Search: amino nitrogen

Medium and high molecular weight proteins are precipitated by phosphomolybdic acid. The nitrogen is determined in the filtrate. Therefore, the results express the amount of low molecular weight proteins.

Low molecular weight nitrogenous substances, especially amino acids in the wort, have an influence on the course of fermentation and the formation of fermentation by-products. For a beer’s aroma profile, the concentration and composition of the amino acids are therefore of considerable significance due to their reactivity with reducing sugars (Maillard reaction), especially during kilning in the malthouse and during mashing and boiling in the brewhouse. The products from these reactions influence the redox potential, the color and the aroma of a beer.

Aside from the quantitative determination of the individual amino acids (methods using an ion exchanger, HPLC, GC), cumulative methods of determination are customary. However, these methods also measure NH₄⁺ ions and amines to some degree.

With methods involving color reactions, the amino acids display color at different levels of intensity. Therefore, the reaction is based upon a “standard amino acid”; glycine usually serves as the standard amino acid for comparison.

With the ninhydrin method, the color yield varies with the individual amino acids between 70 and 105 %, based on glycine. Up to approx. 30 % of ammonium salts are quantified using this method and up to approx. 7 % of proline. 

The ninhydrin reaction is the most well-known of the color reactions employed for use with amino acids. Ninhydrin is an oxidant and brings about the oxidative decarboxylation of amino acids, producing CO₂, NH₃ and the formation of an aldehyde. The aldehyde produced in this reaction possesses one less carbon atom than the original amino acid that served as the reactant. Reduced ninhydrin then reacts with non-reduced ninhydrin and the NH₃ that was liberated to generate a blue pigment (all amino acids except for proline) or in the case of proline, a yellow pigment. Fructose also takes part in the color reaction as a reducing compound. Potassium iodide present in the solution used for dilution preserves the ninhydrin in an oxidized state and thus inhibits undesirable secondary reactions. The solution to be analyzed is heated together with ninhydrin at a pH of 6.7 and the resulting color is measured at 570 nm. 

This method quantifies the amino acids, ammonia and also the terminal α-amino groups of the peptides and proteins. Proline is also measured in part at the wavelength employed in this method.

The method is non-specific for α-amino nitrogen, because γ-amino butyric acid found in wort also generates a color reaction in the presence of ninhydrin.

The determination of nitrogen content according to Kjeldahl is divided into the following steps:

a. Digestion of the sample (oxidation of substances H₂O, CO₂, NH₃)

b. Distillation (distillation of NH₃ over into a boric acid solution)

c. Titration (determination of the amount of NH₃ present in the receiver after distillation)

a. Digestion → 2 NH₃ + H₂SO₄ → (NH₄)₂SO₄

b. (NH₄)₂SO₄ + 2 NaOH → Na₂SO₄ + 2 NH₃ + 2 H₂O
3 NH₃ + H₃BO₃ → (NH₄)₃BO₃

c. 2 (NH₄)₃BO₃ + 3 H₂SO₄ → 3 (NH₄)₂SO₄ + 2 H₃BO₃

Wort is produced according to the Congress mash method (fine grind), and then the nitrogen content of the wort is determined.

The amount of soluble nitrogenous substances in Congress wort is determined using spectrophotometry at wavelengths of 215 and 225 nm.