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Acetic acid (acetate) is converted to acetyl-CoA in the presence of the enzyme acetyl-CoA synthetase (ACS) by adenosine-5'-triphosphate (ATP) and coenzyme A (CoA).

\(\text{(1) Acetate + ATP + CoA }\space ^{\underrightarrow{\text{ACS}}} \space \space\text{Acetyl-CoA + AMP + Pyrophosphate}\)

Acetyl-CoA reacts with oxaloacetate in the presence of citrate synthase (CS) to form citrate.

\(\text{(2) Acetyl-CoA + Oxalacetate + H}_2\text{O }\space ^{\underrightarrow{\text{CS}}} \space \space\text{Citrate + CoA}\)

The oxaloacetic acid required for reaction (2) is produced from malic acid and nicotinamide adenine dinucleotide (NAD) in the presence of malate dehydrogenase (MDH). In doing so, NAD is reduced to NADH:

\(\text{(3) L-Malate + NAD}^+ \space ^{\underrightarrow{\text{L-MDH}}} \space \text{Oxalacetate + NADH + H}^+\)

The formation of NADH+H⁺ forms the basis of this analysis, which is measured as an increase in the absorbance at 340 nm. Since this concerns a previous indicator reaction, the quantity of NADH+H⁺ is not linearly proportional to the acetic acid concentration.

Glucose and fructose are phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P) and fructose 6-phosphate (F-6-P):

\(\text{Glucose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{G-6-P + ADP}\)

\(\text{Fructose + ATP} \space ^{\underrightarrow{\text{HK}}} \space \text{F-6-P + ADP}\)

In the presence of the enzyme glucose-6-phosphate dehydrogenase (G6P-DH), G-6-P is oxidized from nicotinamide adenine dinucleotide phosphate (NADP⁺) to gluconate-6-phosphate. Reduced nicotinamide adenine dinucleotide phosphate (NADP + H⁺) is formed:

\(\text{G-6-P + NADP}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{Gluconate-6-phosphate + NADPH + H}^+\)

The amount of NADP + H⁺ formed during the reaction is equivalent to the amount of glucose. NADPH + H⁺ is a measurand and is determined based on its absorbance at 340 nm.

After the reaction is complete, F-6-P is converted to G-6-P by phosphoglucose isomerase (PGI):

\(\text{F-6-P} \space ^{\underrightarrow{\text{PGI}}} \space \text{G-6-P}\)

The amount of NADPH + H⁺ formed during the reaction is equivalent to the amount of fructose. NADPH + H⁺ is the parameter being measured and is determined on the basis of its absorption at 340 nm.

G-6-P reacts in turn with NADP⁺ to form gluconate-6-phosphate and NADP + H⁺. The additional amount of NADP + H⁺ formed is equivalent to the amount of fructose and is determined photometrically based on its absorption at 340 nm.

Note:

Alternatively, NAD⁺/NAD + H⁺ can be used instead of NADP⁺/NADPH + H⁺:

\(\text{G-6-P + NAD}^+ \space ^{\underrightarrow{\text{G6P-DH}}} \space \text{Gluconate-6-Phosphate + NAD + H}^+\)

The measuring principle of the micro viscometer is based on the Höppler "Falling Ball Viscometer". A sphere of known dimension rolls through a closed capillary inclined at a defined angle. Inductive sensors detect the ball over a precisely defined distance of approx. 100 mm or 25 mm. The rolling time is measured by the device and used for dynamic viscosity calculation. The temperature is precisely controlled by thermoelectric Peltier elements and checked by a Pt100 temperature sensor. A temperature accuracy of ± 0.02 °C and a repeatability of ± 0.005 °C can be achieved.

Non-alcoholic beverages (NABs) can be divided into three main groups:

Juices and nectars

Soft drinks (e.g. spritzers, fruit juice drinks, lemonades, fizzy drinks, flavoured waters, sports and energy drinks, mixed drinks, etc.)

Waters (mineral water, table water, medicinal water, etc.)

Raw materials for non-alcoholic beverage production (fruit juice concentrates, base materials, flavourings, sugar syrups, etc.) are also listed.

Hot and cold drinks such as coffee, tea and milk are not included here.

With the exception of waters, this regulatory classification is based on their composition: mainly due to their juice content (0-100 %), flavouring (natural, natural-identical, artificial) and various ingredients such as caffeine, vitamins, minerals, etc.

