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How much protein is in your beans?

September 11, 2008

Feature

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In Depth

Healthy high protein, low fat foods such as beans are becomingly increasingly prevalent in the food market with people taking a growing interest in their dietary health. Here we learn of a new protein level detection technique that has bean gaining acclaim

Beans are proven to boost muscle glycogen levels, lower the risk of prostate or breast cancer and deliver vitamins and minerals while giving you serviceable protein1. Protein is found in every cell of the body and is essential for their manufacture, growth and repair. As one of the most important nutrients, the monitoring of the amount of protein, through the determination of nitrogen, must be accurate to assess the nutritional quality of these products. The protein content determination is also fundamental in evaluating the value of bean feed in the farming trade.

The two key methods in identifying the amount of protein contained within foodstuffs are the Kjeldahl and Dumas combustion. The more traditional Kjeldahl method of protein/nitrogen determination has many recognised flaws whereas the lesser-known Dumas combustion method has been greatly improved with new pioneering technology making it a superior protein content analysis method. This article discusses the Kjeldahl method and investigates the Dumas combustion method as an improved alternative for measuring protein/nitrogen levels in beans.

Johan Kjeldahl, a Danish industrial chemist developed his nitrogen/ protein

Table 1: N/protein determination in beans

determination method whilst working in the innovative laboratory team at the Carlsberg Brewery. The Kjeldahl method relies upon the chemical breakdown of all the protein bonds into ammonium ions. In order to make this reaction rate industrially viable, a combination of acid, heat, salt and catalysts are used. Highly concentrated sulfuric acid is raised above its boiling temperature of 338ºC, with the addition of salt, to the critical temperature for decomposition at 373ºC. A mercury, selenium and/or copper catalyst is used to speed the reaction even further. The ammonium ions are freed from their salt complex with sodium hydroxide and the free volatile ammonia formed is then carried out of the solution and condensed into a collection vessel containing boric acid buffer solution. The amount of nitrogen and protein can then be calculated through pH measurement, back-titration and calculations.

The original Kjeldahl method involved large amounts of strong acids with very hot

Table 2: N/protein determination in red beans

digests that emitted acid and corrosive fumes2. It was time-consuming and not very ecological, requiring a highly specialised scientist to carry out determinations in a facility with anti-corrosion tables and appropriate fume-compartments. Although some improvements have been made, the Kjeldahl methodology is becoming increasingly inadequate in meeting international safety regulations. In addition, the Kjeldahl method suffers from high waste cost, the inability to operate continuously, and is highly dependant on the user's experience and capabilities.

The protein/nitrogen determination method developed by Professor Jean-Baptiste

Table 3: N/protein determination in lentils

André Dumas originated in 1833. Based on complete oxidation of the protein in a sample being burned in an oxygen rich atmosphere at high temperatures (about 1000oC), the resulting gases are then analysed. The combustion products (mainly CO2, H2O, NxOy, and N2) are collected and allowed to equilibrate. An aliquot of the gas mixture is passed over hot copper to remove any oxygen and converts all nitrogen oxides to nitrogen gas (N2). The sample is then passed through a trap that removes carbon dioxide and water. The remaining nitrogen is measured by thermal conductivity and the protein content of the sample is calculated.

Originating in academia this method was initially spurned by users in the industrial environment because of complications in achieving complete oxidation and contamination of the gaseous products with air. The comparative rapidity (minutes rather than hours), safety and economic advantages of the Dumas combustion method, as well as vast technological developments, have brought it back to the forefront of nitrogen/protein determination. It overcomes all concerns which tarnish the Kjeldahl method, including reduced sample preparation. This, in turn eliminates safety concerns, operator experience requirements, costs of materials and most significantly, the time involved.

Many official regulative organisations including the Association of Official Analytical Chemists (AOAC), the American Oil Chemist Society (AOCS) and the American Association of Cereal Chemists (AACC) support the new modified combustion method, as an alternative to the well-known Kjeldahl method. An example of this alternative method includes the Thermo Scientific FLASH 4000N/Protein analyzer, based on dynamic flash combustion, which features the safety and efficiency of the Dumas combustion method.

The experiment outlined below was carried out to validate new technologies, such as the Thermo Scientific FLASH 4000 analyzer and demonstrates the comparative efficacy to the Kjeldahl method.

The conditions of the experiment are detailed below with Figure 1 depicting the setup of instrumentation for the flash combustion method. The AOAC indicates that the suitable fineness of grind must be determined (for each different material analyzed) to achieve precision that gives RSD (equal or less) of 2% for ten successive determinations of nitrogen.

Temperature left reactor: 950 °C
Temperature right reactor: 840 °C
Temperature oven: 50 °C
Standard: 500 mg EDTA (9.59 %N)
Sample weight: 600 mg - 1.6 g

Different beans were chosen to validate the system in terms of accuracy and reproducibility according to the pre-treatment of the sample. The protein content is calculated automatically by the Thermo Scientific Eager Xperience Software using the default protein factor of 6.25. The protein factor can be changed in accordance with the food type.

Table 1 shows the analysis of ten consecutive determinations of bean samples with different homogenisation, 1mm and 2mm particle size. The data obtained indicate that it is not necessary for a fine homogenisation to reach a RDS% (equal or less) to 2% as suggested in the official methods.

Table 2 shows the nitrogen and protein data obtained from the red beans

Figure 1: Analytical Configuration within the Thermo Scientific FLASH 4000N/ Protein Analyser

samples using different sample weights. Excellent reproducibility was obtained in the range 600 - 1600mg. No memory effect was observed against changing the sample weight. In the case of lentils, see Table 3, the sample was analysed after homogenization at 1 and 2mm particle size and without homogenisation. The average of the data obtained is comparable with the results of the homogenised lentil sample at the different particle size and the RSD% obtained is according to the official methods, indicating that depending on the sample nature it can be analyzed directly, without pre-treatment.

the above experiment demonstrates that current leading dynamic flash combustion instruments provide nitrogen/protein determination within a matter of minutes as opposed to hours. In addition the improved combustion method achieves this in a more environmentally-friendly manner than the Kjeldahl method with fewer safety concerns. Further benefits of these newer technologies include continuous operation through the use of automation and dual regenerating carbon dioxide filters operating alternately. The increased ease-of-use ensures that they do not require a specialist in the field to operate them.