.6%
10 2 0.8441 3 13.7 24.6 10.9 21.62%
11 3 0.9545 2 24.6 37.1 12.5 24.5%
12 5 1.0649 0 37.1 51.2 14.1 27.38%Yeast
extract acid hydrolyzed
13 1.0mL 0.2438 4 51.2 53.4 2.2 5.96%
14 2 0.2093 3 53.4 55.1 1.7 5.06%
15 3 0.1403 2 55.1 55.8 0.7 3.26%16 5 0.1955 0 55.8 57.3 1.5 4.7%
Titration Curve
0 0.5 1 1.5 2 2.50
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
f(x) = 0.0690184049079755 x + 0.092638036809816R² = 0.13077171456248
Standard Curve (Biotin)
Concentration (µg/mL)
Vol.
of N
aoH
Regression Equation: y = 0.069x + 0.092
Test tubes 1-6
Test tubes 7-12
Test tubes 13-16
Lactobacillus casei was inoculated in assay tubes containing pure distilled water (test tubes 1
and 2), increasing volume of biotin standard with an increasing amount of biotin and decreasing volume
of distilled water (test tubes 3,4,5,6 and 7), pure biotin standard with the highest amount of biotin
without distilled water (test tube 8), an increasing volume of yeast extract without biotin and decreasing
volume of distilled water (test tubes 9,10 and 11), pure yeast extract without biotin and without distilled
water (test tube 12) , an increasing volume of yeast extract acid hydrolyzed without biotin and
decreasing volume of distilled water (test tube 13,14 and 15) and pure yeast extract acid hydrolyzed
without biotin and without distilled water (test tube 16). Test tubes were incubated for 72 hours at 30
degrees Celsius, test tubes 1-8 showed a clear solution while test tubes 9-16 showed a yellowish (turbid)
solution after incubation. Test tubes were titrated with 0.1 N NaOH using a bromthymol blue as
chemical indicator. Upon titration with .1 N NaOH (titrant), sample solutions of test tubes 1,2,3,4,5,6,7
and 8 gave a clear light blue solution, while sample solutions of test tubes 9,10,11 and 12 gave a clear
yellow green solution and sample solutions of test tubes 13,14,15 and 16 gave a clear light blue solution.
The titration volume of test tubes 1-8 showed an alternation of increasing and decreasing volume. Test
tubes 9-12 showed an immense increase in titration volume of 12mL to 14.1 mL with a sudden decrease
of 10.9mL at test tube 10. And test tubes 13-16 showed a decrease in titration volume from 2.2mL to
1.5mL with a sudden increase of 1.5mL at test tube 16. The percent lactic acid produced for test tubes 1-
8 gave an alteration of increasing and decreasing values. Test tubes 9-12 gave an increasing amount of
23.6% to 27.38% lactic acid with a sudden decrease of 21.62% at test tube 10. And test tubes 13-16 gave
a decreasing percent lactic acid of 5.96% to 4.7% with a sudden increase of 4.7% at test tube 16.
DISCUSSION
In microbiological assays, the growth of certain microorganism in an extract of a vitamin-containing
sample is compared with the growth of test microorganism in the presence of known amounts of
vitamins. Growth can be measured in terms of acid production (acidimetry). This study employed
Lactobacillus casei, a non-starter lactic acid bacterium (NSLAB) suitable for microbiological assay of
biotin (Vitamin B7 or Vitamin H) which falls under the B complex vitamin group. Additionally,
Lactobacillus casei is known to be as a fastidious bacterium since it requires the presence of most B
vitamins like biotin in particular, in order to sustain its growth, although one study shows that
Lactobacillus casei is viable in biotin-free media but with Dethiobiotin or Diaminobiotin media which are
both precursors of the Biotin compound (Bowman&DeMoll, 1993).
Furthermore, in order to determine how much biotin was utilized by Lactobacillus casei, the
amount/concentration of lactic acid that has been a produced by bacterial biosynthesis of biotin
concentrations from each tube, Acid-Base Titration was employed which makes use of the neutralization
reaction that occurs between acids and bases and the knowledge of how acids and bases will react if
their formulas are known. Basically, biotin functions as a protein-bound coenzyme for bacterial cells like
Lactobacillus casei , assisting primarily in reactions in which enzymes transfer carbon dioxide to
compounds to create carboxylic acids (carboxylation reactions) like lactic acid. Furthermore,
bromthymol blue was used as a chemical indicator giving a yellow color at acidic pH and blue color at
basic pH with a transition range of pH 6.0-7.6. For optimal accuracy, the color difference between the
two species should be as clear as possible, and the narrower the pH range of the color changes the
better.
