-
- QUESTION
DETERMINATION OF THE GLUCOSE CONTENT OF BANANAS
This is a “fictional” laboratory session. You are provided with a lab schedule, data from the experiment performed and instructions to guide you in writing your lab report. Be sure to read these carefully before you begin.
INTRODUCTION
The sweetness of fruit is due to the presence of a number of sugars. Some sugar in fruit is termed 'invert sugar' as it is derived from the conversion of sucrose to its constituent monosaccharides by the action of the enzyme invertase. Invert sugar is an equimolar mixture of glucose and fructose. In some fruits during the ripening process, starch in the fruit is converted initially to sucrose and then to glucose and fructose.
The concentration of invert sugar can be determined by methods which measure total reducing sugars (both glucose and fructose have reducing properties). However, in this practical only the glucose component will be measured, using a highly specific enzymatic method. The so-called ‘GOD-PAP’ assay (glucose oxidase peroxidase aminophenazone phenol) is based on 2 coupled enzyme reactions with a colorimetric end-point:
glucose oxidase
D-glucose + O2 + H2O H2O2 + gluconate
peroxidase
aminophenazone + phenol + H2O2 a red dye + H2O
[Note that a product of the first reaction (H2O2) acts as a substrate for the second].
Under controlled conditions, the intensity of the colour produced (measured as absorbance in a spectrophotometer) will be proportional to the amount of glucose present.
You will be provided with a standard solution of glucose (concentration 2mg/ml) and you should use this standard glucose to construct a calibration curve. You will then use this calibration curve to convert absorbance to concentration for extracts of 2 bananas, at different stages of ripening.
Preparation of extracts of bananas A and B:
You will be provided with 2 bananas. Banana A (peeled weight 120g) is under-ripe. Banana B (peeled weight 105g) is over-ripe. Weigh out accurately about 2 g of each banana (record the exact weight), place the banana pieces in the blender with 20 ml distilled water and homogenise for a few minutes. You must prepare each banana sample separately in order to make 2 different extracts.
Transfer all of the homogenate to 50ml plastic centrifuge tubes, making sure there is an equal volume in each tube, and centrifuge for 3 min at 3000 rpm. Carefully decant (i.e. pour off) the supernatant through glass wool in a funnel and into the 25 ml volumetric flasks provided. Make up to the mark with distilled water and mix thoroughly. Remember again here that you are preparing each banana extract separately.
Dilution of banana extracts
Because it is not known at this stage whether the concentration of glucose will give an absorbance off the top of the calibration curve, a range of dilutions of the extracts must be prepared before carrying out the GOD-PAP assay.
Label small microcentrifuge tubes (also called “microfuge” or “Eppendorf” tubes) as follows and make up dilutions of the banana extracts as follows:
Dilution: X Y
---- ----
Banana extract (ml): 0.5 0.2
Distilled water (ml): 0.5 0.8
Dilution factor: 2 5
Mix each dilution by vortexing ( ‘Whirlymixing’). Remember to make dilutions for each extract separately. Keep the remaining undiluted banana extracts as you will also assay these.
Assay for glucose by the GOD-PAP method
Set up a series of cuvettes as shown in Table 1 below. The samples marked S0-S5 are the calibration standards and these should be set up in duplicate (ie S0a/S0b, S1a/S1b etc). Use the 2mg/ml glucose standard provided to set up the calibration series.
The unknowns (Banana extract A –Undiluted, 1 in 2 diluted and 1 in 5 diluted and Banana extract B- Undiluted, 1 in 2 diluted and 1 in 5 diluted)) should also be assayed in duplicate (label replicates as a and b).
You should have a total of 24 cuvettes.
‘S0’ represents the ‘zero standard’ and serves as the reagent blank, since it contains only the assay reagent, with water replacing the glucose solution. This will be used to set the spectrophotometer reading to zero before measuring the other solutions.
Table 1: Estimation of glucose by the GOD-PAP method-setting up your cuvettes
Cuvette
S0
a/b
S1
a/b
S2
a/b
S3
a/b
S4
a/b
S5
a/b
Banana A
Undiluted
a/b
Banana A
1 in 2 dilution
a/b
Banana A
1 in 5 dilution
a/b
Banana B
Undiluted
a/b
Banana B
1 in 2 dilution
a/b
Banana B
1 in 5 dilution
a/b
Glucose conc (mg/ml)
0.0
0.25
0.5
1.0
1.5
2.0
?
