Molecular and functional flour analytics
f2m_science_Microscope

Cereal commodities are subject to quality variations resulting from genetic differences between varieties as well as cultivation and storage conditions. However, cereal processing companies aim at delivering products with constant properties. Therefore, they need appropriate methods for assessing quality variations in their raw materials to be able to take appropriate measures.

Authors: Dr. Julien Huen, ttz; Dr. Tina Bohlmann, ttz; Jessica Wiertz, Brabender

In order to characterize cereal grains and flour, two approaches may be used: either functional analysis that describes the behavior of samples in a simulated application (e.g. lab kneading test), or molecular analysis that aims at quantifying the single groups of substances contained in the sample.

This article describes both approaches based on selected examples of analytical procedures and discusses in which context each approach may be most appropriate. Functional analysis may be seen as the most pragmatic approach for routine quality control in processing companies, because of the simplicity and robustness of the methods. Molecular analysis, in contrast, is often more difficult to implement but delivers insights on the mechanisms explaining functionalities. Therefore, it is of particular value for solving complex quality problems or establishing new recipes and processes.

  1. Functional analysis

The objective of functional analysis is to assess in the lab how a sample will perform in a certain application. In most cases, only little sample preparation is required. Grains have to be ground prior to measurement. The modalities of grinding vary according to the type of grains. Roller mills with sifters can be used, for example, which use cylindrical rollers to grind the grains, or break mills with adjustable milling gaps, consisting of a stationary upper block with three reversible cutting plates and a rotating block with four more reversible cutting plates. Other plant-based materials, such as beans or pulses, can be ground with e.g. a break mill, which is a standard mill Brabender uses for analytics processing of flour and bran fractions. The grains should be free of foreign materials (e.g., stones, soil, etc.) before grinding.

Often, the moisture content of the sample has to be determined, as the quantity of sample used in the measurement is adjusted accordingly. When measuring water absorption in a Farinograph test, 300g is the reference weight to be used. If the moisture content is different than 14%, however, the system software automatically corrects the weight of the flour required.

There are several physical methods to test a flour, which replicate the processes in an industrial bakery. Brabender’s approach is based on three steps of phases:

Phase 1: mixing

Brabender measures the water absorption of the flour, which gives a good indication regarding the types of products for which it can be used. Biscuits and wafers require flour with low water absorption, while bread uses flour with high water absorption, since the starch and proteins are able to retain it. In addition to this, the mixing characteristics of the flour are also measured. How the dough behaves during the mixing process gives an indication of its tolerance to mixing on an industrial scale and the energy required to be put into the dough.

Phase 2: extension

Dough is formed and shaped similarly to industrial operations, at first to a round piece of dough, then to elongated pieces. The dough is allowed to rest before being stretched to test its extensibility. This test indicates if the dough is able to expand and hold the gas during the proofing stage, to obtain a high-volume loaf of bread. If the dough breaks quickly during this test, it will not be able to hold the gas, making it better suited for products in the ranges of biscuits or wafers.

Phase 3: pasting

The flour is gelatinized, a procedure similar to a pure starch sample analysis. Water is mixed with flour and heated up to 93℃ to observe the height of its gelatinization peak. This will help gain a better understanding of how the starch will behave during the baking process. As the crumb is heated up, the starch and the proteins continue taking up water and the gelatinization process takes place inside the bread loaf. If the viscosity is too low, the starch is not assimilating enough water. While for the flour, it is interesting to observe the heating process, while for the starch activity, the cooling process is also investigated, due to the broader application range of starch.

The Farinograph and Extensograph give an indication of the mixing and extension properties of wheat flour (phases 1 and 2). In addition, Brabender uses the GlutoPeak as the main method of gluten characterization. The GlutoPeak can also be used in industrial mills when flour is received. Flour is mixed with water at high speed (over 2,000rpm), so that the gluten within the slurry of water and flour aggregates and forms a network causing a torque peak. The quantity and quality of the gluten influence the readings and anticipates how the flour will perform: a big amount of strong gluten translates into a short and high peak, while the absence of enough gluten to aggregate and/or a low protein flour will determine the peak to be late and very small.

