Lab 3
Lactate Fermentation
Lactate Fermentation Associate Professor K. Loomes
To o follow the production of lactate and the effect of dicholoroacetate using yoghurt as a Aim: T model system. Background :
Yoghurt is a dairy food in which the milk sugar lactose is (partly) converted to lactic acid by pathway. This conversion of sugar into lactic acid is accompanied a homolactic fermentation pathway. by a pH decrease, and when the pH nears the isoelectric point of the main milk protein casein (pH 4.6 to 4.7), the casein micelles precipitate to give the familiar curd. The temperature for yoghurt manufacture is usually about 43ºC, to prevent the growth of potentially pathogenic bacteria which might grow at 37 ºC. In this experiment the yoghurt bacterium Streptococcus salivarius subspecies salivarius subspecies thermophilus thermophilus will will be used as the inoculum. It is a thermophile (growth temperature optimum 39 ºC) and it makes L(+)-lactic acid. The formation of L(+)-lactic L(+)-lactic acid can be measured using L(+)-lactate dehydrogenase. Experimental: You need to read through the whole experiment and make yourself a ow diagram to help you with the experiment. experiment. You You can use the diagram on the next page to help you with this. Reagents
Yoghurt (containing S. salivarius inoculum) salivarius inoculum) Homogenized low fat milk This milk has been pasteurised; then kept at 90 ºC for 30 min (i) to lower the dissolved oxygen level, and (ii) to inactivate antibacterial proteins in the milk (lactoferrin, immunoglubulins, lysozyme, etc); and then cooled to 43ºC . (In industrial yoghurt production, the milk is supplemented with milk non-fat solids – mainly casein and lactose – to increase the thickness of the nal product). Before you start:
Remove 0.1 mL from the inoculated milk and put into a test tube. Add 9.9 mL water. Mix. Place each sample on ice. (This is for your blank for the L(+)Lactic acid assay) Now:
1)
Into In to eac each h of tw two o 50 mL fal falco con n tube tube (on (onee of wh which ich is lab label eled ed,, dich dichlo loro roac aceta etate) te),, add add 6 mL of yoghurt and 24 mL of homogenized low fat milk.
2)
Place Pl ace th thee falco falcon n tube tubess cont contai aini ning ng 30 30 mL of ino inocu culat lated ed milk milk in a wate waterb rbath ath at 43º 43ºC. C.
3)
Incu In cuba bate te bo both th th thee fal falco con n tu tube bess con conta tain inin ing g th thee yo yogh ghur urtt sa samp mples les..
4)
Forr the Fo the samp sample le con contai taini ning ng the the dich dichlo loro roac aceta etate te carr carry y out out the the foll follow owin ing g 2 assa assays ys at at 0 min, 30 min, 60 min, and 90 min. (i) pH and (4 readings) readings) (ii) L (+) lactic acid concentration (4 x 0.1 mL samples)
LABORATORY LABORATOR Y GUIDE
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BIOSCI 203
Lab 3
5)
Lactate Fermentation
For the sample that does not contain dichloroacetate, carry out the following 3 assays at 0 min, 30 min, 60 min, and 90 min. (i) pH
(4 readings)
(ii) soluble protein in solution (4 x 2 mL samples) (iii) L(+) lactic acid concentration (4 x 0.1 mL samples) On the diagram below indicate the type, size and time you will take samples from each tube. Show your demonstrator before you start the experiment. Sample WITH dichloroacetate
Sample WITHOUT dichloroacetate
How to carry out each of these determinations.
(i) pH determination. To determine each pH you will need to pour your sample into a small beaker, take your mea surement and then return your sample to the falcon ask. Failure to do this will mean you will run out of milk solution. LABORATORY GUIDE
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BIOSCI 203
1)
Calibrate your pH meter, using pH 6.0 phosphate buffer.
2)
Wash the electrode with water.
3)
Place the electrode of your pH meter in the inoculated milk, and record the pH. (The pH of fresh milk should be about pH 6.5, and this will decrease as the lactic acid accumulates).
(ii) Soluble protein determination. [Carry out these protein assays frst, and once the Biuret incubation is proceeding start and complete the lactic acid assay. Then come back to reading the results of the Biuret assay]. i)
Prepared each protein sample as follows
1)
Remove a 2 mL sample from the inoculated milk at each time point, and centrifuge at 1000 x g for 3 min. (Any precipitated milk proteins should form a pellet at the bottom).
2)
Pipette a 0.25 mL sample from the top of the centrifuged milk into a test tube.
3)
Add 0.1 mL of 0.2% (v/v) Tween 20 detergent (to disrupt casein micelles). Mix.
4)
Place the sample on ice temporarily.
ii)
Make up two protein standards
1)
Pipette 0.125 mL and 0.25 mL of protein standards (containing 40 mg protein of casein per mL) into test tubes.
2)
Add 0.125 mL water to the former.
3)
Add 0.1 mL Tween 20 to both. Shake.
