The Ninhydrin Reaction Revisited: Optimisation and Application for Quantification of Free Amino Acids.

The ninhydrin reaction is commonly used for the detection of amino acids. However, in the literature, different conditions with respect to the buffer system, its pH and concentration, type of organic solvent, incubation time, and temperature, as well as the concentrations of the reagents, are described. To identify the most suitable conditions, colour development with reagents of varying compositions and different reaction temperatures and times were investigated using asparagine as a model amino acid. Asparagine was selected since it is one of the most abundant free amino acids in many types of samples. The optimal reaction mixture consisted of 0.8 mol L-1 potassium acetate, 1.6 mol L-1 acetic acid, 20 mg mL-1 ninhydrin and 0.8 mg mL-1 hydrindantin in DMSO/acetate buffer 40/60 (v/v) (final concentrations). The best reaction condition was heating the samples in 1.5 mL reaction tubes to 90 °C for 45 min. Afterwards, the samples were diluted with 2-propanol/water 50/50 (v/v) and the absorbance was measured at 570 nm. The proteinogenic amino acids showed a similar response except for cysteine and proline. The method was highly sensitive and showed excellent linearity as well as intra-day and inter-day reproducibility.

Adding pulse flours to cereal-based snacks and bakery products: An overview of free asparagine quantification methods and mitigation strategies of acrylamide formation in foods.

Thermal processing techniques can lead to the formation of heat-induced toxic substances. Acrylamide is one contaminant that has received much scientific attention in recent years, and it is formed essentially during the Maillard reaction when foods rich in carbohydrates, particularly reducing sugars (glucose, fructose), and certain free amino acids, especially asparagine (ASN), are processed at high temperatures (>120°C). The highly variable free ASN concentration in raw materials makes it challenging for food businesses to keep acrylamide content below the European Commission benchmark levels, while avoiding flavor, color, and texture impacts on their products. Free ASN concentrations in crops are affected by environment, genotype, and soil fertilization, which can also influence protein content and amino acid composition. This review aims to provide an overview of free ASN and acrylamide quantification methods and mitigation strategies for acrylamide formation in foods, focusing on adding pulse flours to cereal-based snacks and bakery products. Overall, this review emphasizes the importance of these mitigation strategies in minimizing acrylamide formation in plant-based products and ensuring safer and healthier food options.

Metabolome analysis for pancreatic cancer risk in nested case-control study: Japan Public Health Center-based prospective Study.

Discovery of a high-risk group for pancreatic cancer is important for prevention of pancreatic cancer. The present study was conducted as a nested case-control study including 170 pancreatic cancer cases and 340 matched controls of our population-based cohort study involving 30 239 subjects who answered a baseline questionnaire and supplied blood samples. Twelve targeted metabolites were quantitatively analyzed by gas chromatography/tandem mass spectrometry. Odds ratios (OR) and their corresponding 95% confidence intervals (CI) were calculated using conditional logistic regression models. Statistically significant P-value was defined as P < .05. Increasing 1,5-anhydro-d-glucitol (1,5-AG) levels were associated with a decreasing trend in pancreatic cancer risk (OR of quartile 4 [Q4], 0.50; 95% CI, 0.27-0.93; P = .02). Increasing methionine levels were also associated with an increasing trend of pancreatic cancer risk (OR of Q4, 1.79; 95% CI, 0.94-3.40: P = .03). Additional adjustment for potential confounders attenuated the observed associations of 1,5-AG and methionine (P for trend = .06 and .07, respectively). Comparing subjects diagnosed in the first 0-6 years, higher levels of 1,5-AG, asparagine, tyrosine and uric acid showed a decreasing trend for pancreatic cancer risk (P for trend = .04, .04, .04 and .02, respectively), even after adjustment for potential confounders. We found that the 12 target metabolites were not associated with pancreatic cancer risk. However, metabolic changes in the subjects diagnosed in the first 0-6 years showed a similar tendency to our previous reports. These results might suggest that these metabolites are useful for early detection but not for prediction of pancreatic cancer.

Assessing the variation and genetic architecture of asparagine content in wheat: What can plant breeding contribute to a reduction in the acrylamide precursor?