For gluten-free flour testing, the Farinograph is used as well, with an additional tool called FarinoAdd. Gluten-free flours lack elastic properties given by the gluten network, meaning they have a different consistency (more plastic), and require attention to mixing. The same procedure is followed beyond this stage to test gluten-free flours. For wheat flour dough, a consistency of 500 FU is defined; for gluten-free flour, individual values have to be defined in advance, depending on the type of flour.

There are clear trends toward faster tests for bakery applications, looking into gluten functionality, on the one hand, and improvements in the gluten-free segment, on the other. GlutoPeak analysis is a quick measurement that can be performed in around 5 minutes, for example. In addition, Brabender developed two further methods: the analysis of pure, dried gluten that can be added to flours and bakery mixtures and gain control of its quality (in collaboration with ttz Bremerhaven and CSM), and a method for wafer flour analytics (in partnership with Bühler) to solve challenges in the industrial processing of dough for wafers – as this is a type of dough should have a very low protein level since it tends to form lumps because the gluten aggregates when pumped through nozzles. 

Starch performance is easily seen in the quality of the baked goods. If the starch gelatinizes properly, it reflects in the characteristics of the crumb. If not, the starch is unable to absorb all the water added to the dough and there will be a certain amount of moisture in the crumb, which might be visible as a water streak. The type of flour and its corresponding starch composition plays an important role in the baking outcome. For a more crispy or sandy structure, starch is added into the composition that typically uses a flour with a low protein content – as in the case of sand cakes, biscuits or wafers, for example; whereas bread would not require additional starch to the quantity normally found in flour. In contrast, gluten is rather added to the flour.

For starch analysis, sample preparation is also a simple procedure: a slurry is made combining starch and water and placed in a mixing bowl to rotate continuously. The sample is then ready for rheological measurement. It undergoes heating to certain temperatures to simulate the processes the starch undergoes in industrial applications. Gelatinization can be observed in this way (increased torque), as the starch absorbs water and the slurry becomes more viscous. It is also interesting to see how the starch behaves in the cooling phase and its final viscosity, because many applications use it in lower temperatures, such as desserts or puddings. Enzyme activity should also be taken into consideration. If enzymes have affected the starch and broken it down, this starch is unable to gelatinize, resulting in a lower peak.

In terms of gelatinization properties, the functional analysis is an established process, done according to ICC standards usually. Modified, cold-swelling starches are interesting cases that require different methods of analysis. They can be obtained through various methods, either chemical or physical, by heat and shear treatment or extrusion. These types of starches have a high viscosity, to begin with, which will immediately further increase when submerged in water. They are commonly used in desserts and sweet products, in general.

2. Molecular analysis

The objective of molecular analysis is to quantify the substances contained in the sample. Cereal flours are mainly composed of starch grains (themselves made of amylose and amylopectin), storage proteins (gluten in the case of wheat) and non-starch polysaccharides (arabinoxylans, fructans and beta-glucans), as mono-, di- and oligosaccharides, fats, minerals, vitamins and enzymes as minor constituents.

Sample preparation for molecular analysis often involves extracting the components of interest from the overall matrix. This may imply dissolving in appropriate solvents and separating phases by centrifugation, filtration or evaporation. Mono-, di and oligosaccharides as well as some fructans and arabinoxylans, for instance, are soluble in (hot) water. Gluten constituents are soluble in different solutions used in Osborne fractionation, i.e. NaCl-Na2HPO4 solution, ethanol (60%) or DTT solution, with the exception of glutenin macropolymer, at the exception of glutenin macropolymer. Fat may be extracted with petroleum. Starch granules may be separated from the other components by filtration and/or centrifugation. Further purification can be achieved by the use of enzymes that will specifically degrade substances that are not in the focus of the analysis.

The extracted substances may then be weighted for quantification and/or further processed in order to separate them into subgroups for more precise description. In this step, chromatographic methods are of particular interest: high pressure liquid chromatography may be used to separate protein fractions, triglycerides, arabinoxylans, beta-glucans, fructans and vitamins according to their molecular weight. For each application, appropriate columns, solvents, detection systems and parameters (pressure, flow, temperature) need to be defined. In addition, reference substances with known molecular weight need to be measured to calibrate the method. IR spectroscopy of mass spectrometry can be used, in addition, to further characterize the molecular composition of the separated substances. The application of these methods requires a high degree of expertise, but will deliver very detailed insights on composition. It has the potential to distinguish in a very fine way between different samples.

