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The sample is membrane-filtered, incubated and analysed.

Titratable acidity represents the sum of the free acids present in a beverage, with the exception of the dissolved carbon dioxide (carbonic acid). In fruit juices and the beverages prepared from them, they usually consist of malic acid, citric acid and tartaric acid.

The titration of the degassed beverage sample (freed from carbonic acid) is carried out potentiometrically using 0.25 mol/l sodium hydroxide solution either to a pH of 7.0 calculated as tartaric acid or to a pH of 8.1 calculated as citric acid.

The representatives of the genus Alicyclobacillus spp. are acidophilic and thermophilic, spore-forming bacteria that can spoil juices, nectars, drinks containing juice, sports drinks, iced tea and even flavoured waters by forming an off-flavour (guaiacol; 2,6-di-bromophenol; 2,6-di-chlorophenol). The beverage itself remains visually flawless. There is no gas formation, discolouration, clarification or sedimentation. Even slight contamination can lead to sensory issues for the product.

The ubiquitous soil inhabitant is usually introduced into the production process of e.g. juices (e.g. NFC) or fruit juice concentrates during the fruit harvest. Other sources of contamination include raw materials such as sugar, stabilisers and binding agents (pectin, starch, etc.) or special additives (cereals, herbs, protein powder, seeds, etc.), usually with pH values that differ from the juice. 

The spores of the bacteria can survive common pasteurisation conditions and then germinate again under favourable conditions (presence of oxygen, warm temperatures, low pH value). To date, only Alicyclobacillus acidoterrestris, A. acidophilus, A. acidocaldarius and A. herbarius have the potential to form off-flavours. Therefore, the sole detection of Alicyclobacilli is not a clear indication of the risk of beverage damage, but must be verified in a second step using a suitable test, e.g. the enzymatic guaiacol detection kit, to determine the risk potential of off-flavour formation.

Internationally, detection in 10 g samples is recommended, see also IFU method MM12 (International Fruit and Vegetable Juice Association) [1].

There are three different methods of analysis:

Quantitative detection from non-filterable samples using the pour-plate method

Quantitative detection from filterable samples using membrane filtration

Qualitative detection (presence/absence test) by means of pre-enrichment (higher sensitivity)

Fruit juices:

The acid spectrum typical of certain types of fruit are used, along with other criteria, as a basis for recognizing unadulterated fruit juices. Tartaric acid, citric acid and L-malic acid are recorded here, which, with a few exceptions, determine the total acidity of the fruit.

Citric acid occurs as the primary acid in citrus juices and other juices. Orange juice usually contains 3–17 g/l citric acid (AIJN).

In citrus juices, an addition of citric acid can be detected via the citric acid/D-isocitric acid ratio, as this lies within relatively narrow limits. In orange juice, values below 130 are found.

D-isocitric acid is partly present in fruit products as a lactone. The lactone must first be saponified prior to enzymatic determination in order to detect the total D-isocitric acid content.

Malt, wort and beer:

Citric acid is an organic acid and is present in malt and wort and is also produced during fermentation.

Citric acid (citrate) is converted to oxaloacetic acid and acetic acid catalyzed by the enzyme citrate lyase (CL):

Citrate oxaloacetic ^{\(^{\underrightarrow{CL}}\)} acid + acetate

In the presence of the enzymes malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), oxaloacetic acid and its decarboxylation product pyruvic acid are reduced to L-malic acid and L-lactic acid, respectively, by reduced nicotinamide adenine dinucleotide (NADH):

Oxaloacetate + NADH + H^{+ \(^{\underrightarrow{L-MDH}}\)} L-malate + NAD⁺

Pyruvate + NADH + H^{+   \(^{\underrightarrow{L-LDH}}\)}L-lactate + NAD⁺

The sum of the quantity of NADH consumed during the reaction is equivalent to the quantity of citric acid. The absorbance is determined photometrically at 334, 340 or 365 nm.

Glucose is phosphorylated by the enzyme hexokinase (HK) and adenosine 5'-triphosphate (ATP) to glucose 6-phosphate (G-6-P).

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

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

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

The amount of NADPH formed during the reaction is equivalent to the amount of glucose. NADPH is determined based upon its absorbance at 340 nm.

Note:

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

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

The analysis of clear beverages, e.g. lemonades, fizzy drinks, as well as samples from intermediate stages such as rinsing water samples, should cover the relevant beverage pathogens, in particular fermentable yeasts, respiratory yeasts, moulds as well as lactic and acetic acid bacteria.

The corresponding procedure is described in the methods:

M-534.60.502 Klare, alkoholfreie Getränke mit pH-Wert < 4,3 – Nachweis von Getränkeschädlingen mittels Gussplattenverfahren

M-534.60.513 Klare, alkoholfreie Getränke mit pH-Wert < 4,3 – Nachweis von Getränkeschädlingen mittels Membranfiltration

Anaerobic incubation is recommended for carbonated beverages in order to increase the selectivity of the analysis for this type of beverage.