Effect of source of starch on action of starch blockers

Does the percentage inhibition activity of starch inhibitors depend on the type of the starch solution used, determined by measuring the absorbance of the solution at 620 nm after adding amylase and Iodine solutions?

Biology is a branch of science that allows an individual to learn the facts and principles which governs, controls and dictates our daily life activities and provides an empirically scientific explanation of the same. Starting from what food to eat to which lifestyle change can offer more risk to health, this subject can satiate our doubts and clarifications in all of these sectors. As there are diabetic patients in my family, I have been exposed to the biological factors behind this disease for a long time. Off late, the dietician has recommended my father to use starch blocking capsules. Being inquisitive in nature, the interest was to explore are they really useful or not. These capsules are carb blockers or more precisely alpha amylase blockers which can inhibit the action of enzymes that helps in digestion of carbohydrates and thus reduce the amount of glucose that the body absorbs after consumption of carbohydrates. However, the most intriguing part was when the dietician mentioned that the starch blockers does not work equally for all kinds of carbohydrates that one consumes. Often it is recommended to reduce consumption of potato and consume corn instead of that as according to research reports corn works better with starch blockers instead of potatoes. Thus, the intent was to understand if the action of starch blockers depends on the source of the starch used. So, the investigation is based on how the efficiency of percentage inhibition activity of starch blocker depends on the source of starch samples used.

Foods high in starch include: Starchy vegetables; like peas, corn, lima beans and potatoes, Dried beans, lentils and peas; such as pinto beans, kidney beans, black eyed peas and split peas and Grains; like oats, barley and rice. Starchy foods are our main source of carbohydrates and have an important role in a healthy diet. Starchy foods should make up just over a third of the food you eat.

Different types of starches have different structures, varying in size, shape and Amylose : Amylopectin ratios. Different starch granules come in different sizes, starting from 3 microns all the way to 100. Some starches also have mixtures of large and small particle size. This is the case in, for example, wheat starch. The shape of a starch granule can also vary in shape. They can be in symmetrical spheres, asymmetrical spheres, symmetrical disks and asymmetrical disks. Some starch granules also have smooth surfaces, versus faceted surfaces. Amylose : Amylopectin ratios also vary from starch to starch. The length of these amylose molecules also differ.

The length, size and ratios of each starch extract are as following;

Corn: 25% amylose, 75% amylopectin; size ranges from 5 microns to 20 microns, irregularly shaped polyhedron-shaped granules.

Tapioca starch: 15% to 18% amylose, 85 to 82% amylopectin; granules are smooth, irregular spheres, 5 to 25 microns in size.

Wheat starch: 25% amylose, 75% amylopectin;  5 to 15 microns, 36 microns in diameter; smooth, round shape; bimodal, other granules have diameters of only 2 to 3 microns.

Potato: 20% amylose, granules are large with a smooth round oval shape, granules range in size from 15 to 75 microns.

Enzymes are biological catalysts. The way they work can be explained using the lock and key model. They have an active site where they bind with the substrate and allow the reaction to occur. Following this, the product is formed and are released from the site to regenerate the enzyme and make it work again with another molecule of the substrate. The actions of enzyme can be inhibited in two ways – competitive inhibition and allosteric inhibition.

In competitive inhibition, a molecule is added that has an active site exactly identical in shape to the active site of the enzyme and has higher affinity towards the substrate than the enzyme. They bind with the substrate even before the enzyme can bind it and thus blocks the substrate from interacting with the enzyme. In allosteric inhibition, the foreign molecule added as an inhibitor will bind with the enzyme at a location different from the active site known as the allosteric site.

As a result, the shape of the active site changes and thus the enzyme is not able to bind with the substrate anymore. Starch blockers acts as allosteric blockers. They bind with the enzyme amylase and inhibit them from acting on starch to hydrolyse them.

Starch blockers are commercially available allosteric inhibitors. Chemically, they are mainly alpha amylase blockers. They bind with the enzymes alpha amylase at allosteric sites and inhibit them from acting on starch molecules to hydrolyse them. Starch or carbohydrates after consumption are broken down into simple sugar units like glucose using the amylase class of enzymes through the process of acid hydrolysis. This is an example of catabolism as it involves breaking down the long polymeric chain of starch into simpler monomeric units. As the enzymes are blocked, the hydrolysis occurs at a slower rate and thus the intake of glucose into blood stream happens at a controlled rate and is delayed too. This reduces the chances of hikes in blood glucose level which is extremely risky for diabetic patients.