?
?
?
?
?
Add the following reagents:
2mg/ml Glucose standard (μl)
0
12.5
25
50
75
100
-
-
-
-
-
-
Distilled water (ml)
100
87.5
75
50
25
0
-
-
-
-
-
-
Banana extract (ml)
-
-
-
-
-
-
100
100
100
100
100
100
GOD-PAP reagent
Add 1.0 ml to ALL cuvettes using automatic dispenser
Mix all cuvettes using a cuvette stirrer.
Incubate at room temperature for 25 min.
Read the absorbance at 500 nm using S0 sample as the reagent blank (i.e. set
spectrophotometer to zero using one of the S0 samples). Then just read the absorbance of all other standards and samples.
Look at the Results Tables (Tables 2 and 3) for the exact weights of each extract and the absorbance readings for all standards and samples.
Using the readings for S0 to S5, plot a calibration curve. Call your calibration curve Figure 1. Remember to plot all points and draw the line of best fit. Using the calibration curve, convert the absorbance values into concentration of glucose for the banana extracts. Use only those dilutions (undiluted, 1 in 2 or 1 in 5) which give an absorbance within the range of the calibration curve. Record these glucose concentrations on your results sheet.
Your answer will give you the concentration of glucose in mg/ml in the two banana extracts. However you now need to calculate the following:
- Glucose content per 100g of banana
- Glucose content per whole banana
To do this you must take into account the weight of banana used to make the extract, the volume that the extract was prepared in, any dilution of the banana extracts, plus the weight of the whole banana.
Results Tables:
Table 1: Absorbance values of standards and samples
Glucose Concentration.
(mg/ml)
Absorbance at 500nm
Replicate A
Replicate B
0.0
0.0
0.0
0.25
0.08
0.06
0.5
0.13
0.16
1.0
0.31
0.30
1.5
0.45
0.44
2.0
0.57
0.59
Banana Extract A:
Undiluted
0.40
0.38
1 in 2 dilution
0.19
0.22
1 in 5 dilution
0.07
0.08
Banana Extract B
Undiluted
0.76
0.88
1 in 2 dilution
0.55
0.58
1 in 5 dilution
0.29
0.28
Table 2: Weights of bananas used to make extracts
Banana
Exact weight used (g)
A
2.32
B
2.21
Full Lab Report (CW2 ) GUILDE
The lab report must be submitted by the deadline of 23rd June 2015 .
Your lab report must be written in the standard format and style Harvard.
(Note the guidance below for appropriate content of your lab report, and the weighting of marks for different sections, when writing your report.
Section
Instructions
Marks weighting
Introduction
Title- your own
Background information
- Sugar content of fruit
- GOD-PAP assay
- Spectrophotometry
Aims of experiment
20%
Methods
Skip the methods. You don’t need to write them down on your lab report. After introduction, just go next to results.
N0 marks
Results
Description of results- TEXT
Table of Absorbance data (draw your own version, do not just copy the schedule here)
Calibration curve- Hand drawn or use EXCEL
Tables and Figs must be numbered and must have appropriate labels/titles
Calculation of concentration of glucose per g of banana and per whole banana (for both banana samples)
30%
Discussion
Comment on calibration curve
Is Beer-Lambert law obeyed?
Comments on spectrophotometry as a means of determining concentration
Comments on accuracy of results
Comments on precision of results
Comments on sources of error in the experiment.
Comment on results obtained- glucose content of 2 bananas – explanation of any differences? Further information on sugar contents of fruits (think about sugars other then glucose)
Overall conclusions
35%
Presentation and referencing
Appropriate style (past tense, 3rd person)
Grammar, scientific English, spelling
References (listed correctly and cited in text)
| Subject | Biology | Pages | 6 | Style | APA |
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Answer
Introduction
Fruits play a significant role in boosting of favorable amounts of water content, fiber composition and a slew of minerals and vitamins. Despite the fact that fruits contain only of natural sugars and a very healthy choice in moderations, it is important to maintain the exact sugar content taken in the body and the origin of the glucose. As a result therefore, the best fruit with least amount of glucose content should at all times be considered for human consumption. For instance, per 100 grams of any fruit that is edible, oranges that are juicy contains approximately 9.35 grams of the natural sugars. Apples contain about 10.39 grams of the glucose content and bananas weigh approximately 12.23 grams of the natural sugar as postulated byOyenuga and Fetuga 2005, p. 20).
