Europe’s knowledge
platform for legumes

Whole faba beans   Outcome This article provides useful information on how to denature flavour-affecting enzymes when developing food products from faba beans.   Off-flavours in faba bean Volatile compounds (e.g., aldehydes, alcohols, alkanes, ketones and aromatic hydrocarbons) are the sensory elements that affect the flavour perceptions of faba beans. Some of these compounds cause the undesirable flavour notes in faba bean foods produced using aqueous (wet) processing or fermentation. They emerge when the lipids undergo a process of degradation and oxidation catalysed by lipase, lipoxygenase (LOX) or peroxidase (POX). Lipid oxidation is important to consider as it affects the shelf life of food products. The process starts during harvesting, early processing and storage, when seeds are exposed to temperature, pH and moisture variations along with physical damage that degrade the physical barrier between the enzymes and the fatty acids (free or esterified), glucosides and amino acids within the cells of the bean.   Heat treatment Heat treatment is an efficient way to inactivate or denature enzymes in any material made from faba bean. The treatment needs to be mild so it denatures these heat-sensitive enzymes without cooking the rest of the protein, as the cooked protein cannot be extracted to make a milk analogue or protein isolate. The target temperature is around 65–70°C. Possible heat treatments include microwaving, conventional ovens, steaming or kilning in the production line. Dehulling and milling increase the surface area of lipids exposed to the air and break the cells, increasing the access of enzymes to the lipids. This boosts the formation of unwanted flavour notes. Heat treatment applied prior to dehulling and milling is therefore beneficial. If the beans were dehulled prior to the heat treatment, then the heat-treatment step needs to follow immediately afterwards to prevent the formation of undesirable flavours. Steaming Hot steam denatures the enzymes of faba beans. The steam penetrates the cotyledons of the bean effectively. Pre-treatment of seeds with hot dry steam is an option for smaller mills. It is regularly used to inactivate the lipases of oats. It is therefore an existing process in many smaller mills that can be applied to faba beans. There are industrial-scaled steamers in Europe available for pre-treatment of grains. The settings on a flow-through oven have been optimised for this purpose in Finland. The timing and temperature have to be determined for each individual oven. Microwaving Microwaves vibrate the water molecules and the vibration energy transforms to heat. The microwave waves penetrate the cotyledon even more effectively than steam. Research conducted at the University of Helsinki showed that microwave heating (at 950 W for 1.5 min) of small batches of faba beans inactivated the peroxidase and lipoxygenase. Achieving the same result on an industrial scale depends on the size of the equipment and sample size. Like conventional oven heating, the timing and energy level have to be determined for each individual oven. Microwaving has a short processing time and is able to spread high temperatures throughout the cotyledons, faster than conventional oven heating. The application of a microwave treatment for faba beans at an industrial scale would require a microwave-based conveyor belt system. This is not commonly used for pre-treatment of grains in Europe.   Testing for enzyme activity In order to check whether the flavour-affecting enzymes have been denatured, the peroxidase activity can be tested. The minimum heat treatment resulting in inactive peroxidase will result in a product with optimal protein performance and without objectionable flavour. Peroxidase activity is more heat tolerant than lipase and lipoxygenase. If peroxidase activity is successfully inactivated, it is safe to assume that the lipase and lipoxygenase are as well. Peroxidase activity is generally analysed with a guaiacol-H2O2 method. The light absorbance of two solutions, one as the reacting solution and the other as blank, is measured using a spectrophotometer and the enzyme activity is calculated from the result. In the absence of a spectrophotometer, the enzyme activity can be visually assessed. This requires colour models to determine the colour development indicating the strength of the enzyme activity. Such a visual assessment is normally part of a miller or mill technician’s skillset.   Spectrophotometer model 1   Key practice points There are several ways to denature flavouraffecting lipoxygenase and other endogenous enzymes. Millers provide important know-how for implementing the treatment effectively. Lipase, lipoxygenase and peroxidase activity can be tested using a guaiacol-H2O2 method by spectrophotometer or visual assessment   Further information Sharan, S., Zanghelini, G., Zotzel, J., Bonerz, D., Aschoff, J., Saint-Eve, A. and Maillard, M. N., 2021. Fava bean (Vicia faba L.) for food applications: From seed to ingredient processing and its effect on functional properties, antinutritional factors, flavor, and color. Comprehensive Reviews in Food Science and Food Safety, 20, 401–428.