Another approach to molecular analysis consists of the use of enzymatic test kits. This allows for the quantification of substances that may be specifically degraded by a certain enzyme. The application of these methods still implies extraction steps, but further processing requires much less equipment and qualification as chromatography, as it is usually based on colorimetric measurements that only require a photometer. Enzyme test kits are available e.g. for amylose and amylopectin, fructans, arabinoxylans, beta-glucans as well as mono- and disaccharides (glucose, fructose, saccharose, maltose).

The total content of minerals may be determined gravimetrically after ashing, but detailed analysis involves atomic absorption spectroscopy (AAS).

How functional properties and molecular composition are related to one another 

It is well understood that functional properties must be related to their chemical composition. Table 1 gives examples of such relationships. Water absorption, for instance, which can be measured by the Brabender Farinograph, mainly depends on three categories of substances that are able to bind water: arabinoxylans (which are present at low quantities but have the highest water-binding properties), gluten (which binds different quantities of water depending on its composition) and starch (which binds more water if it is mechanically damaged, e.g. as a consequence of the milling process). Based on this, it is obvious that the same level of water absorption may be achieved by different combinations of gluten, starch and arabinoxylan composition. This explains why flour batches showing the same values in functional analysis still may exhibit different behavior in baking applications.

Table 1: Examples of relationships between physical properties and molecular composition in the case of wheat flour

Functional analysis test

Physical properties

Molecular composition

Farinograph

water absorption

composition of gluten (gliadins, glutenins, glutenin macropolymer)

amount of total starch and damaged starch

amount and type of arabinoxylans

ViscoQuick, Micro Visco-Amylo-Graph (MVAG), Viscograph

pasting properties

amount of total starch and damaged starch

proportions of amylose and amylopectin

GlutoPeak, Farinograph dough development time

gluten aggregation properties

gluten composition (gliadins, glutenins, glutenin macropolymer)

ascorbic acid

 

It results from the above that functional and molecular analysis are two different ways to look at the same materials, and that they deliver data that complement each other. When the objective is to understand how functional properties arise and why different batches of flour behave differently, molecular characterization of the relevant components is necessary. This is especially the case because molecular composition can, in turn, be interpreted in the light of the knowledge on the genetic properties of the single varieties (ability to synthesize specific substances) and on environmental factors of cultivation (soil composition, use of fertilizers).

Ongoing research

In a recent research project (AiF 20283N), ttz Bremerhaven and Leibniz LSB[i] have worked at investigating the relationship of molecular gluten composition and baking performance of wheat flour. A main hypothesis of the project was that, in the context of the reduction of nitrogen fertilization, the level of protein in commercial wheat flour may decrease. As the available protein quantity gets lower, it becomes more important to understand which protein quality i.e. composition is required in the different applications to obtain satisfactory results. Optimizing protein quality for single applications may also help reduce the use of additives, thus satisfying the demand for clean label products. The project was supported by a large group of industrial companies from the whole supply chain, under the coordination of Brabender.

One of the main conclusions of the work was the importance of the degree of polymerization of glutenins in the flour for functional properties. The optimal degree of polymerization was different depending on the application. The detailed results of the project will be published in separate articles.

Importance of microstructure

One may argue, finally, that not only the molecular composition of a sample is important, but also the spatial distribution of the constituents in the sample. Hence, biological materials are not perfect blends of the single molecules they are made of. Investigating raw materials, intermediate and final products by microscopy techniques reveals gives further insights on the mechanisms of functionality (see figure 1c).

Conclusion

While daily quality control may be well performed by the use of functional analysis, molecular analysis helps with the understanding of the mechanisms at the origin of quality features. Some of the corresponding methods, especially the colorimetric tests, can be implemented quite easily, while others, especially the chromatographic ones, require expensive equipment and extensive knowledge. This makes them more appropriate for implementation in specialized laboratories than in processing companies.

[I] The Leibniz Institute for Food Systems Biology at the Technical University of Munich has a unique research profile at the interface between food chemistry & biology, chemosensors & technology, and bioinformatics & machine learning. 

This article was published in Baking+Biscuit International, Issue 2- 2022

Photo credit: ttz Bremerhaven