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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:

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

The amount of NADP + H⁺ formed during the reaction is equivalent to the amount of glucose. NADP + 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):

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

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⁺/NADP + H⁺:

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

Determine the SG_A20/20 obtained from pycnometry or the density measured with a precision hydrometer or another device for measuring the density. Using the value from the SG_A20/20 measurement or the density from the sugar, alcohol, original gravity and correction table according to GOLDINER/KLEMANN, BLOCK, KÄMPF or a polynomial, determine the apparent extract content of the sample.

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 D-glucose content is determined before and after enzymatic hydrolysis of sucrose. D-fructose is measured following D-glucose determination.

D-glucose/D-fructose determination before inversion:

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

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

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

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:

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

The amount of NADP + H⁺ formed during the reaction is equivalent to the amount of glucose. NADP + H⁺ is 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):

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

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 absorbance at 340 nm.

Enzymatic inversion:

Sucrose is hydrolyzed to glucose and fructose by the enzyme β-fructosidase (invertase) at pH 4.6:

\(\text{Saccharose + H}{_2}\text{O} \space ^{\underrightarrow{\text{β-Fructosidase}}} \space \text{Glucose + Fructose}\)

The D-glucose determination after inversion (total D-glucose) is carried out as described above.

The sucrose content is calculated from the difference between the glucose concentration before and after enzymatic inversion.

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.

Intermediate products such as fruit juice concentrates or sugar syrups indicate a high level of intrinsic microbiological protection due to their usually very low water activity (low A_w value) and high osmotic pressure (high Brix value). This means that predominantly osmophilic and osmotolerant bacteria are to be expected. Only a few can have a harmful effect on the product after re-dilution.