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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.

Detection of cracked kernels is based upon the reaction of iodine with starch. Unprotected starch grains located in the cracks are dyed with iodine, thereby producing a vivid shade of blue, making the cracks easily discernible.

The analysis sample (e.g., Congress wort or wort from above the grain bed during lautering) undergoes a chemical reaction with an acetic acid/thiobarbituric acid solution; the resulting product is yellow in color and is measured spectrophotometrically. 

Determination of low-molecular weight nitrogenous compounds in wort and beer (< 2600 Da) after precipitating the high and middle-molecular weight nitrogenous compounds with molybdate

The nitrogen content in the filtrate is determined using Kjeldahl or Dumas.

Precipitation with phosphomolybdic acid provides information regarding the medium molecular weight nitrogenous substances present in samples of wort and beer. Nitrogenous substances with a molecular weight greater than 2600 Da are precipitated [1]. However, a quantitative elimination of all nitrogenous substances of an equivalent molecular weight does not occur, which indicates specificity toward certain nitrogenous substances.

Nitrogenous substances of a medium molecular weight can be calculated as the difference between the nitrogenous substances precipitated with molybdic acid and those precipitated with magnesium sulfate [2, 3].

The reagent phosphomolybdic acid itself is not necessary for the analysis; a solution of sodium molybdate is sufficient. Through the addition of sulfuric acid, enough phosphoric acid is released from the phosphates present in wort and beer to form phosphomolybdic acid.

Zinc in wort is measured using the AAS technique by directly aspirating the diluted sample into an acetylene oxygen flame or through electrothermal atomization; the measurement is made at 213.9 nm.

The hot break material (trub) and any hop particles which may be present in the wort, must first be removed. After the wort has been cooled to 2 °C, it is filtered through a glass fiber filter. The residue remaining on the filter is dried and then weighed.

Cold break material or cold trub refers to all material that settles out in the process of chilling wort after separation of the hot trub or hot break material. Cold trub can be filtered out of the wort and primarily consists of proteins (48–57 %), tannins (11–26 %) and carbohydrates (20–36 %). The amount of cold break material in wort depends on the quality and composition of the raw materials, brewhouse equipment and wort handling. In academic and professional circles, opinions regarding the significance of cold break material for downstream processes and for the quality of the finished beer are strongly divided [1, 2, 5]. Under certain circumstances, the quantity of cold break material in wort may exceed 250 mg/l, especially where accelerated fermentation is practiced. Ultimately, this can detract from the flavor of the finished beer [3]. Breweries, where removal of the cold break material has been practiced successfully, determine the quantity of cold break in their pitching wort at regular intervals, in order to evaluate the efficacy of their separation equipment.