A fish farm in Greece   Outcome The operation of an efficient market for grain legumes in aquaculture value chains helps growers and suppliers of grain legumes who are interested in supporting the fish feed industry. Understanding the requirements is the foundation of tailoring crop production and processing for this growing market. With this attention to quality requirements, European legumes can support the sustainable development of the European aquaculture sector. The sector has specialised requirements dictated by the physiology of farmed fish which, if met, can support premiums for farmers who meet these needs. This article presents the needs of the Mediterranean marine farmed fish, currently the top farmed fish produced in the European Union.   Basic nutritional requirements Mediterranean marine farmed (MMF) fish species are mainly carnivorous in nature and as such have high requirements for proteins and fats. Proteins are the main components of the fillet and fats are needed to cover the energy needs as well as the essential omega-3 and omega-6 fatty acids as much as possible. To achieve this, fish feed is typically 42–48% crude protein and 14–22% crude fat depending on the fish species and on the growth stage of the fish. MMF fish have a low capacity to digest carbohydrates and hence low requirements, which can be covered only by gelatinised starch from cereals. Crude fibre is indigestible and it is a critical limiting factor in selecting the raw material for fish feed (Figure 1). With these requirements met and with a proper feeding management on the farm, a high feed conversion rate (FCR) of 1.6 to 1.8 kg of feed fed per kg of fish produced is achieved in Mediterranean aquaculture today.     Grain legumes can significantly contribute to the protein needs and to the starch fraction of the fish feed, reducing the inclusion of cereals such as wheat. In the early days of aquaculture, fishmeal provided the foundation of the protein component of the diet. Replacing the fishmeal with legume-derived protein is a cornerstone of the sustainable development of the sector. With this shift to legumes, which in contrast to fishmeal, all contain starch, the starch processing characteristics are important, especially for grain legumes such as faba bean and pea. Furthermore, content and quality of starch are critical points for the extrusion process as fish feeds are extruded feeds. In practice, this means that legume grains containing 25–45% protein and up to 40% starch could be included from about 8% to 25% in fish feed (Figure 2).       In addition to a high protein content, a balanced amino acid profile that meets the nutritional requirements of the fish is crucial for replacing fishmeal. Of the 20 amino acids, 10 are essential that fish cannot synthesise. Therefore, so-called essential amino acids must be supplied by the feed (Figure 3). Comparing the amino acid profile of fishmeal as a benchmark to that of a number of legumes, it is clear that legumes can supply varied quantities of essential amino acids. However, the concentrations are lower than in fishmeal. Among the essential amino acids, lysine and methionine are the first limiting amino acids.     The special role of antinutritional factors (ANFs) Legumes have chemical constituents which form a defence mechanism in the plant against diseases and consumption by animals. While they are beneficial in protecting the plant itself and some contribute to the flavour for human consumption, these substances have negative effects on the performance of livestock. These are what we call ‘antinutritional factors’ (ANFs) with ‘protease inhibitors’ (PIs) being the main part. The most important PI is trypsin inhibitor (TI). Table 1 presents the trypsin inhibitor activity of different legume seeds. PIs impair protein digestibility and reduce the bioavailability of amino acids by inhibiting protein digestive enzymes. They can severely affect vital functions resulting in significant mortalities of farmed fish populations. In addition to direct impacts on the fish, there is a negative environmental impact due to increased nutrient emissions into the sea water. Indigestible plant components, such as non-starch polysaccharides present in high concentrations, increase faecal production and alter faecal properties.     In addition to PIs, a variety of other ANFs are found in legume seeds (Table 2). ANFs such as non-starch polysaccharides (raffinose, stachyose), phytic acid, saponins and alkaloids derived from legumes and from cereal glutens may reduce palatability and feed consumption. Once consumed, they absorb water and increase intestinal motility and feed passage, resulting in reduced nutrient uptake and increased nitrogen excretion into seawater. Processing is necessary to meet the requirements Grain legumes must be processed to tailor them for use in fish feed. Dehulling removes the outer coat of the seeds and with it a large proportion of indigestible fibre and anti-nutritional tannins in the case of faba bean (Figure 4). At the same time, this increases the protein concentration of the raw material. The oil content of soybean is higher than required and oil extraction is performed, which further increases the protein concentration of soybean meal. Different processes have been developed to inactivate or to reduce ANFs