Null: The percentage inhibition activity of the starch blocker is same for all the four different types of starch solution used.

Alternate: The percentage inhibition activity of the starch blocker is not same for all the four different types of starch solution used.

A confounding variable is a variable that can question the accuracy and generalizability of the result of the investigation and is beyond the control of the experimenter. The starch blockers are capsules prescribed by doctors and dieticians in the market. These capsules may contain many certain impurities and the composition or biological assay may not be sufficiently correct as reported. However, assessing the authenticity of the biological assay was not done in this investigation due to the lack of the apparatus and devices and materials needed to execute those biochemical quantitative analysis.

• None of the materials must be consumed.
• Hair must always be tied up.
• Solutions must be prepared under strict supervision of an expert.
• Protective laboratory clothing’s like a lab coat, safety gloves must be used.

Any animals or forbidden chemicals were involved in the investigation.

All the waste chemicals were disposed of into the waste bin and largely diluted before disposal.

• A clean and dry weighing glass was taken and placed on a digital mass balance and the reading was set to 0.00 ± 0.01g.
• Using a spatula, amylase powder was transferred to the watch glass until it reads 1.00 ± 0.01 g.
• The weighted amylase powder was transferred from the watch glass to a 100 cc glass beaker.
• Using a graduated measuring cylinder, distilled water was added to the same beaker till the mark of 100 cc.
• A glass rod was used to stir the solution and dissolve the solid.

The starch extracts were made by adding the same mass of source sample to 100 cc distilled water. 10.00 ± 0.01 g of wheat was weighted using a watch glass and a digital mass balance. The weighted solid was added to a 100 cc glass beaker. A graduated measuring cylinder was used to add 100 cc of distilled water to the same beaker. The beaker was placed on a water bath for 30 minutes at a temperature of 60.00℃. After 30.00 minutes, the beaker was taken away from the water bath and filtered using a filter paper and a funnel. The filtrate was collected in a conical flask and the residue was discarded. For potato, it was first peeled off and cut off into pieces before the extract is made.

Mass of Iodine = molar concentration × Volume × Molar mass

= 0.01 × \(\frac{100}{1000}\) × 126.90 = 0.13 g

Mass of KI = moles of KI  molar mass of KI = 0.001  166.00 = 0.16 g

• A watch glass, a spatula was taken to weigh 0.13 ± 0.01 g of I2 using the digital mass balance.
• A watch glass, a spatula was taken to weigh 0.16 ± 0.01 g of KI using a digital mass balance.
• Both the weighted solids were transferred to a 100 cc glass beaker.
• Using a graduated measuring cylinder, distilled water was added till the mark of 100 cc.
• A glass rod was used to stir the solution and dissolve the solid added.

The starch blocker capsules were used as inhibitors. A mortar and pestle was used to grind the capsules and make fine powder of it. A digital mass balance, a spatula and a watch glass was used to weigh the powder. In all trials, 1.00 ± 0.01 g of the powder was weighed and added to the solution.

• A clean and dry test tube was taken.
• Using a graduated measuring cylinder, 1.00 ± 0.05 cm of 1.00% amylase solution to the test tube.
• A watch glass was taken and placed on a digital mass balance.
• The powdered capsules of starch blocker was transferred to the watch glass until the balance reads 1.00 ± 0.01 g.
• The powdered weighted mass of the starch blocker was added to the same test tube.
• Using a graduated measuring cylinder, 1.00 ± 0.05 cc of buffer 7.00 solution was added.
• The test tube was kept for incubation at 37.00℃ for 45 minutes. A stop-watch was used to monitor the timings.
• Using a graduated measuring cylinder 1.00 ± 0.05 cc of the starch extracts to the same test tube.
• Using a graduated measuring cylinder, 1.50 ± 0.05 cc of 0.01 mol dm^-3 of Iodine solution to the same test tube.
• The spectrophotometer was calibrated using distilled water at 620 nm.
• A clean and dry cuvette was taken and the solution in the test tube was transferred to the cuvette.
• The cuvette was transferred to the spectrophotometer and the optical density of the solution was recorded at 620 nm.
• The same process was repeated for two more times.
• The same steps were executed for other starch extracts – corn starch, potato starch, wheat starch.