KEY MESSAGE: A large genetic variation, moderately high heritability, and promising prediction ability for genomic selection show that wheat breeding can substantially reduce the acrylamide forming potential in bread wheat by a reduction in its precursor asparagine. Acrylamide is a potentially carcinogenic substance that is formed in baked products of wheat via the Maillard reaction from carbonyl sources and asparagine. In bread, the acrylamide content increases almost linearly with the asparagine content of the wheat grains. Our objective was, therefore, to investigate the potential of wheat breeding to contribute to a reduction in acrylamide by decreasing the asparagine content in wheat grains. To this end, we evaluated 149 wheat varieties from Central Europe at three locations for asparagine content, as well as for sulfur content, and five important quality traits regularly assessed in bread wheat breeding. The mean asparagine content ranged from 143.25 to 392.75 mg/kg for the different wheat varieties, thus underlining the possibility to reduce the acrylamide content of baked wheat products considerably by selecting appropriate varieties. Furthermore, a moderately high heritability of 0.65 and no negative correlations with quality traits like protein content, sedimentation volume and falling number show that breeding of quality wheat with low asparagine content is feasible. Genome-wide association mapping identified few QTL for asparagine content, the largest explaining 18% of the genotypic variance. Combining these QTL with a genome-wide prediction approach yielded a mean cross-validated prediction ability of 0.62. As we observed a high genotype-by-environment interaction for asparagine content, we recommend the costly and slow laboratory analysis only for late breeding generations, while selection in early generations could be based on marker-assisted or genomic selection.

[Development of LC-MS and LC-MS/MS Methods for Free Asparagine in Grains].

New analytical methods for the determination of free asparagine （Asn）, which is a precursor of acrylamide, in grains were developed using LC-MS and LC-MS/MS. Asn was extracted from a sample with 5％ （w/v） aqueous trichloroacetic acid solution, appropriately diluted with 0.1％ （v/v） formic acid solution, and then analyzed by LC-MS or LC-MS/MS. HPLC separation was performed by isocratic elution on a Penta Fluoro Phenyl （PFP） column using 0.1％ （v/v） formic acid and acetonitrile mixture as the mobile phase. The calibration curve was linear in the range of 0.005-0.1 µg/mL. The mean recoveries from potato starch, non-glutinous rice flour and whole wheat flour ranged from 95.4 to 100.9％, repeatability （RSD） ranged from 0.9 to 6.0％, and within-laboratory reproducibility （RSDwr） ranged from 2.8 to 7.1％. Limits of quantitation （LOQs） were 7 mg/kg for potato starch, and 5 mg/kg for non-glutinous rice flour. In addition, an inter-laboratory study was performed in 10 laboratories using 5 kinds of grains （non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour）, which naturally contained free asparagine. The HORRATR values ranged from 0.4 to 1.0. These results are within the range of the procedural manual of the Codex Alimentarius Commission, confirming the effectiveness of the developed procedures.

[Inter-laboratory Study of an HPLC-UV Method for Free Asparagine in Grains].

In order to validate an HPLC-UV method for the determination of free asparagine, which is a precursor of acrylamide, in grains using dansyl derivatization, an inter-laboratory study was performed in 9 laboratories using 5 kinds of grains (non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour), which naturally contain free asparagine. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDr) were in the ranges of 0.5-2.2 and 2.3-5.9%, respectively. The HorRat values ranged from 0.4 to 0.6. These results were within the range of the procedural manual of the Codex Alimentarius Commission, and therefore the method is effective.

PrionW: a server to identify proteins containing glutamine/asparagine rich prion-like domains and their amyloid cores.

Prions are a particular type of amyloids with the ability to self-perpetuate and propagate in vivo. Prion-like conversion underlies important biological processes but is also connected to human disease. Yeast prions are the best understood transmissible amyloids. In these proteins, prion formation from an initially soluble state involves a structural conversion, driven, in many cases, by specific domains enriched in glutamine/asparagine (Q/N) residues. Importantly, domains sharing this compositional bias are also present in the proteomes of higher organisms, thus suggesting that prion-like conversion might be an evolutionary conserved mechanism. We have recently shown that the identification and evaluation of the potency of amyloid nucleating sequences in putative prion domains allows discrimination of genuine prions. PrionW is a web application that exploits this principle to scan sequences in order to identify proteins containing Q/N enriched prion-like domains (PrLDs) in large datasets. When used to scan the complete yeast proteome, PrionW identifies previously experimentally validated prions with high accuracy. Users can analyze up to 10 000 sequences at a time, PrLD-containing proteins are identified and their putative PrLDs and amyloid nucleating cores visualized and scored. The output files can be downloaded for further analysis. PrionW server can be accessed at http://bioinf.uab.cat/prionw/.