4)
Place these standards on ice temporarily.
iii)
To all your soluble protein samples (you should have 4 samples and 2 standards)
1)
Add 3.65 mL of Biuret reagent. Mix.
2)
Incubate at room temperature for 30 min (or a longer time if it ts better). While you are waiting start your lactic acid assays.
3)
Read the absorbances at 550 nm against a blank comprising 0.25 mL water, 0.1 mL of Tween 20 and 3.65 mL of Biuret reagent.
4)
Using the protein standards calculate the concentrations of (soluble) protein still present in the centrifuged, inoculated milk. (Fresh milk contains about 34 mg protein per mL.)
(iii) L(+)Lactic acid production assay . [Carry out the lactic acid assays after you have started the Biuret incubations. Then return to reading the Biuret assays after completing the lactic acid assays]. So much lactic acid should be produced in yoghurt, that samples will have to be diluted before assay. Potentially the lactic acid concentration could reach 0.3 M (300 μmol per mL) if all the glucose-component in the lactose molecule were to be converted to L(+)-lactic acid. In practice the acid production stops the fermentation at a much lower concentration. Our assay has a measuring range of 0 - 0.1 μmol lactic acid.
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BIOSCI 203
Assay
1)
Remove 0.1 mL from the inoculated milk at each time point. Add 9.9 mL water. Mix.
2)
Place each sample on ice temporarily.
After you have collected all your samples (there should be 8 samples)
1)
Pipette 0.03 mL of this diluted milk into an eppendorf tube.
2)
Add (i) 0.77 mL of 50 mM sodium glycine buffer, pH 8.5, (ii) 0.1 mL NAD+ solution (1 μmol per mL), and (iii) 0.1 mL of bovine heart L(+)-lactate dehydrogenase (20 units/mL). Mix.
3)
Incubate for at least 15 min at room temperature.
4)
Read the absorbance at 340 nm (in a 1 mL cuvette), using a blank ( you should have prepared this blank at the beginning of the lab) containing 0.03 mL diluted milk, and 0.97 mL sodium glycine buffer, pH 8.5, (but no enzyme).
Given that 0.1 μmol NADH formed in the 1 mL incubation will have an absorbance of 0.622
,
you can calculate the amount of lactic acid present in each of your assays. Multiple this result by 33.33, to calculate the amount of lactic acid in 1 mL of diluted milk. And multiple this result by 100, to calculate the amount of lactic acid in 1 mL of undiluted inoculated milk. (In practice you will only do one multiplication by 3,333. The units will be μmol L(+)lactic acid per mL of undiluted inoculated milk. The value should increase with time of incubation.
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BIOSCI 203
Lab 3
Lactate Fermentation
Recording results.
1.
Record your results in a Table (see overleaf). Attach to your Hand-in Sheet.
2.
Plot 3 separate graphs of each of the three parameters (pH, soluble protein remaining, concentration of L(+) lactic acid. Overlay the pH and lactic acid data for the dichloroacetate sample on the pH and lactic acid graphs respectively (i.e. you will have two data sets on the same graph). Attach to your Hand-in Sheet.
3.
Once you have completed the table and graphs, explain, in no more than 10 lines, what the results show, and comment on any deciencies or any problems encountered.
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BIOSCI 203
BIOSCI 203 Lab 3: Lactate Fermentation
Surname First Name AUID Stream Lab Group No.
Lin Mouhan (William) 6737564 WED (PM) 25
Results Tables
Low Fat Milk + dichloroacetate Time pH
Lactic acid concentration in
(min)
the presence of dichloroacetate (mmol L(+) lactic acid per mL)
0 30 60 90
6.1 5.9 5.7 5.6
383 298 277 260
Low Fat Milk Time pH
Soluble protein
Lactic acid concentration
(min)
(mg protein per mL)
(μmol L(+)lactic acid per mL)
0 30 60 90
6.1 5.8 5.6 5.5
LABORATORY GUIDE
8.38 8.23 8.21 8.07
287 273 300 313
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BIOSCI 203
Lab 3
Lactate Fermentation
BIOSCI 203
Surname First Name AUID
Stream Lab Group No.
Laboratory 3: Lactate Fermentation: Hand-in Sheet 1.
What is the difference between homolactic fermentation and heterolactic fermentation?
2
If another yoghurt-making bacterium, Lactobacillus delbruekii subspecies bulgaricus (which makes D(-) lactic acid, had been used instead of the Streptococcus salivarius subspecies thermophilus, what would have happened in the lactic acid assay using bovine heart lactate dehydrogenase?
3.
Write the equation of the reaction catalyzed by lactate dehydrogenase. What makes it proceed in the direction of NADH formation.
4.
How is the build-up of lactic acid produced by muscle counteracted in animals?
5.
What was the aim of adding dichloroacetate
6.
What is the mechanism of action of dichloroacetate?
7.
How are the actions of dicholoroacetate relevant to the treatment of cancer?
8.
Did the addition of dichloroacetate have any effect on pH? Suggest reasons for your observation.
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