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Sunday, July 22, 2012

Production of Wine from Grapes & Estimation of Percentage of Alcohol, Total Acidity, Volatile Acidity in wine

The making of wine involves the fermentation of soluble sugar of juices of grapes into carbon dioxide and ethanol. After grapes are harvested they are crushed to produce a raw juice or "must", a highly acidic liquid containing 10 - 25% sugar by weight. The mixed yeast flora on the grapes can serve as the inoculum for the fermentation or the true wine yeast, Saccharomyces cervisae. Must from both the red & white wine grape is white & results in white wine. Since the colour of red grapes is in the skin, and red wine are made by fermentation. Following fermentation, new wine must be clarified, aged and stabilized to produce a satisfactory final product. These process require months & for high quality red wine even years.During the first year, many wines undergoes a second spontaneous fermentation, malo-lactic acid fermentation which can be caused by a variety of lactic acid bacteria. This fermentation converts malic acid to lactic acid & CO2. thus converting a dicarboxylic acid to monocarboxylic acid, hereby reducing the acidity of wine.

The different types of the wine are:
  1. Fortified Wine
  2. Sparking Wine
  3. Champagne
  4. Dry Wine

Wine Production : Procedure
  1. Clean the grapes by the removing stems & wash under tap water.
  2. Grapes are then soaked in 5% NaCl solution and then washed with distilled water.
  3. 250 grams of grapes are then weighed and crushed into pulp in a blender along with 25g of sugar and 200ml of water and transferred into a brown bottle.
  4. 4-5 granules of yeast granules are re-suspended in water and added to the grape extract as inoculum.
  5. The bottle is then tightly covered and incubated overnight at room temperature.
  6. The remaining grapes are then crushed along with 50g of sugar & 300ml of water. This is taken in a 200ml bottle fitted with a one-holed cork.
  7. A pinch of potassium meta bi-sulphite is added to this & the inoculum is transferred.
  8. The contents are mixed well & a bent glass tube is inserted into one holed cork.Little water is added to the glass tube to serve as air trap.
  9. The contents are mixed are mixed well & incubated for 7 days.
  10. Presence of air bubble in the glass tube indicative of fermentation.

White Wine Making - Image Source

Estimation of percentage of Alcohol, Total Acidity & Volatile Acidity 

Specific gravity is the density (weight per unit volume) of a substance divided by the density of water. A specific gravity of 1.05 indicates that the substance is 5% heavier than equal volume of water.

Variation with temperature: The densities of water & wort vary with temperature. when using specific gravity, reference temperatures for the wort sample and water must be specified along with the value of specific gravity.

Specific Gravity bottle  - Image Source


  1. The specific gravity bottle is cleaned and dried, its weight (W1) is noted.
  2. The bottle is filled with alcohol and the weight of bottle + alcohol is noted (W2).
  3. Wash and dry the bottle, the fill it with distilled water, weigh of bottle + distilled water is noted (W3)
Specific Gravity of Alcohol =  ( (W2 - W1) / (W3 - W1 ) ) * Density of Water

W2 - W1 -  GIves the weight of alcohol
W3 - W1 - Gives the weight of water

Estimation of Totall Acidity & Volatile Acidity

Acids are very important structural components of wine. if a wine is too low in acid., it tastes flat & dull. if a wine is too high in acid, it tastes too tart and sour. so maintaining proper acid content is important as it has impact on the taste of wine.

Total Acidity

The total acidity (TA) of wine is measured assuming all the acid present in it is tartaric acid. This allows one to determine a value for the total acidity, ie, content. A high TA of 10% most people would find this level of acidity too tart  & Sour for consumption.A low TA result in flat tasting of wine.
The principle acids found in grapes are tartaric acid, potassium hydrogen tartarate, malic acid and potassium hydrogen malate.

Volatile Acidity
Both tartaric and malic acids are non-volatile which means that they don't evaporate or boil off when the wine is heated. This is to be distinguished from volatile acidity (VA) in wine that represents acetic acid. Acetic acid does boil off and high VA is undesirable in wine.A VA of 0.3 - 0.6 % is produced during fermentation and is considered as normal level.

  1. 10ml of alcohol is taken in a conical flask.
  2. 10 ml of distilled water is added to this along with few drops of Phenolphthalein as indicator.
  3. This solution is titrated against 0.1N NaOH taken in burette.
  4. The end point is determined by change in colour from dark green to permanent pink.
  5. The titration is repeated to get concordant values.

Total Acidity Estimation / Calculation 

Total Acidity can be calculated from the following formula:

Total Acidity  = (Volume of Alkali added * Normality of Alkali * 7.5) / Weight of the sample

Volatile Acidity Estimation / Calculation

Volatile Acidity(Volume of Alkali added * Normality of Alkali * 6) / Weight of the sample

Friday, July 13, 2012

Bacterial Endotoxin Test (LAL Test): Principle & Advantages

Bacterial Endotoxin Test (LAL Test)
The Limulus amebocyte lysate (LAL) test is an alternative method to the rabbit pyrogen test focussed on detection of pyrogenic substaces in sterile parenteral drugs. It is an invitro test performed to check the presence endotoxin in sample (eg: Parenteral drugs). LAL (Limulus amoebocyte lysate) test detects the presence of bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane component of Gram negative bacteria. LAL test is used for detecting and quantifying the amount of bacterial endotoxin in the sample. LAL test is FDA approved test for detecting endotoxin content in drugs.

