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regions" are formed from glycosidically bound D-galactoses, L-arabinoses and D-xyloses, which give them high resistance to pectinolytic enzymes. Hairy regionsaccount for max. 5% of the total galacturic acid content of the pectin molecule. In the latter, the galacturonic acid units are rotated by 120° against each other, so that every fourth takes the same position. On the other hand, the dissolved molecule takes on a helical structure in which 3 galacturonic acid units form 1 complete turn (by virtue of steric factors and above all, stabilizing hydrogen bridge bonds). 

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Art. 5

Rhamnose Sub-bond

As a rule, two neighboring helixes (in the solution) are arranged exactly parallel to one another. But the regular helix structure is interrupted by the rhamnoses, which then leads the linear pectin structure to buckle and thus negatively influences the hydration ability and the gelling properties. The macromolecular dimerization of the pectin chains influenced in this way disturbs the formation of important adhesion zones and favors the immobilization of solvents (usually water). As a result, the rhamnoses responsible for this must be made enzymatically ineffective. This is because in the end: For gel-forming properties, the degree of galacturonic acid molecule esterification of the carboxyl groups is decisive. However, these are partly neutralized with methanol and partly (or completely) neutralized by negative cations (e.g. Na+and Ca++). Pectins are high and/or low methylated. (Highly methylated pectins have a degree of esterification of more than 50%). There are no further subdivisions; only the group of medium methylated pectins, with a degree of esterification that varies between 45 - 65%, should be mentioned briefly. A detailed description of the enzymatic-microbial rhamnose sub-bond is left out due to IP.

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Art. 6

Gel formation according to the degree of esterification

During gel formation, the degree of esterification of the pectin is closely related to the gel formation rate and the texture of the gels themselves. In other words: highly methylated pectins gel faster, and do this at higher temperatures. To form the gel itself, a sugar content of at least 55% by weight and a pH value of 1 to 3½ is required. The acid ensures that the pectins are not negatively charged and repel each other as a result. In the meantime, the sugar binds the water, which would otherwise be absorbed by pectins. In this way, binding between the molecules is prevented. If the ratio of pH-value and acidity is correctly adjusted, a sufficient number of pectin molecules appear, after which the pectin chains can be connected with one another (with the help of corresponding hydrogen bonds). Pectins with a degree of esterification below 50% require the additional use of calcium, but sugar is no longer needed. In this reaction, the pH range (by adding calcium) is between 1 and 7. Pectic acid itself is pectin with a degree of esterification of less than 5%. However, pectic acid is able to form gels under the same conditions (equal to the low methylester pectins). At high pH values (due to the high content of polyvalent cations) the pectic acid even precipitates as pectate (salts of unesterified pectin acids). However, contrary to general assumptions, sugar beet pectic acid (C41H60O36) has theproperty of being prepared enzymatically and microbially in such a way that it is able to form solid gels (like higher methylester pectins). Details are also left out here due to IP.  

 

Art. 7

Pectinolytic enzymes

Enzymes are bio/protein polymers, which are formed by cells and work as so-called "biocatalysts". They consist of one or more subunits composed of amino acids (20 in total). These in turn form a protein-specific sequence and are linked to each other via peptide bonds. Peptide bonds in turn are formed by carboxyl/amino groups of individual amino acids (with elimination of water). If there is a compound of more than 100 amino acids, it is referred to as a protein. If the compound contains between 10 and 100 amino acids, it is considered a polypeptide. In the enzyme, proteins take on a specific structure and are spatially arranged so as to form a three-dimensional structure with a pocket-like depression (the center of action). This specific, 3-dimensional structure is a prerequisite for enzymes being able to form an "enzyme-substrate complex" with very specific substances (referred to as substrates). The dynamic, spatial arrangement in the action center determines the "key-lock principle" of the enzyme. The power of this arrangement hardly requires any activation energy, which leads to an immense increase in the speed of the actual reaction process. Bio/chemical reactions are therefore increased 1010 - to 1020 - fold. The molecular weight of each enzyme is between 10,000 and several million daltons. However, these enzymatically catalyzed reactions are also subject to typical saturation kinetics. Since enzymes (in comparison to chemical catalysts) can only catalyze one or very few reactions at a time, one speaks of reaction-specific catalysts, whereby the specificity of this enzyme catalysis does not concern the substrate as a whole molecule, but only the chemical groups of the substrate. This makes the enzymatic research of LP all the more precise.

