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UNIVERSITY OF KWAZULU-NATAL (WESTVILLE CAMPUS)

DISCIPLINE OF BIOCHEMISTRY

BIMI200: BIOCHEMISTRY AND MICROBIOLOGY FOR OPTOMETRY
PRACTICAL 1: STARCH ISOLATION & HYDROLYSIS

INTRODUCTION TO STARCH ISOLATION AND HYDROLYSIS

Background

  • The most important storage polysaccharides in nature are:
    • Starch in plant cells
    • Glycogen in animal cells

Composition of Starch

  • Starch consists of two types of glucose polymers:
    • Amylose:
    • Composition: Long unbranched chains of D-glucose units
    • Linkages: Connected by α(1-4) linkages
    • Amylopectin:
    • Composition: Branched chains of D-glucose
    • Linkages:
      • Internal bonds: α(1-4) linkages
      • Branch points: α(1-6) linkages occurring every 24 to 30 residues
  • Glycogen:
    • Structure: Similar to amylopectin but has more frequent branching (every 8 to 12 residues)

Enzymatic Hydrolysis

  • Saliva contains α-amylase, an endosaccharidase that breaks down starch and glycogen.
  • Hydration of Polysaccharides:
    • Polysaccharides can be heavily hydrated due to exposed hydroxyl groups, facilitating adsorption of water.
    • Heating is critical for efficient digestion.
  • Hydrolysis Reaction:
    • Enzyme: Salivary and pancreatic α-amylase
    • Substrates: Starch and glycogen
    • Products:
    • Disaccharide maltose
    • Trisaccharide maltotriose
    • α-limit dextrins (average of 8 glucose units with α(1-6) bonds)
  • Limitations:
    • α-amylase cannot hydrolyse the α(1-6) linkages at branch points.
  • β-Amylase:
    • Found in plants, acts on starch to remove maltose units stepwise from the non-reducing end.
    • It produces β-maltose and β-limit dextrins and is also unable to act on α(1-6) bonds.

Test Principle

  • Iodine Interaction:
    • In neutral or acidic solution, iodine forms complexes with amylose, amylopectin, glycogen, and dextrins.
    • Color Reactions with Iodine:
    • Starch: Intense blue due to the formation of the starch-iodine complex; occurs as amylose winds into a left-handed helix (6 glucosyl residues per turn).
    • Amylopectin: Yields less intense colors due to interruptions in helical structure.
    • Glycogen: Produces pale reddish-brown complexes because of extensive branching.
      • Note: NaCl enhances this color reaction by fulfilling ionic requirements.
    • Dextrin: Yields a pale color similar to glycogen.
    • Maltose and Glucose: No complex formation with iodine, retain normal iodine color.

Factors Affecting Reaction Conditions

  • Excess Heating:
    • High temperatures can denature α-helical coils in amylose, displacing iodine and resulting in pale-to-colorless solutions.
  • pH Conditions:
    • Acidic or alkaline conditions will prevent iodine-starch interaction.
    • Example: Adding H₂SO₄ (acidification) or NaOH (alkalinization)
  • Addition of Sodium Thiosulfate:
    • Will lead to the disappearance of the blue-black color due to the following reaction:
    • I_2 + 2S_2O_3^{2-}
      ightarrow 2I^- + S_4O_6^{2-}

Caution

  • Iodine: Harmful if inhaled; avoid contact with skin and eyes.
  • NaOH: Corrosive; avoid contact with skin.

EXPERIMENTAL PROCEDURE

PART 1: Isolation of Starch from Potato
  1. Collect approximately 20 g of freshly ground potato and strain through cheese cloth into a 400 ml beaker.
  2. Add 50 ml of water to the pulp on the cheese cloth and collect the filtrate in the beaker.
  3. Add another 50 ml of water to the beaker and allow the starch granules to settle.
  4. Decant the supernatant fluid and repeat the washing process.
  5. Suspend the starch in a total volume of about 25 ml of water.
  6. Slowly pour this mixture into approximately 25 ml of boiling water with stirring; continue boiling for 3 minutes.
    • If the solution becomes extremely viscous, additional water maybe required for convenient pouring.
  7. This starch solution will be used for subsequent tests (Iodine test and Starch Hydrolysis).
PART 2: Iodine Test
  1. Prepare 3 clean test tubes with the following:
    • Tube 1: 3 ml of water (negative control)
    • Tube 2: 3 ml of previously prepared starch solution (from Part 1)
    • Tube 3: 3 ml of 1% glycogen
  2. Add 4-5 drops of 0.01 M iodine solution dropwise to each tube and observe color changes.
  3. Add a small amount of NaCl (half a spatula) to tube 3 containing glycogen + iodine, noting that NaCl intensifies color due to optimal ionic conditions for α-amylase activity.
  4. Divide the blue-black mixture in tube 2 into three separate tubes for treatment:
    • Tube 2A: Warm gently
    • Tube 2B: Add a few drops of 10% NaOH
    • Tube 2C: Add a few drops of 1% thiosulfate solution

PART 3: Starch Hydrolysis by Salivary Amylase

  • Objectives: Digest or hydrolyse starch using salivary amylase; use iodine and Benedict's tests to track progress.
  1. Measure 30 ml of isolated starch solution into a test tube and incubate in a water bath at 37-40°C.
  2. Collect a few ml of saliva in a clean test tube and also incubate at 37-40°C.
  3. Mix the saliva with the starch solution and start timing.
  4. At 2-minute intervals, transfer a few drops from the starch-saliva mixture to a well with iodine solution.
    • A negative (yellow) iodine test indicates complete starch breakdown.
  5. To confirm complete digestion, perform the Benedict's test by:
    • Transferring 1 ml of the starch-saliva solution to a test tube with Benedict's reagent.
    • Heating the test tube for several minutes.
    • A red precipitate indicates glucose or maltose presence.
  6. Use provided videos to understand the hydrolysis process and the effects of temperature and pH on digestion.