The kinetics of the inhibition of acrylamide by glycine in potato model systems.

BACKGROUND: Acrylamide (AA) is a potential carcinogen which widely exists in heat-processed foods. The addition of glycine (Gly) has been shown to reduce the formation of AA. The objective of this work was to investigate the kinetics of the inhibition of AA by Gly in both asparagine (Asn)/glucose (Glc) and Asn/Glc/Gly potato model systems during heating at 160 °C, 180 °C, and 200 °C. RESULTS: The simplified two consecutive first-order kinetic model fitted well to the changes of AA in both systems. No significant difference in rate constant (kF) and apparent activation energy (EaF) was observed for AA formation between the two systems (P > 0.05). Whereas EaE and only kE at 200 °C for AA elimination in the Asn/Glc/Gly system was significantly higher than Asn/Glc system (P < 0.05). The elimination reaction between Gly and AA was confirmed by the identification of their major reaction product 2-((3-amino-3-oxopropyl)amino)acetic acid in the Asn/Glc/(15) N-Gly system. CONCLUSION: The reduction of AA by Gly is predominantly attributed to the elimination reaction between Gly and AA.

Asparagine 326 in the extremely C-terminal region of XRCC4 is essential for the cell survival after irradiation.

XRCC4 is one of the crucial proteins in the repair of DNA double-strand break (DSB) through non-homologous end-joining (NHEJ). As XRCC4 consists of 336 amino acids, N-terminal 200 amino acids include domains for dimerization and for association with DNA ligase IV and XLF and shown to be essential for XRCC4 function in DSB repair and V(D)J recombination. On the other hand, the role of the remaining C-terminal region of XRCC4 is not well understood. In the present study, we noticed that a stretch of ∼20 amino acids located at the extreme C-terminus of XRCC4 is highly conserved among vertebrate species. To explore its possible importance, series of mutants in this region were constructed and assessed for the functionality in terms of ability to rescue radiosensitivity of M10 cells lacking XRCC4. Among 13 mutants, M10 transfectant with N326L mutant (M10-XRCC4(N326L)) showed elevated radiosensitivity. N326L protein showed defective nuclear localization. N326L sequence matched the consensus sequence of nuclear export signal. Leptomycin B treatment accumulated XRCC4(N326L) in the nucleus but only partially rescued radiosensitivity of M10-XRCC4(N326L). These results collectively indicated that the functional defects of XRCC4(N326L) might be partially, but not solely, due to its exclusion from nucleus by synthetic nuclear export signal. Further mutation of XRCC4 Asn326 to other amino acids, i.e., alanine, aspartic acid or glutamine did not affect the nuclear localization but still exhibited radiosensitivity. The present results indicated the importance of the extremely C-terminal region of XRCC4 and, especially, Asn326 therein.

Sequential capillary electrophoresis analysis using optically gated sample injection and UV/vis detection.

We present sequential CE analysis of amino acids and L-asparaginase-catalyzed enzyme reaction, by combing the on-line derivatization, optically gated (OG) injection and commercial-available UV-Vis detection. Various experimental conditions for sequential OG-UV/vis CE analysis were investigated and optimized by analyzing a standard mixture of amino acids. High reproducibility of the sequential CE analysis was demonstrated with RSD values (n = 20) of 2.23, 2.57, and 0.70% for peak heights, peak areas, and migration times, respectively, and the LOD of 5.0 µM (for asparagine) and 2.0 µM (for aspartic acid) were obtained. With the application of the OG-UV/vis CE analysis, sequential online CE enzyme assay of L-asparaginase-catalyzed enzyme reaction was carried out by automatically and continuously monitoring the substrate consumption and the product formation every 12 s from the beginning to the end of the reaction. The Michaelis constants for the reaction were obtained and were found to be in good agreement with the results of traditional off-line enzyme assays. The study demonstrated the feasibility and reliability of integrating the OG injection with UV/vis detection for sequential online CE analysis, which could be of potential value for online monitoring various chemical reaction and bioprocesses.