Microbiological sensitivity of non-alcoholic beverages

The microbiological susceptibility of non-alcoholic beverages should be considered separately due to the diversity of the test matrix. It is characterised by the following key selective criteria:

Beverage ingredients:

The microbiological sensitivity of a beverage is expressed via the specific growth and inhibition substances. These include nutrient-rich substances such as carbohydrates, amino acids, minerals and vitamins etc., which provide the basis for microbiological growth. The presence of fruit acids, essential oils although also the lack of nitrogen sources, inhibit growth and thus provide additional inherent protection for non-alcoholic beverages.

pH value:

The pH value of a beverage plays a central role in beverage production and microbiological analysis. The acidification of the beverage is intended to prevent the growth of pathogenic microorganisms. However, there is no absolute limit value. In practice, a pH value of < 4.3 is considered sufficient protection, depending on the nature of the beverage and its ingredients. Vegetable juices, for example, are an exception.

Aerobiosis/anaerobiosis:

The carbonation of beverages creates an anaerobic environment to largely suppress the growth of aerobic microorganisms, for example. Depending on the type of packaging, sufficient CO₂ must be added to compensate for gas losses due to migration during the shelf life. For example, at least 3-4 g/l CO₂ is recommended when using lightweight PET bottles.

In general, the groups of acidophilic and acid-tolerant, aerobic and facultative anaerobic as well as anaerobic microorganisms are identified as potentially harmful to beverages in the NAB industry, provided that the pH value of the products is in the acidic range (pH value < 4.3). For NAB with more critical pH values (> 4.3), this increases the relevance of mesophilic and thermophilic microbes and potentially pathogenic microorganisms.

Osmophilic/osmotolerant microbes must also be taken into consideration. These can occur as spoilers, especially in highly concentrated raw materials for beverage production. These include, for example, fruit juice concentrates, fruit pulp, fruit preparations, base products, and also sugar syrups, etc.

The beverage-spoilage organisms in non-alcoholic still drinks are generally all yeasts, acetic acid bacteria, moulds and alicyclobacilli. Depending on the pH value, other germs such as Bacillus sp. become relevant.

In carbonated non-alcoholic beverages, fermentable and fermenting yeasts are particularly important, but also other microorganisms such as lactic acid bacteria. Depending on the pH value, other germs such as Bacillus sp. or potentially pathogenic germs such as Clostridium sp. can occur.

The listed groups of beverage spoilers can be categorised as primary contaminants from the corresponding raw ingredients or as secondary contaminants.

Due to the complexity and diversity in the NAB sector, the microbiological controls and analyses, both in the production process and in the end products, must be considered in a different way according to each product group. The requirements are always based on the specific selective criteria of the products in question.

Ethanol is oxidized by nicotinamide adenine dinucleotide (NAD) in the presence of the enzyme alcohol dehydrogenase (ADH) to acetaldehyde:

\(\text{Ethanol + NAD}^+ \space ^{\underrightarrow{\text{ADH}}} \space \text{Acetaldehyd + NADH + H}^+\)

The equilibrium of this reaction favors the side with ethanol and NAD. In an alkaline medium, the equilibrium can be shifted to favor the substances on the right side of the equation.

The amount of NADH+H⁺ produced in the reaction is equivalent to the amount of ethanol and is measured photometrically due to its absorption at wavelengths of 340 nm.

Specificity of the determination [1]

The influence of aldehydes and ketones is eliminated by the order of reagent addition during the test. Methanol is not converted due to unfavorable KM values (Michaelis-Menten constant) of the enzymes used.

n-propanol and n-butanol are quantitatively converted under test conditions, higher primary alcohols lead to sample-dependent creep reactions. Secondary, tertiary and aromatic alcohols do not react. Glycerin does not interfere with the test even at higher concentrations.

Acetaldehyde does not interfere below a concentration of 3000 mg/l. Sulphite does not interfere below a concentration of 300 mg/l.

The quantity of soluble dry matter is determined refractometrically. This is related to the percent by weight of sucrose in an aqueous sucrose solution, which under defined conditions possesses the same refraction index as that of the product being analyzed. The amount of soluble dry matter is expressed in g per 100 g of solution. The refraction index is not determined directly for non-alcoholic soft drinks, but rather by means of a scale based on the percentage of sucrose by weight (°Brix). Since the °Brix scale uses sucrose, correction factors for other sugars must be taken from tables for each individual type of sugar. The presence of other substances, such as organic acids, minerals and amino acids, has an effect on the refraction index of a product. Due to the high acidity of citrus juices and citrus juice concentrates, correction factors are also necessary for measuring their °Brix values.