Generally, fruits are healthy in the human body and contain a lot of nutrients that are basically the good sources of fibers, minerals, vitamins and the phytochemicals. They also play an important role as explained byKader 2001, p.4, in maintaining a net balance in alkaline-yielding diet. Fruits that are domesticated contain a lot of fibers and are sweeter that the wild fruits.The GOD-PAP assay is a method used to determine the glucose concentration after the process of enzymatic oxidation by the enzyme glucose oxidase. The dye produced at the end point is quinoneimine and represents the colorimetric indicator. Quinoneimine is generated from the compound phenol and 4-aminoantipyrine by hydrogen peroxide as a catalyst (Barham& Trinder 2002, p.143).
Glucose + O2 GOD → Gluconic acid + H2O2
2H2O2 + 4-Aminoantipyrine + Phenol POD→ Quinoneimine + 4H2O
The sweetness of a fruit is as a result of the presence of number of sugars. Some sugar content in the fruit is called invert sugar because it is derived from the conversion of sucrose sugar to its constituent monosaccharide catalyzed by the action of the enzyme invertase. Invert sugar is regarded as an equimolar that is a mixture of fructose and glucose. In some fruits, during the process of ripening, starch found in the fruit is converted initially to sucrose and then to fructose and glucose. Inverse sugar concentration can be determined by various methods that measure total reducing sugars that are both fructose and glucose. In this report, glucose component was measured using a high specific enzymatic method called glucose oxidase peroxidase aminophenazone phenol (GOD-PAP) assay.
Glucose oxidase
D-glucose + O2 + H2O → H2O2 + gluconate
Peroxidase
Aminophenazone + phenol + H2O2→ a red dye + H2O
D-glucose + O2 + H2O → H2O2 + gluconate
Peroxidase
Aminophenazone + phenol + H2O2→ a red dye + H2O
The product of the first reaction that is hydrogen peroxide acts as a substrate for the second product. Under controlled conditions, the intensity of the color produced that is measured as absorbance in a spectrophotometer will be proportional to the amount of glucose present (Marriott, Robinson &Karikari 2001, p.1022). Spectrophotometry is a chemical method used in measuring how much a chemical substance can absorb lightthrough measuring light intensity as the beam of light passes through a given sample. The fundamental principle behind the method is that each compound can transmit or absorb light over a given range of wavelength. The measurement can, therefore be used to measurean amount of chemical substance such as the glucose that is known.
Aim
To determine the concentration of glucose in bananas using spectrophotometry
Results
Table 1: Absorbance values of standards and samples from spectrophotometry
|
Glucose Concentration. (mg/ml) |
Absorbance at 500nm |
|
|
Replicate A |
Replicate B |
|
|
0.0 |
0.0 |
0.0 |
|
0.25 |
0.08 |
0.06 |
|
0.5 |
0.13 |
0.16 |
|
1.0 |
0.31 |
0.30 |
|
1.5 |
0.45 |
0.44 |
|
2.0 |
0.57 |
0.59 |
|
Banana Extract A: |
|
|
|
Undiluted |
0.40 |
0.38 |
|
1 in 2 dilution |
0.19 |
0.22 |
|
1 in 5 dilution |
0.07 |
0.08 |
|
Banana Extract B |
|
|
|
Undiluted |
0.76 |
0.88 |
|
1 in 2 dilution |
0.55 |
0.58 |
|
1 in 5 dilution |
0.29 |
0.28 |
Table 2: Weights of bananas used to make extracts
|
Banana |
Exact weight used (g) |
|
A |
2.32 |
|
B |
2.21 |
Graph1: concentration of glucose against absorbance of replicate A standard solution
Graph 2: concentration of glucose against absorbance of replicate B standard solution
Calculations
From graph 1 above, concentration of the glucose against absorbance produced a linear relationship between the two variables. This results into the equation of the line.