Application of a portable infrared instrument for simultaneous analysis of sugars, asparagine and glutamine levels in raw potato tubers.

The level of reducing sugars and asparagine in raw potatoes is critical for potato breeders and the food industry for production of commonly consumed food products including potato chips and French fries. Our objective was to evaluate the use of a portable infrared instrument for the rapid quantitation of major sugars and amino acids in raw potato tubers using single-bounce attenuated total reflectance (ATR) and dial path accessories as an alternative to time-consuming chromatographic techniques. Samples representing a total of 84 experimental and commercial potato varieties harvested in two consecutive growing seasons (2012 and 2013) were used in this study. Samples had wide ranges of sugars determined by HPLC-RID (non-detectable (ND)-7.7 mg glucose, ND-9.4 mg fructose and 0.4-5.4 mg sucrose per 1 g fresh weight), and asparagine and glutamine levels determined by GC-FID (0.7-2.9 mg and 0.3-1.7 mg per 1 g fresh weight). Infrared spectra collected from 64 varieties were used to create partial least squares regression (PLSR) calibration models that predicted the sugar and amino acid levels in an independent set of 16 validation potato varieties. Excellent linear correlations between infrared predicted and reference values were obtained. PLSR models had a high correlation coefficient of prediction (rPred >0.95) and residual predictive deviation (RPD) values ranging between 3.1 and 5.5. Overall, the results indicated that the models could be used to simultaneously predict sugars, free asparagine and glutamine levels in the raw tubers, significantly benefiting potato breeding, certain aspects of crop management, crop production and research.

Analysis of deamidation artifacts induced by microwave-assisted tryptic digestion of a monoclonal antibody.

The thorough characterization of biopharmaceuticals is essential for ensuring their quality and safety since many potential variations can cause changes to the properties of a drug that may be detrimental to the patient such as decreased efficacy, shorter half-life or increased immunogenicity. Prior to approval and release, protein-based drugs are subject to a battery of analyses to assess the nature of those parameters that are considered critical quality attributes. In some cases the analytical method used may itself cause modifications that are impossible to distinguish from those induced by the intended test conditions (e.g. storage time/temperature, light exposure) which are used to assess drug stability. It is therefore important to develop and utilize analytical methods which impose as few artifactual modifications as possible. Asparagine deamidation is a common protein modification and it is known to be induced during tryptic digestion. Therefore we examined common tryptic digestion protocols and compared their propensities towards asparagine modification. Since microwave assisted hydrolysis techniques are often used to shorten digestion times and the effect on deamidation is unknown we sought to compare this method against alternate digestion protocols.

Genetic variation and possible SNP markers for breeding wheat with low-grain asparagine, the major precursor for acrylamide formation in heat-processed products.

BACKGROUND: In products made from wheat (Triticum aestivum) flour, acrylamide formation is almost exclusively determined by the level of free asparagine in the grain. Genetic variability for grain asparagine content was evaluated in order to assess the potential for acrylamide mitigation by breeding. RESULTS: Free asparagine levels in the grains of 92 varieties varied from 137 to 471 mg kg⁻¹, representing an approximate threefold difference between the low- and high-asparagine genotypes. Heritability was low, with a value of 32%, indicating that breeding cultivars with inherently low grain asparagine would be a challenge. A genome-wide scan with single-nucleotide polymorphism (SNP) markers identified nine SNPs that were significantly (P < 0.001) associated with variation in free asparagine. The significant SNPs were localized on chromosome 5A, and explained between 14% and 24% of the observed variation. These putative SNPs are candidates for further studies to develop molecular markers. CONCLUSION: Significant genetic variation exists for reducing acrylamide precursors in wheat flour, indicating that breeding and genetics could play an important role in mitigating the acrylamide risk in wheat products. The study identified a region on chromosome 5A that could provide a basis for further research to develop functional markers.