Mean absorbance at 620 nm without inhibitor = \(\frac{Trial-1\ +\ Trial-2\ +\ Trial-3}{3}\)

Standard deviation (SD) = \(\frac{\sum(trial\ value-mean\ value)^2}{number\ of\ values}\)

Sample calculation:

For corn,

Mean = \(\frac{1.112\ +\ 1.024\ +\ 1.114}{3}\) = 1.083

Standard deviation (SD) = \(\frac{(1.112-1.083)^2\ +\ (1.024-1.083)^2\ +\ (1.114-1.083)^2}{3}\) = 0.0051

Percentage inhibition activity = \(\frac{absorbance\ at\ 620\ nm\ with\ inhibitor-absorbance\ at\ 620\ nm\ without\ inhibitor}{absorbance\ at\ 620\ nm\ without\ inhibitor}\) × 100

Sample calculation:

For corn,

Percentage inhibition activity

= \(\frac{1.298-1.083}{1.083}\) × 100  = 19.582

Impact of uncertainty:

For corn:

Percentage inhibition activity

= \(\frac{(1.298±0.001)-(1.083±0.001)}{1.083±0.001}\) = \(\frac{(1.298-1.083)±(0.001+0.001)}{1.083±0.001}\) = \(\frac{0.215±0.002}{1.083±0.001}\)

Uncertainty in % inhibition

= \(=\bigg(\frac{±0.002}{0.215}\ +\ \frac{±0.001}{1.083}\bigg)\)× 100 =  ±(0.009 + 0.0009) × 100 = ±1.022

The graph above is a bar graph comparing the percentage inhibition activity of the four different types of starch solution used as a substrate here.

There is no significant difference between the absorbance in the two groups – with inhibitors and without inhibitors. Any significant contradictions between the two groups can be ascertained as an outcome of a systematic limitation of the investigation.

There is a significant difference between the absorbance in the two groups – with inhibitors and without inhibitors. Any significant contradictions between the two groups can be ascertained as an outcome of a systematic limitation of the investigation.

Significance level (α) = 0.05%

Null hypotheses (H_o): The mean of the two groups – with inhibitor and without inhibitor are equal

Alternate hypotheses (H₁): The mean of the two groups- with inhibitor and without inhibitor are not equal.

Significance level = 0.05

Number of groups (a) = 2

Total number of values (N) = 8

Number of values in each group (n) = 4

Degrees of freedom between \(\bigg(df_{between}\bigg)\)= 2-1 = 1

 Degrees of freedom within\(\bigg(df_{within}\bigg)\) = N – a = 8 – 2 = 6

Degrees of freedom total \(\bigg(df_{total}\bigg)\) = N -1 = 8 – 1 = 7

Sum of square between (SS between)

= \(\frac{(1.083+0.703+0.885+0.811)^2+(1.298+0.894+1.420+1.269)^2}{4}\) - \(\frac{(1.083+0.703+0.885+0.811+1.298+0.894+1.420+1.269)^2}{8}\)

= \(\frac{12.12+23.82}{4}\) - \(\frac{69.93}{8}\) = \(\frac{35.94}{4}\)  -8.74 = 8.98 - 8.74 = 0.24

Sum of square within (SS within)

= [(1.083)² + (0.703)² + (0.885)² + (0.811)² + (1.298)² + (0.894)² + (1.420)² + (1.269)²] -  \(\frac{ (1.083+0.703+0.885+0.811)^2+(1.298+0.894+1.420+1.269)^2}{4}\) 

=  9.21- 8.98 = 0.23

Test statistic (F) = \(\frac{\frac{SS_{between}}{df_{between}}}{\frac{SS_{within}}{df_{within}}}\) = \(\frac{\frac{0.24}{1}}{\frac{0.23}{6}}\) = 6.26

Critical value = 5.98

Test statistic is greater than critical value.

Thus, null hypotheses is rejected and alternate hypotheses is accepted.

Figure 10 shows that the percentage inhibition is maximum for wheat and minimum for corn, the values being 60.45 % and 19.85% respectively. This shows that, in presence of inhibitor, it takes maximum time to break the starch molecules in wheat and the least time to break the starch molecules in corn . Using the values displayed in the graph, if the action of the inhibitor is arranged in ascending order, the order is corn, potato, tapioca and starch. This means that if the different types of starch solution used in the investigation is arranged in ascending order of the ability of the inhibitor to delay the hydrolysis of starch, the order would be corn, potato, tapioca and wheat.

