Non-protein amino acids in Australian acacia seed: Implications for food security and recommended processing methods to reduce djenkolic acid
Introduction
The seed of certain species of Australian acacia have been shown to have significant potential to improve rural livelihoods and reduce malnutrition in semi-arid regions of Africa. Acacias provide a range of benefits including environmental services, such as nitrogen fixation and wind speed reduction; they produce valuable fuelwood and building materials and they produce edible seed (Adewusi et al., 2006, Cunningham et al., 2008, Harwood et al., 1999, Rinaudo, 2001, Rinaudo and Cunningham, 2007, Rinaudo et al., 2002, Thompson et al., 1996, Yates, 2010).
The use of acacia seed as a food source is not new, with up to forty species known to have been eaten regularly by Aboriginal people in Australia for thousands of years (Devitt, 1992, Latz and Green, 1995, Lister et al., 1996, Midgley et al., 1991, O’Connell et al., 1983, Orr and Hiddens, 1987). At the present time, acacia seed is produced and marketed as ‘wattleseed’ by a small, but growing industry in Australia. Australian acacia seed of a number of species have been analysed by several groups, and shown to contain crude protein at between 22% and 27%, carbohydrate between 21% and 57%, and fats between 7% and 15% (Yates, 2014). Acacia seed is rich in the amino acid lysine, making it an excellent complement to the predominantly cereal based diets of semi-arid Africa (Adewusi et al., 2003, Yates, 2010). The seed of Acacia colei was the subject of a human volunteer trial in Niger in 1995 (Adewusi et al., 2006), and has been consumed regularly in the Maradi district since that time.
Acacia seed is known to contain several antinutritional factors, which if not removed through appropriate processing or cooking could reduce absorption of nutrients. The presence of trypsin inhibitors, oxalate, phytate, saponins and tannins are reported by Adewusi et al., 2011, Adewusi et al., 2003 and Ee and Yates (2013), though not in concentrations likely to be injurious to human health. Non-protein amino acids (NPAAs) also occur in acacia seed and may have potentially antinutritional or toxic effects (Bell, 2003). For example, Falade, Adewusi, and Harwood (2012) show that (S)-carboxyethyl cysteine in acacia seed interferes with the absorption of methionine in rats.
This study screened and quantified the levels of a variety of NPAAs in five species of acacia seed in order to assess the health risk that these compounds may pose if the seeds become a regular part of the human diet. Amino acids, NPAAs, biogenic amines and many other amines can be selectively derivatised and quantified using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (Aqc) (Boughton et al., 2011). The initial screen comprised the NPAAs: (S)-carboxymethylcysteine (CMC), lanthionine, djenkolic acid (DJK), mimosine and canavanine.
In the first part of the study NPAAs were measured in the seeds of five species: A. colei, Acacia elacantha, Acacia torulosa, Acacia tumida and Acacia saligna. The A. saligna seed was tested in two preparations, as a raw seed and as a roasted seed. A sample of soybean was also tested as a negative control for comparative purposes. When concerning levels of djenkolic acid were found, commercially sourced Djenkol bean was also tested as a positive control, then a second part of the study sought to determine the most effective means of processing for reduction of this compound in three of the five species of acacia and the effect of processing methods on essential amino acids content.
The species selected for testing are all under investigation for their potential contribution to agricultural systems in semi-arid regions of Africa. A. colei has been used as a human food in Niger for over 15 years. A. torulosa, A. elacantha and A. tumida have performed well in agronomy trials and show potential for good seed yields in Niger and other parts of the Sahel (Cunningham & Abasse, 2005). A. saligna has been planted widely in the dry highlands of Tigray, in northern Ethiopia, with seed production representing a significant potential food resource (Hagazi, 2011, Yates, 2010). The species trialled in the Sahel could be expected to perform similarly in the Ethiopian lowlands, where many of the livelihood and land degradation problems seen in Niger are also evident.
Section snippets
General
All chemicals and solvents used for analysis were purchased from Sigma Aldrich (Australia) and were either of analytical or mass spectrometric grades. The Aqc reagent was synthesized accordingly (Cohen & Michaud, 1993) and deionised water (18.2 MΩ) was produced using a Synergy UV Millipore System (Millipore) and was used throughout. An Eppendorf 5415R refrigerated mircrocentrifuge (Eppendorf AG, Hamburg, Germany) cooled to 0 °C was used for centrifugation. External calibration curves within the
Derivatisation of non-protein amino acids and detection by LC-QQQ-MS
The NPAAs canavanine, (S)-carboxymethylcysteine (CMC), djenkolic acid (DJK), lanthionine and mimosine each possess at least one reactive amine group that can be selectively derivatised using 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (Aqc) under standard conditions (Boughton et al., 2011). The NPAAs canavanine, CMC and mimosine contain only a single reactive amine group and were successfully derivatised and when analysed by LC-QQQ-MS in the positive ion mode the corresponding Aqc
The risks of djenkolic acid
Djenkolic acid was first identified as the cause of acute kidney disease in people who ate djenkol bean (Archidendron jirringa), which is considered a delicacy in south-east Asia (van Veen & Hyman, 1933). Patients afflicted by ‘djenkolism’ complain of nausea, acute pain in the loins and kidney region and haematuria. The toxicity is due to the tendency of DJK to precipitate in the acidic environment of the kidney, forming needle-like crystals that cause mechanical damage to the kidney (
Conclusion
Results from screening five species of Australian acacia for toxic non-protein amino acids in seed provided surprisingly high concentrations of the nephrotoxin djenkolic acid. The high levels of djenkolic acid observed raise serious concerns for human health if raw acacia seed meal is consumed or the levels are not reduced by food processing methods. Under resource poor (famine) or extended exposure a significant risk of the development of djenkolism exists. Exploration of appropriate food
Acknowledgements
The authors wish to acknowledge Ms. Nirupama Jayasinghe, Ms. Alice Ng and Ms Terra Stark, Metabolomics Australia, for expert laboratory assistance. The authors also thank the School of Botany node of Metabolomics Australia (MA) at The University of Melbourne, a member of Bioplatforms Australia Pty. Ltd. which is funded through the National Collaborative Research Infrastructure Strategy (NCRIS), 5.1 Biomolecular Platforms and Informatics and co-investments from the Victorian Government.
Funding
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