Experiment 7 Biotin Assay GROUP 4 Mary Ann Claudine Avena Jerome Bedao Keesha Dimaano Gillian Anthony Mamuric

Jane 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 CoenzymeR. 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 Test tubes 0.1/10 N NaOH

50% and 95% ethyl alcohol Bromthymol blue 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.

Tests incubated for 6572 hrs were titrated with 0.1 NaOH just to a definite blue color.

Assays that have been incubated were read on a spectrophotometry at an absorbance of 540nm

the inoculated assay was incubated at 30 Degrees Celsius

A standard curve values were obtained by titration or turbidity measurement.

RESULTS mL of biotin (0.4g/mL) 0 0 0.5 1.0 1.5 2.0 3.0 5.0 0.1 g biotin per tube 0 0 0.2 0.4 0.6 0.8 1.2 2.0 Distilled water 5 5 4.5 4 3.5 3 2 0 TITRATION Final volume 0.5 0.6 0.8 1.0 1.1 1.2 1.6 1.7

Tube # 1 2 3 4 5 6 7 8 Yeast Extract 9 10 11 12 Yeast extract acid hydrolyzed 13 14 15 16

Initial volume 0 0.5 0.6 0.8 1.0 1.1 1.2 1.6

Titration volume 0 0 0.2 0.2 0.1 0.1 0.4 0.1

% Lactic Acid 0% 0% 2.36% 2.36% 2.18% 2.18% 2.72% 2.18%

1.0mL 2 3 5

0.92 0.8441 0.9545 1.0649

4 3 2 0

1.7 13.7 24.6 37.1

13.7 24.6 37.1 51.2

12 10.9 12.5 14.1

23.6% 21.62% 24.5% 27.38%

1.0mL 2 3 5

0.2438 0.2093 0.1403 0.1955

4 3 2 0

51.2 53.4 55.1 55.8

53.4 55.1 55.8 57.3

2.2 1.7 0.7 1.5

5.96% 5.06% 3.26% 4.7%

Titration Curve

Standard Curve (Biotin)

0.45 0.4 0.35 Vol. of NaoH 0.3 0.25 0.2 0.15 0.1 0.05 0 0 0.5 1 1.5 2 2.5 Concentration (g/mL) y = 0.069x + 0.0926 R = 0.1308

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 18 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 theres 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 wasnt 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: ( )

Test tube 10: Test tube 11: Test tube 12:

( ( (

) ) )

Yeast Extract Acid Hydrolyzed Concentration: Test tube 13: Test tube 14: Test tube 15: Test tube 16: ( ( ( ( ) ) ) )

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