 

Art. 8

Enzyme nomenclature 

With regard to their nomenclature, it is also noticeable that for a large number of different enzymes several names are often used at the same time (which is certainly due to the fact that earlier enzymology arbitrarily gave the enzymes names). It was not until much later that enzyme names were created by adding the suffix "-ase" to the substrate converted by the respective enzyme. As a result, starch-splitting enzymes were soon called "amylases" and fat-splitting "lipases". According to their respective functionality, various enzyme groups also received trivial names such as "oxidases", "dehydrogenases", "decarboxylases", some of which have survived to this day. A precise nomenclature is provided by the IUB, which describes the mechanism of the reaction more precisely... Further details are omitted, since the addressee(s) may also be familiar with the complex enzyme classification. According to IUB, each enzyme is assigned a key number, which is identified as an EC number, and its first number reveals the main class of enzymes. There are a total of six main enzyme groups. For example, the pectinesterase has the key number "PE, EC 3.1.1.11", where the 3 stands for the third main group and therefore indicates that this enzyme is a hydrolase. Cofactors or coenzymes in enzymatic reactions are no less important and the non-protein molecules are mainly required by the enzymes of main groups 1, 2, 5 and 6. 

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Art. 9

Coenzymes

Coenzymes that participate in reactions (like a 2nd substrate) are co-substrates. One of the most important coenzymes is the NADP+molecule, since it is hydrogen-transferring. These coenzymes are usually firmly linked to the actual enzyme, and are occasionally also linked by covalent bonds with the protein. The resulting enzyme and coenzyme complex is called a holoenzyme, which has a protein component called an apoenzyme. This presence of coenzymes is therefore decisive for enzyme activity (cf. Art. 1, endo-polygalacturonase activity control). There are still many other factors influencing enzyme activity (pH value, concentration of dissolved salts, reaction temperature, pressure, etc.), which are assumed to be known to the addressee(s). As a result, no further details are provided. It should also be noted that by quantifying the increase in reaction product of the decisive pectinolytic enzyme "exo-polygalacturonase” also determines the activity of this enzyme by photometrically measuring it, in addition to pectin esterase and endo-14-polygalacturonase, which are determined on the basis of its activity.

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Art. 10

Pectin esterases

Pectin esterases, also known as pectin methyl esterases, like polygalacturonases belong to the large main group of hydrolases. On closer inspection, however, they belong to the subgroup of carboxyester hydrolases. Of course, these enzymes hydrolyze the methyl ester groups of pectin. As a result, high methylester pectin results in low methylester pectin, which is formed by the action of pectin methyl esterases on the methyl ester groups of the pectin to form pectic acid and methanol. In this case as well, LP (in the logical reverse) enzymatic-microbially transfers the low esterification into high esterification. As usual, IP-related details are also (and especially here) omitted. Only this in advance: the polygalacturonases catalyze the cleavage of the α-1-4 glycosidic bonds between the galacturonic acid molecules of the pectin. The endo-polygalacturonase (EC 3.2.1.15) cleaves the pectin chain quite randomly, whereas exo-polygalacturonases (EC 3.2.1.67) cleaves monogalacturonic acid units (dimers) from the non-reducing end of the chain. (Exo-polygalacturonases and endo-polygalacturonases occur in all higher plants and numerous microorganisms). Furthermore, since the formation of an enzyme-substrate complex can only occur in the immediate proximity, free cayrboxyl groups are used without exception via hydrolysis. Along the pectin chain, therefore, ever larger sections are formed in which pectin is de-esterified - a basic prerequisite for further hydrolytic cleavage by polygalacturonases! In accordance with the enzymatic "key-lock-principle", the reverse conclusion is also drawn here from de-esterification to esterification (which in practice is much more complicated than it sounds here). 

