Not too little, not too much

Dr Liliana Krotz and Dr Guido Giazzi discuss optimising protein analysis in animal feed

The composition of most forage and by-product animal feeds is variable, with feedstuffs differing widely in nutrient concentration due to many determinants including location, harvest date, maturity, soil nutrients and other management factors. The content and quality of feed can have a significant impact on livestock productivity, as animal nutrition is the largest variable cost associated with livestock production. As a result, reliable analysis of nutritional content is essential to ensure a balanced feeding program and profitability of any livestock operation.

Undertaking feed analysis not only enables farmers to optimise nutrient utilisation in animal feeds but also allows researchers to relate animal performance to feed characteristics. One of the most important nutrients in animal feed is protein, as its intake provides the building blocks needed by livestock to produce their own proteins in order to grow, maintain or produce muscles, enzymes, hormones, milk and wool. Animal protein requirements can vary with stage of production, size of the animal and expected performance. However, the quantity of protein required by animals is very precise; excess can generate unnecessary amounts of energy and too little can lead to amino acid deficiency. In addition, not all types of protein are of nutritional value to animals and certain types can pose serious animal and human health risks, making reliable analysis of feed vital.

Protein in livestock diets is commonly expressed as crude protein, typically calculated through the determination of nitrogen in feed. Nitrogen value is multiplied by 6.25 because proteins typically contain 16 percent nitrogen (1/0.16 = 6.25). Rapid analytical tests have been developed over the last few years to ascertain levels of protein in feed samples and are essential when faced with a need for quick decisions or when confronted by a large number of samples.

The Kjeldahl method has traditionally been used for quantitative determination of nitrogen/protein content in food. The technique consists of five main steps: sample digestion in boiling sulphuric acid; neutralisation with sodium hydroxide solution; distillation of the resulting ammonia gas into a trapping solution; titration with an acid solution and determination of nitrogen content by calculation. Although this technique is capable of providing precise and reproducible results, its effectiveness is limited by a number of shortcomings, including that it is a very time-consuming method, with a complete analytical cycle lasting more than four hours. Efficient implementation of the Kjeldahl method also requires constant operator interaction throughout the entire process. Additionally, the technique poses considerable health risks to operators since it requires the use of concentrated sulphuric acid at high temperature, while the use of mercury or selenium as catalysts for digestion generates toxic waste.

These disadvantages have ended the dominance of the Kjeldahl method as the preferred technique for nitrogen/protein determination in food, paving the way for new methodologies and techniques.

Designed to offer an alternative to the Kjeldahl method, the Dumas combustion method overcomes some of the limitations of the traditional technique. This is an easy to use, automated method capable of providing measurements of the nitrogen/protein concentration in food samples more rapidly and without the use of toxic chemicals or catalysts. However, the technique involves a high initial cost and like the Kjeldahl method it does not offer measurements of the true protein content and various correction factors are necessary to account for amino acid sequences. A further shortcoming of the Dumas combustion method is that it uses a small sample size making it difficult to obtain a representative sample.

As the demand increases for improved sample throughput, reduced operational costs and a reduction in human error, it has become increasingly important to have a simple automatic technique which enables fast nitrogen/protein analysis combined with excellent reproducibility. By adding a stage to the Dumas combustion method where samples undergo flash combustion, the method’s safety, accuracy, speed and dependability are increased significantly.

The dynamic flash combustion method features a unique CO2 adsorber regenerating technology, which is automatically activated to adsorb the CO2 generated during the combustion. Unlike traditional technologies, the use of a self-cleaning filter eliminates the need for it to be changed several times a week, saving time and money and enhancing autonomy while reducing instrument maintenance. The method also does not require sample digestion of toxic chemicals, eliminating health hazards.

Dynamic flash combustion incorporates a Peltier water elimination device and a safe, sensitive and reliable thermal conductivity detector (TCD) which covers quantitative nitrogen/protein determination from low ppm to high percentage concentrations. A much higher sample throughput is achieved within five to seven minutes depending on the nature of the samples, while a large sample size capacity enables maximum accuracy and reliability of results, reduces sample handling time and minimises matrix effects.