LAL Test Principle

The sample to be analysed for bacterial endotoxin is added to the lysate, which is taken from the haemolymph cells of horse shoe crab(limulus polymhemus) (crab is not killed).
The principle of the LAL test is based on the physiological affect. The blood of horseshoe crabs contains a type of blood cell, called the amebocytes. These cells are important in exhibiting defence mechanism against pathogens. Amebocytes contain granules with a clotting factor known as coagulogen, this clotting factor is released outside the cell when bacterial endotoxin is encountered. The resulting coagulation is thought to contain bacterial infections in the animal's semi-closed circulatory system.

Biochemical studies on the principle of Limulus test indicate that the amebocytes contain several serine protease zymogens, which constitute a coagulation cascade triggered by endotoxins, and that there is a (1,3)-β-D-glucan-mediated coagulation pathway which also results in the formation of gel.The rate of reaction depends upon conc. of endotoxins, pH, temperature and presence of clotting enzyme system and clottable proteins from lysate.

Before the LAL test was developed, Pyrogens were tested on rabbits. Sample to be analyzed will be injected on rabbit and temperature elevation will be monitored.

Advantages of LAL Test

  1. Simple and Easy to perform.
  2. Results obtained in few hours.
  3. Animals (Rabbits) not used in testing.
Paper Publications  / Protocols

List of Commercial Limulus Amebocyte Lysate Quantification Kits

Pierce - LAL Chromogenic Endotoxin Quantitation Kit

Lonza - Limulus Amebocyte Lysate (LAL) QCL-1000TM

Endotoxin detection assay kits - Genscript

E-TOXATE™(Limulus Amebocyte Lysate) test kit - Sigma Aldrich

Biochemical Principles of Limulus test for detecting bacterial endotoxins

Wednesday, July 4, 2012

Immobilized Metal ion Affinity Chromatography (IMAC) Principle & Applications

Immobilized Metal ion Affinity Chromatography (IMAC)- Principle & Applications

Affinity Chromatography Principle

Affinity chromatography is a technique used to separate biochemical molecules (eg: proteins) from a mixture (cell lysate or mixture of proteins) making use of the specific interaction of the molecule, interaction of antigen-antibody, interaction of protein to a specific ligand etc.

immobilized metal affinity chromatography

Affinity chromatography has got number of applications,
  1. Nucleic acid purification.
  2. Protein purification from cell free extracts.
  3. Purification of Proteins from Serum.
Immobilized Metal ion Affinity Chromatography (IMAC)

Immobilized metal ion affinity chromatography (IMAC) is based on the specific interaction of certain amino acids with the metals, especially histidine. Proteins which has affinity towards certain metals can be used for separating out target protein from a mixture of proteins. Nickel or Copper are used for purifying His-Tagged proteins and phosphorylated proteins are separated out using iron or zinc metal ions.

Chelators most widely used for IMAC are Nitrilotriacetic acid (NTA) or Iminodiacetic acid (IDA).

Ni-Affinity Chromatography is one such affinity chromatographic technique which utilizes the interaction of Nickel and Histidine for purifying the Histidine Tagged proteins (6X His Tag).

Polystidine tag is mostly used in E.coli expressed recombinant proteins, His tag will be present at the C or N terminal of the protein which is makes it easier in purification by affinity chromatography step. Mostly 6X His-Tag is used in recombinant proteins.

Nickel, Cobalt and Copper are most widely used metal ions for the purification of His-Tagged proteins.Nickel provides good binding efficiency to His-Tagged proteins but tend to have non-specific binding also. Cobalt provides more specific binding to histidine tags, cobalt based resin is used when protein is required in high purity. copper ions binds His-Tags more strongly but specificity is very low. purity of the separated protein can be checked using SDS-PAGE or Western Blotting.

Ni-Affinity Chromatography Principle and Uses

Ni-Affinity Chromatography uses the ability of Histidine (His) to bind to nickel. Six histadine amino acids (Polyhistidine tag / 6X His Tag) at the end of a protein (either N or C terminus) is known as a 6X His tag.

Nickel is bound to an agarose bead by chelation using nitrilotriacetic acid (NTA), NTA binds Ni2+ ions by four coordination sites.

Nickel ion chromatography
Qiagen Nickel ion chromatography: Image source: Qiagen

The general procedure involves batch binding of the protein, the protein mixture is mixed with the sample which allows the His-Tagged proteins to bind to Nickel of the chromatographic matrix, the slurry is then poured into a chromatographic column.

Lower concentrations of phosphate and imidazole are used to remove low affinity bound proteins, increasing the imidazole concentration results in the elution of all bound proteins from the column depending on their strength of binding.

Affinity chromatographic matrix / resin is commercially available, even prepacked columns are available (HisTrap HP from GE, Ni-NTA Agarose from Qiagen, etc)

Immobilized Metal ion Affinity Chromatography (IMAC) Methods

Sample Preparation:
Cells are harvested and lysed by mechanical or enzymatic methods, suitable lysing conditions are selected and need to be optimized for better results. In HisTrap™ FF from ge, cell lysate can be directly loaded onto the column.

Choice of Buffer:
Binding is done at near neutral pH.

Elution can be done using increased imidazole concentration. Suitable concentration of imidazole need to be used for optimum results.

Regeneration of Matrix:

The beads can be regenerated with 10 column volumes of the following:
  1. MES Buffer wash at pH 5.0,
  2. Wash with water,or20% EtOH.
Long term storage of Matrix / Resin can be done in 20% Ethanol (EtOH).

Thermo Scientific Technical Resources.
Ni-NTA Purification System, Life Technologies.
Bio-Techniques, International Journal of Life Science Methods.
Nickel Affinity Chromatography Protocol/Guide, DragonTech.