below threshold limits. The thermal treatments have become the most used since they can gradually and precisely reduce trypsin inhibitor levels. However, heat treatment is costly and may damage the nutrients, including protein. Alternative options have been developed such as fermentation, ultrasound, gamma irradiation, germination and soaking. In general, thermal treatments such as cooking or/and extrusion as well as chemical and biotechnological approaches such as fermentation are the most effective treatments currently used. Finally, milling homogenises the material, increases digestibility and improves the quality of feed that is fed as pellet. Plant breeding also offers a solution to the challenge of ANFs. Cultivars of soybean with low TI content have been developed. The successful use of unprocessed soybean varieties with reduced content of TIs creates additional options for fish feed manufacturers while reducing the costs for thermal treatment.   Figure 4. Raw, dehulled and milled species Vicia faba (left) and species Lupinus albus (right)   A range of grain legume species can be used Based on these requirements, the aquaculture sector can use a range of protein sources. All the major grain legume species can be used successfully. Soybean is the main crop used. Soybean products (soybean meal, soy protein concentrate etc.) are the ingredients that have successfully reduced the use of fish meal in fish feed diets over the last twenty years. Faba bean meal and pea (mainly as protein concentrate) are also used in substantial quantities along with smaller quantities of lupin and chickpea. Pea, chickpea and faba bean successfully replaced wheat in seabass diets resulting in improved growth performance.   Basic quality requirements The quality requirements of MMF fish feed are determined by market status, processing mill specifications and nutritional performance. In particular, the quality parameters of legumes required for use in fish feed are: Protein content: Protein content is the most important characteristic in determining the competitiveness of a raw material compared with other protein sources. Legume grains and products with a high protein content are preferred. Protein quality: selection of species/cultivars with a balanced profile of essential amino acids and high protein bioavailability, i.e., protein that is easily hydrolysed in the presence of water and the digestive enzymes of the MMF fishes. Low levels of anti-nutritional factors: low levels of PIs to ensure good function of digestive enzymes and high dietary protein bioavailability. Content and quality of starch: This is a critical point for the use of legumes with protein concentrations below 35% e.g., faba bean and pea meal. Such legumes with high starch contents can be used in fish feed that include high protein raw materials like animal by-products to balance the protein level while covering the demand for starch for the extrusion process. Moisture content: Grain moisture content of legumes delivered to a feed mill should not exceed 12%. In addition to reducing the stability of the grain in store, the moisture content has a large effect on dehulling, which is necessary to remove the indigestible fibres contained in the seed coat. Impurities (foreign matter) content: The level of impurities in each batch (load) should not exceed 1.5% by weight. Impurities include fragments of the other parts of the crop, sand or stones, other seeds, etc. 1.5% is the upper limit to prevent price reductions due to contamination. In addition, most fish feed producers exclude GMOs from the supply chain. Only non-GM legume crops are permitted for cultivation in the EU and so all crops legally grown in the EU meet this requirement.   Transportation, warehousing and delivery Transportation is done by truck, either in bulks or in big-bags of 1-tonne each. The following should be ensured: The load meets basic physical standards of moisture content and impurities. Proof that the truck and loading equipment has not been used for genetically modified soya in at least the last three shipments. Protection in store with temperatures below 22°C and relative humidity less than 75%. In accordance with the domestic and European legislation, quality management systems and feed safety (ISO 22000:2005 HACCP) accompanying documents or certificates of analysis for heavy metals, mycotoxins & aflatoxins, dioxines & PCBs, pesticides residues and microbiological content are required before the first delivery.   Further Information Feedipedia. Animal feed resources information system, www.feedipedia.org  