The efficiency of the dynamic flash combustion method has been recognised by the Association of Official Analytical Chemists (AOAC) in method 990.031, which approves and adopts the technique for nitrogen/protein determination in animal feed. The AOAC method specifies the use of combustion for nitrogen identification analyses in all types of fodder, According to the method, analysis must be performed by an instrument which is equipped to provide certain conditions, including a furnace to maintain a minimum operating temperature of 950°C for combustion of the sample in pure oxygen. The analyser must be capable of measuring nitrogen in feed materials containing 0.2 to 20% nitrogen. It should also incorporate an isolation system to separate liberated nitrogen gas from other combustion products for subsequent measurement by a thermal conductivity detector. A detection system will ensure that the detector response is interpreted as percentage nitrogen (weight/weight).

Located in Rimini, Italy, the Gruppo CSA SpA Research Institute houses a food laboratory division which performs a broad range of analyses including nitrogen/protein determination in animal feed and human food samples. The institute has seen a surge in the number of animal feed analyses that it performs due to a recent European Commission initiative that provides a financial incentive for farmers producing dehydrated alfalfa with levels of protein above 15%. In response to customer demand, Gruppo CSA is required to conduct rapid nitrogen/protein analysis on high quantities of dehydrated alfalfa in adherence with the Association of Official Agricultural Chemists (AOAC) 992.23 method. The method indicates that the suitable fineness of grind must be determined to achieve precision that gives RDS of less than 2% for 10 successive determinations of nitrogen. To address the growing analytical requirements of the agricultural community while also remaining compliant with industry regulations, Gruppo CSA has implemented the Thermo Scientific FLASH 4000 nitrogen/protein analyser.

Prior to implementing new instrumentation, Gruppo CSA utilised the Kjeldahl method for its nitrogen/protein analyses. However, the use of acid at extremely high temperatures had a corrosive effect on the institute’s instrumentation. In addition, a chimney was required within the laboratory to remove the waste, which incurred a high cost and raised the problem of pollution being omitted into the environment. Due to these factors, the Kjeldahl method could not be operated continuously within the laboratory which reduced sample throughput and meant that customer costs for these services were high.

Working with the Dumas combustion method for protein analysis, the new instrumentation overcomes the challenges, increasing overall productivity and cost-efficiency while also eliminating the use of corrosive acids, which improves health and safety conditions within the institute. Since implementing the new system, Gruppo CSA’s team is able to process up to 50 samples in a single cycle, while the four-hour cycle time has been dramatically reduced to just 10 minutes. With the high sample capacity and cost-efficiency of the new system, Gruppo CSA has been able to provide animal feed analysis as a key service to its customers for the first time. In contrast to the Kjeldahl method, which requires constant monitoring, the new system can also run unattended and overnight, saving time for the institute’s technicians and further increasing throughput. In addition, the elimination of dangerous acids and toxic reagents has meant that the instrument is much easier to clean and maintain.

Protein is one of the most important nutrients in the diet of livestock and its consumption is essential in order to ensure animal growth and production of products such as milk and wool. However, excessive protein intake can lead to unnecessary levels of energy while too little can result in amino acid deficiency. Therefore, the analysis of protein concentration in animal feed through the determination of nitrogen is vital. The use of a dynamic flash combustion analyser offers a safe and accurate alternative to the traditional Kjeldahl method, accelerating throughput and improving the reliability of results. The method also eliminates sample preparation while minimising matrix effects. 

References
1. National Forage Testing Association, Protein (Crude) in Animal
Feed: Combustion Method. (990.03), Official Methods of Analysis, 1990, Association of Official Analytical Chemists, 15th Edition, www.foragetesting.org/lab_procedure/sectionB/3/part3.3.htm

Contact:
t: +1 800-532-4752,
e: analyze@thermofisher.com
w: www.thermoscientific.com/flash4000

Authors:
Dr. Liliana Krotz, Product Specialist, OEA and Dr. Guido Giazzi, Product Manager, OEA, Thermo Fisher Scientific, Milan, Italy