On the other hand, the tubes were divided into three groups in such a way that each correspond
different concentrations according to its nutrient contents; Biotin, Yeast extract, and Yeast Extract Acid
Hydrolyzed.
Tubes 1 and 2 are purely distilled water and that the inoculated Lactobacillus casei can neither
utilize any biotin in order to sustain its growth nor produce biosynthetic products like lactic acid in
absence of a non-carbohydrate precursor like biotin. Theoretically, the ideal % Lactic acid of these tubes
must be 0% since there are no carboxylation reactions that occurred in the first place in order to
synthesize biotin, a precursor to lactic acid product formation, thus it is not recommended to perform
any titration procedures for these tubes since it is expected that there’s no presence of lactic acid from
each tube. In addition, the clear light blue solution observed, was affected by the NaOH to the distilled
water resulting to a basic pH.
Test tubes 3 to Test tube 8, ideally, the pH must be observed in a way that the pH is dramatically
increasing however the pH gets constant at a point it reaches its maximum as the system leads to
saturation, meaning the amount of biotin (substrate) is too many to be catalyzed than the enzymes
produced by Lactobacillus casei. Moreover, a clear, faint, light blue solution was observed from these
test tubes indicating a basic pH that might be very close to the neutral ph which indicates a weak acidic
pH of the solution first hand; perhaps Lactobacillus casei partially utilized biotin. Possibly, this is brought
by the age of the bacterial cells that are in their lag phase, creating a slow utilization process, in contrast
when bacterial cells are at log phase and stationary phase. Once more, the ideal % Lactic acid yield was
not met just like what is expected theoretically due to imprecise titration procedures, thus the
maximum amount of biotin required in order for the Lactobacillus casei to form lactic acid wasn’t
quantified as it suppose to reach its optimum concentration.
Test tubes 9-12, show a clear, yellow-green solution indicating an acidic pH. Yeast extract is rich
in various B complex vitamins, in particular Vitamin B1, Vitamin B2, Vitamin B3, Vitamin B6, Vitamin B12
and folic acid which enable Lactobacillus casei to have a multisubstrate reaction. Thus, production of
lactic acid and other organic carboxylic acids (e.g. Propionic acid) must be abundant. This is evident
through the yellow-green solution result which means that the system within the solution after
incubation is neutralized as it is being titrated with NaOH, thus the solutions in each tube are already
acidic in the first place, indicating that there was indeed synthesis of carboxylic acids. Furthermore, it is
evident that as the concentration of yeast extract increases and so as the production of lactic acid;
however, the concentration will sooner meet its maximum as the system gets saturated.
Test tubes 13-16 showed a clear light blue solution indicating a basic pH. A yeast extract acid
hydrolyzed implies the loss of B complex vitamins as they are destroyed by the acid. As yeast extract is in
an increasing concentration in a acid hydrolysate, this suggests that the lesser the amount of yeast
extract the more is the possibility that they are destroyed and that Lactobacillus casei has nothing to
synthesized; the greater the amount of yeast extract the more chance of having a trace amount of B
complex vitamins from yeast extract since not all are completely destroyed by acid, thus this must be
evident through the concentration of lactic acid production by Lactobacillus casei.
Once more, several factors affect the obtained results, such as getting inaccurate and imprecise
titration volume of NaOH needed to give the clear blue appearance of each solution, and a basis of color
spectrum of how dark and how light the blueness of each solution affects the way we quantify the
amount of biotin synthesized by Lactobacillus casei in order to produce Lactic acid.