Downy mildew on soybean seed.   Biology The plant pathogen P. manshurica exists currently in more than 30 different races. There is little relevant difference between races in virulence and response to management. This might change due to the ability of P. manshurica to rapidly adapt to the resistance genes in commercial cultivars. Resting spores survive from over-winter on leaf debris. They are also carried on seed. In most situations where soybean is rotated with other crops, the main infection route is infected seeds. An infected seed results in systemic infection of the whole plant causing stunting and mottling of the leaves. Symptoms are evident at all growth stages. Spores spread by wind in the growing crop. Initial disease development is favoured by a combination of frequent rainfall, heavy morning dew, high relative humidity, and moderate temperatures of 18–22°C. If this phase is followed by a prolonged dry spell, the infection risk is further increasing.   Symptoms The most visible symptoms are 2–4 mm angular spots on the leaves. The initial symptoms are small pale green or yellowish spots which merge with time. On the reverse side of the leaf, a tan to grey covering forms, especially under wet and humid conditions. The early symptoms are present at the two (V2 grown stage) to three (V3 grown stage) trifoliate leaf stages. Younger leaves are more susceptible and infected leaves are commonly seen on the top of the plants. Pods may be infected without any outside symptoms, but the seeds inside are partly or completely encrusted with white mycelia and oospores.   Peronospora manshurica lifecycle on soybean seeds.     Impact on crop development Infested seeds might suffer minor adverse effects during germination. Several experiments at different sites worldwide have investigated the potential yield losses by comparing fungicide-treated and untreated plots. Across those trials, severe disease infestation resulted in yield losses in susceptible cultivars of about 10% in extreme cases. There is also evidence that crop quality from fields which were planted with infested seeds is reduced in terms of seed vitality and protein content. In farming practice however, yield losses caused by downy mildew solely are difficult to measure accurately since yield losses are usually caused by several interacting factors (e.g. weather, pests).   Key practice points to manage the disease risk Downy mildew is currently not a major disease in European soybean crops. This is in most cases due to the relatively small soybean area and due to the short history of soybean production. Incidence is low even in regions with a relatively high proportion of soybean areas (e.g., Eastern Austria). Where it occurs, yield losses are low. However, this may change as the crop expands. The following measures will help preserving this positive situation:   Pathogen-free seeds The use of healthy seed is the most important control measure. Seed infection levels can be tested by accredited laboratories and only pathogen-free seed should be used (see also below).   Downy mildew on the front side of leaves.   Cultivar choice The Austrian Descriptive Variety List provided by the Austrian Agency for Food Safety (AGES) provides a downy mildew resistance assessment for about 70 common soybean cultivars for growing in Europe. Crop rotation Generally, there should be at least two years, or better three years, between successive soybean crops in the same field to reduce the risk for disease infection through infested crop residues. This practise helps to manage downy mildew as well as other common soybean diseases unless infested seeds are planted. Soil tillage Tillage that speeds up the decomposition of infected material reduces the risk of a spread to future crops. Sowing dates Sowing soybean very early in the season at cool soil temperatures (<9°C) may result in a slow and poor establishment. This weakens the vitality of plants and increases potentially the risk of an attack by pests and diseases. Avoiding mechanical damages Cracks in the soybean hull are potential entry points for fungi. Hence, mechanical damages during harvesting and transportation shall be avoided. Most essential are therefore proper harvester settings and gentle conveying applications in the storage. Fungicide management Fungicide use is rarely economic in case of downy mildew in soybean. Experiences from studies in USA indicate that this disease is better managed through preventive measures (see above).   Downy mildew on the back side of the leaf.   Resistance scoring of cultivars The Austrian Agency for Health and Food Safety (AGES) is the responsible body for seed certification to place it on the national and international markets of the European Union, the OECD market and the national seed market of Austria (Institute for seed and propagating material). As part of the three-year registration procedure, AGES assesses the performance of cultivars in the field in terms of yield, quality and resistance towards diseases and pests. AGES provides the data in the “Austrian Descriptive Variety List” which includes assessment for about 70 registered soybean varieties with a score system from 1 (trait strongly expressed feature) up to 9 (trait is not expressed). Link: https://www.ages.at/en/topics/agriculture/varieties/   Testing opportunities AGES in Vienna and in Novi Sad (Serbia), the Institute of Field and Vegetable Crops provide an analysis of seeds or leaves for Peronospora manshurica. Prices for an analysis may be up to 100 Euro per sample (excl. VAT). One kilogram of seed is needed for this testing procedure. Essential to achieve valid results is that a representative sampling has been conducted. Therefore, it is recommendable to take the sampling procedures of official seed certifications as a guidance. Links: Austrian Agency for Food Safety: https://www.ages.at/en/service/services-agriculture/analysis-of-seed-and-propagating-materia/ Institute of Field and Vegetable Crops Novi Sad: https://ifvcns.rs/en/research/laboratories/laboratory-for-seed-testing/