 

Art. 11

Extraction of pectinolytic enzymes

Pectinolytic enzymes occur only very scarcely in plant tissue. This makes their correct extraction process all the more important, which usually takes place in the following two ways: the first is by precipitating the enzymes using ammonium sulphate, whereas LP prefers ultrafiltration, since this is not so time-consuming and also has a much lesser effect on enzyme activity. However, both manufacturing processes must be carried out in all their steps at 4°C, because enzymes are inherently thermally unstable. Enzyme extraction through ultrafiltration takes place in three process steps: 

1. Digestion of the cell tissue (plant tissue is transformed with the addition of saponin-transferred NaCl solution using a knife mill). Homogenization and further, finer transformation of the plant tissue pieces, e.g. using Ultra-Turrax. The suspension is stirred for two hours, and then passed through a nylon cloth or similar to separate the remaining components for the first time; the remaining filter cake is then wrung out with a nylon cloth. Centrifuge filtrate at 4°C. 

2. Clear supernatant is concentrated with ultrafiltration; enzyme solution then becomes salts, reducing sugars and any substances that could still pass through the membrane, together with water. As soon as the enzyme solution is concentrated to a certain volume, the membrane is rinsed, to the intended degree, with a NaCl solution added to the retentate and tempered to 4°C. 

3. The remaining enzyme extract is freed from low molecular weight soluble substances by means of a dialysis bath containing a dialysis solution of deionized water, NaCl and very low sodium azide. Sodium azide is meant to prevent microbial growth. The enzyme extract is located underneath in a semi-permeable membrane (cellulose "dialysis tube"). The dialysis tube is boiled before filling with enzyme extract for disinfection. Filled dialysis tube leaded (at constant 4°C) twice in dialysis solution for at least 4 hours. Extract is centrifuged again to remove residual small precipitates. 

 

Finally, the enzyme extract can be used to determine the activity of the endo-polygalacturonases and pectinesterases (cf. again Art.1, casus cnactus)! Furthermore, the consideration of the colloidal influencing parameters is considered an essential measurement criterion. 

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Art. 12

Quality measurabilities

State-of-the-art rapid methods for the analysis and guarantee of food quality increasingly require the search for chemical-physical quantities that can be measured as quickly as possible and reliably meet the quality requirements. One of the most recent methods is rheological (rheology = science of material deformation/flow behavior, sub-discipline of elasticity/plasticity theory together with fluid mechanics of non-Newtonian fluids). In general, it is responsible for the research of "continuum mechanical" problems, i.e. the evaluation and conclusion of material laws required for this purpose, namely from the microscopic or nanological basic structure of different material precipitates (suspensions, macromolecular systems, etc.). 

 

However, the general literature so far hardly reveals any material-scientific information on the fluid-dynamic behavior of raw materials and intermediate/final/byproducts of all industrial manufacturing processes. As a result, LP attached particular importance to an all-encompassing, material-scientific examination of the processing technologies. Before LP there was already marginalresearch work on the fluid dynamic behavior of concentrated solutions in the evaporation station, although the fluid dynamic characteristic values of the intermediate products were largely only determined by LP: Its material scientific and rheological investigations led to some unexpected conclusions about the chemical and physical processes in, for example, subsequent process steps:

 

1. Determination of physical-mechanical properties of the raw material in relation to ingredients.

2. Evaluation of the rheological behavior of native extraction juices directly after crushing beets.

3. Creation of a viscosity matrix for the processing of aged beets.

4. Determination of the rheological combination behavior of pure molasses, vinasse, sucrose solution... as a
   function of temperature (30 to 130°C), dry matter and lime salt content, pH value, etc.

5. Rheological characterization of molasses and vinasse in relation to dry matter and their chemical consistency. 

6. Determination of the influence of viscosity by pectin and dextran in raw extract. 

 

Furthermore, since the rheological behavior of vinasse (a derivative of molasses fermentation) prior to LP has hardly been investigated, LP was the first to find out that the dependence of the flow behavior (Newton/non-Newton) is largely determined by the previous process – but not by the dry matter. LP also found out that – contrary to expectations – density and specific heat capacity of the thermal conductivity coefficient (together with specific boiling point increase) do not necessarily have to be taken as a basis for the thermal physical characteristic values, which considerably simplifies the process overall. However, the thermal-physical characteristic values served LP as orientation data for the rheological vinasse measurement that it had newly developed.