CONCLUSION
REFERENCE
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1197656/pdf/biochemj00910-0050.pdf
http://www.umm.edu/altmed/articles/vitamin-h-000342.htm
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC276355/pdf/jbacter00416-0159.pdf
http://www.healthynutritionguide.info/vitamins.htm#H
http://www.diet.com/g/biotin
http://www.livestrong.com/article/156332-what-is-difference-between-d-biotin-and-biotin/
http://www.nrel.gov/docs/gen/fy01/NN0017.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC277592/pdf/jbacter00433-0143.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC206929/pdf/jbacter00065-0206.pdf
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC276353/pdf/jbacter00416-0147.pdf
Appendix I
Stock solution
1mL = 1:50 dilution
1/50= 0.02mL
1mL- 0.02mL = 0.98mL diluent
Measure out .98mL of 50% EtOh, add 0.02mL d.biotin
Bromthymol blue (chemical indicator)
*1% bromthymol blue in 50% alcohol solution (ethyl alcohol)
=.1 grams of bromthymol blue in 10 ml of alcohol solution
Sterile saline solution
0 .8 __ × 100mL = 0.08 g
100
0 .8 __ × 99mL = 0.792 g
100
Titration Volume
Final volume- Initial volume= Titration volume
Test tube 1- 0
Test tube 2- 0
Test tube 3- .8m1- .6mL = .2mL
Test tube 4- 1.0ml- .8mL = .2mL
Test tube 5- 1.1ml- 1.0mL = .1mL
Test tube 6- 1.2ml- 1.1mL = .1mL
Test tube 7- 1.6ml- 1.2mL= .4mL
Test tube 8- 1.7ml- 1.6mL= .1mL
Test tube 9- 13.7ml- 1.7mL = 12mL
Test tube 10- 24.6ml- 13.7ml = 10.9mL
Test tube 11- 37.1mL- 24.6mL= 12.5mL
Test tube 12- 51.2Ml-37.1mL= 14.1mL
Test tube 13- 53.4mL-51.2mL= 2.2mL
Test tube 14- 55.1mL-53.4mL= 1.7mL
Test tube 15- 55.8mL-55.1mL= .7mL
Test tube 16- 57.3mL-55.8mL= 1.5mL
Concentration of Unknown:
Regression Equation: y = 0.069x + 0.092
Yeast Extract Concentration:
Test tube 9: 0.92µg /mL=0.069 (12mL)+0 .092
Test tube 10: 0.8441µg /mL=0.069 (10.9mL )+0.092
Test tube 11: O ..9545 µg /mL=0.069 (12.5mL )+0.092
Test tube 12: 1.0649µg /mL=0.069 (14.1mL )+0.092
Yeast Extract Acid Hydrolyzed Concentration:
Test tube 13:O .2438µg /mL=0.069 (2.2mL )+0.092
Test tube 14: O .2093 µg/mL=0.06 9 (1.7mL )+0.092
Test tube 15: O .1403µg /mL=0.069 (0.7mL )+0.092
Test tube 16: O .1955 µg/mL=0.06 9 (1.5mL )+0.092
Percent Lactic Acid
%Lactic acid= Normality NaOH × Volume NaOH × milli equivalent of Lactic acid × 100
Volume of Sample
Test tube 1- .1N NaOH x0× 0.090 milli equivalent of Lactic acid × 100= 0
5mL
Test tube 2- . 1N NaOH × 0× 0.090milli equivalent of Lactic acid × 100= 0
5mL
Test tube 3- .1N NaOH × .2mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 2.36%
5mL
Test tube 4- .1N NaOH ×.2mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 2.36%
5mL
Test tube 5- .1N NaOH × .1mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 2.18%
5mL
Test tube 6- .1N NaOH × .1mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 2.18%
5mL
Test tube 7- .1N NaOH × .4mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 2.72%
5mL
Test tube 8- .1N NaOH × .1mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 2.18%
5mL
Test tube 9- .1N NaOH × 12mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 23.6%
5mL
Test tube 10- .1N NaOH × 10.9mL NaOH × 0.090milli equivalent of Lactic acid × 100= 21.62%
5mL
Test tube 11- .1N NaOH × 12.5mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 24.5%
5mL
Test tube 12- .1N NaOH × 14.1mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 27.38%
5mL
Test tube 13-.1N NaOH × 2.2mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 5.96%
5mL
Test tube 14-.1N NaOH × 1.7mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 5.06%
5mL
Test Tube 15- .1N NaOH × .7mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 3.26%
5mL
Test tube 16- .1N NaOH × 1.5mL NaOH × 0.090 milli equivalent of Lactic acid × 100= 4.7%
5mL
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