Due the iodine test, change in colour was observed. The iodine solution darkened as it went through the ascending order of corn, potato, tapioca and wheat, in conjunction with the data . This indicated a difference in rate of inhibition in differing starch types. As the test statistic (6.26)  is greater than the critical value (5.98), the null hypothesis is rejected. This indicates that there is a significant difference between the two groups-absorbance in presence of inhibitor and in absence of inhibitor. Thus, the activity of inhibitor in delaying the hydrolysis of starch is statistically proven. It clearly means that the inhibitor has the biological activity to block the active sites of starch molecules and inhibit the enzymes from acting on them to thereby delay the rate at which hydrolysis of starch occurs. In ascending order of the ability of the inhibitor to delay the hydrolysis of starch, the order would be corn, potato, tapioca and wheat.

Does the percentage inhibition activity of starch inhibitors depend on the type of the starch solution used, determined by measuring the absorbance of the solution at 620 nm after adding amylase  and Iodine solutions?

Thus, there is a significant difference between the two groups-absorbance in presence of inhibitor and in absence of inhibitor. Thus, the activity of inhibitor in delaying the hydrolysis of starch is statistically proven. It clearly means that the inhibitor has the biological activity to block the active sites of starch molecules and inhibit the enzymes from acting on them to thereby delay the rate at which hydrolysis of starch occurs. The percentage inhibition activity for the different types of substrate are – 19.85, 27.16, 60.45 and 56.47 for corn, potato, wheat and tapioca respectively.

Starch inhibitors or blockers acts as enzyme inhibitors. Starch consumed contains both amylose and amylopectin. Digestion of these starch molecules refers to hydrolysis of the starch molecules. The starch molecules are polymers of monomeric sugar units like glucose. During hydrolysis, the glycosidic linkages are broken and the polymeric carbohydrate chain is broken down into simple monomeric sugar units which can be absorbed by the body. This process is really slow and requires the presence of enzymes like amylase to make it occur at speed. Starch blockers are compounds that binds to the enzyme amylase and deforms their geometry. As a result, the shape of the active site of the enzyme is lost and it is not able to bind with the substrate starch anymore. This stops the starch molecules from getting hydrolysed and thus they are not absorbed by the body.

The percentage inhibition is maximum for wheat and minimum for corn, the values being 60.45 % and 19.85% respectively. This shows that the inhibitor used takes maximum time to break the starch molecules in wheat and the least time to break the starch molecules in corn.

If the different types of starch solution used in the investigation is arranged in ascending order of the ability of the inhibitor to delay the hydrolysis of starch, the order would be corn, potato, tapioca and wheat.

Starch blockers are compounds that binds to the enzyme amylase and deforms their geometry. As a result, the shape of the active site of the enzyme is lost and it is not able to bind with the substrate starch anymore. This stops the starch molecules from getting hydrolysed and thus they are not absorbed by the body.

• The investigation adopts a spectrophotometric method to deduce the difference in the activity of starch blockers or inhibitors. Spectrophotometric method is a highly sensitive and accurate analytical method.
• The investigation has a real life significance as starch blockers are commonly used medicines for people suffering from Diabetes. These patients are often given several diet recommendations like to use tapioca instead of potato as a source of carbohydrate in their daily diet. Thus a comprehensive idea about if there is a difference in the way the starch blockers acts on different types of starch will be relevant and of importance.
• The experiments has been performed using a easy and reproducible method.
• The impacts of uncertainty has been considered and the values of percentage error is less. This shows that the results have low random error.

The capsules available in the market have been used as starch blockers. These capsules contains flavourings and food colours. These chemicals may interfere with the way the actual chemical in the capsule blocks the enzyme amylase. This is a methodological limitation.

Cooking methods are also important parameters to be considered while recommending a proper diet for a patient suffering from diabetes. Potatoes can be consumed in multiple ways – boiling in water, roasted, deep fried, pan fried, smashed and roasted. The carbohydrate content absorbed by the food will vary depending on the way it has been cooked. Change in cooking method changes the temperature at which the food is exposed to and this can alter the biological assay of the compound and the nature of the starch molecule present in it. This can alter the chain length and linkages of the monomers in the starch molecules in it. I would like to investigate how the starch content of potato depends on the cooking method used and the type of potato used – white potato and yellow potato. To estimate the quantity of starch, the same concentrated solution of iodine can be added to all of them and the intensity of the colour formed can be measured in a colorimeter in terms of optical density.

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