Therefore, glucose concentrations in banana A that was under-ripped is given by:
From equation y= 0.296x - 0.0007, (x= absorbance average)
Undiluted; [y = 0.296 (0.39) – 0.0007] = 0.11474 mg/mL
1 in 2 dilution; [y = 0.296 (0.205) -0.0007] =0.05998 mg/mL
1 in 5 dilution; [y = 0.296 (0.75) – 0.0007] = 0.2213 mg/mL
Glucose concentration in banana B that was over-ripped is given by:
From equation y = 0.296x – 0.0007 (x= absorbance average)
Undiluted; [y= 0.296(0.82)-0.0007] = 0.24202 mg/mL
1 in 2 dilution; [y = 0.296(0.565) - 0.0007] =0.16654 mg/mL
1 in 5 dilution; [y = 0.296(0.285) - 0.0007] =0.08366 mg/mL
In banana A (under-ripped), average concentration is 0.13200667mg/mL,
Thus, if 0.1320067mg/mL =2g, therefore, in 100g, concentration = 
= 5.6899 mg/mL. The whole banana, 120g, concentration = (120gX 5.6899mg/mL)/100g = 6.8279 mg/mL.
In banana B (over-ripped), concentration= 0.16407mg/mL.in 100g, =
= 7.42398mg/mL.the whole banana,105g =7.7952mg/mL
Discussion
Sugar content in bananas changes as the fruit undergoes the ripening process with the activation of the hormone ethylene. Under-ripped bananas generally are almost all starch in composition and thus have low sugar content. From the results above, the glucose concentration of under-ripped banana was slightly lower reading than the over-ripped banana content. As the ripenning process continues, the content of starch decreases and content of sucrose appears. As the ripening continues, glucose and fructose appears and increases. However, PAYASI and SANWAL (2010) p.699explains that when ripening intensifies, sucrose content reduces because the starch content adds up. Sugar content in bananas that are over-ripped is thus higher than the under-ripped ones.
The Beer-Lambert Law that states that usually there is a linear relationship existing between the concentration and the absorbance of a given sample, generally applied in linear relationships comparison of the two variables above. The results of the two samples were precise meaning they were too close to another in terms of dilution. In the visible spectrophotometry, transmission or the absorption of glucose concentration was determined by the intensity of the observed dye and measured in absorbance levels. Visible spectrophotometers generally uses a prism as explained by Kocsis, Herman and Eke 2006, p.91 to narrow down certain ranges of wavelength for the particular beam to pass through the sample given.
Banana contains approximately 5.88 grams of glucose content commonly referred to as blood sugar. It is mostly the common carbohydrate. Fructose, a common sugar, is found in many fruits such as the banana. When banana is consumed in the human body, the body breaks it down into components of simple sugar to be used as energy source in normal functioning of body cells. Natural sugar such as fructose is being metabolized by the body than any of other forms of natural sugar but in limited amounts (Cordenunsi & Lajolo 2005, p.348).
Durham (2012) p.5, recommends that people who are overweight and are resistant to insulin should limit intake of high sugar fruits such as bananas, sweet cherries, apples, mangos and pineapples to the point body weight normalizes and health status improves. More vegetables should be included in lieu of fruits that are of high sugar. Dried fruits generally contain excessive amount of sugar. For the people who are insulin resistant the consumption of fructose particularly maybe problematic. This is because the composition of metabolic fructose is made up of fructose and sucrose. Along the gut of digestive system, sucrose is broken down to fragments of glucose and fructose before it finally enters the blood stream. The contribution of sucrose therefore must be considered at all times in the total dietary load of fructose.
Conclusively, ripped banana contains approximately 55 percent of total sugar and the natural sugar found in the fruit is ideal in energy supplement in the human body. Additionally, nutrients and vitamins are supplement of the fruit and they significantly play an important role in maintaining the health of a person. About 105 calories is contained in banana and total sugar roughly counts to 14.43 grams. Sugar is generally a carbohydrate and supplies about 4 calories of the total per gram. It therefore means that about 58 grams of the total calories come from the content of sugar (KAYISU, HOOD & VANSOEST 2001, p.1887).
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15% |