Chemical composition and sensory profiling of coffees treated with asparaginase to decrease acrylamide formation during roasting.

Acrylamide is an amide formed in the Maillard reaction, with asparagine as the primary amino acid precursor. The intake of large amounts of acrylamide has induced genotoxic and carcinogenic effects in hormone-sensitive tissues of animals. The enzime asparaginase is one of the most effective methods for lowering the formation of acrylamide in foods such as potatoes. However, the reported sensory outcomes for coffee have been unsatisfactory so far. This study aimed to produce coffees with reduced levels of acrylamide by treating them with asparaginase while retaining their original sensory and bioactive profiles. Three raw samples of Coffea arabica, including two specialty coffees, and one of Coffea canephora were treated with 1000, 2000, and 3000 ASNU of the enzyme. Asparagine and bioactive compounds (chlorogenic acids-CGA, caffeine, and trigonelline) were quantified in raw and roasted beans by HPLC and LC-MS, while the determination of acrylamide and volatile organic compounds was performed in roasted beans by CG-MS. Soluble solids, titratable acidity, and pH were also determined. Professional cupping by Q-graders and consumer sensory tests were also conducted. Results were analyzed by ANOVA-Fisher, MFA, PCA and Cluster analyses, with significance levels set at p ≤ 0.05. Steam treatment alone decreased acrylamide content by 18.4%, on average, and 6.1% in medium roasted arabica and canefora coffees. Average reductions of 32.5-56.0% in acrylamide formation were observed in medium roasted arabica beans when 1000-3000 ASNU were applied. In the canefora sample, 59.4-60.7% reductions were observed. However, steam treatment primarily caused 17.1-26.7% reduction of total CGA and lactones in medium roasted arabica samples and 13.9-22.0% in canefora sample, while changes in trigonelline, caffeine, and other evaluated chemical parameters, including the volatile profiles were minimal. Increasing enzyme loads slightly elevated acidity. The only sensory changes observed by Q-graders and or consumers in treated samples were a modest increase in acidity when 3000 ASNU was used in the sample with lower acidity, loss of mild off-notes in control samples, and increased perception of sensory descriptors. The former was selected given the similarity in chemical outcomes among beans treated with 2000 and 3000 ASNU loads.

An 18O-labeling assisted LC/MS method for assignment of aspartyl/isoaspartyl products from Asn deamidation and Asp isomerization in proteins.

An (18)O-labeling assisted LC/MS method was designed for unambiguous assignment of aspartyl/isoaspartyl products produced by Asn deamidation and Asp isomerization. By preparing the acid- and base-catalyzed deamidation standards in H2(18)O, isomer-specific mass tags were introduced to aspartyl- and isoaspartyl-containing peptides, which could be easily distinguished by mass spectrometry (MS). In contrast to the traditional ways of assigning the isomers on the basis of their elution order in reverse phase HPLC, the new method is more reliable and universal. Furthermore, the new method can be applied to the entire protein digest, and is therefore more time- and cost-effective compared with existing methods that use synthetic aspartyl- and isoaspartyl-containing peptide standards. Finally, since the identification of isomers in the new method only relies on LC/MS analysis, it can be easily implemented using the most basic and inexpensive MS instrumentation, thus providing an attractive alternative to tandem MS based approaches. The feasibility of this new method is demonstrated using a model peptide as well as the entire digest of human serum transferrin.

Comparative analysis of barophily-related amino acid content in protein domains of Pyrococcus abyssi and Pyrococcus furiosus.

Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code.

Mass spectrometric peptide analysis of 2DE-separated mouse spinal cord and rat hippocampus proteins suggests an NGxG motif of importance for in vivo deamidation.

Asparagine deamidation is a common nonenzymatic post-translational modification comprising the conversion of asparaginyl residues to aspartyl and isoaspartyl residues, respectively. As a result an additional negative charge is introduced that can affect the tertiary structure as well as the biological activity of a protein. Since deamidation reduces the protein's pI value, differentially deamidated forms of a protein can be separated in 2D gels. We have analyzed a dataset of 430 protein spots from 2D gels that contained mouse spinal cord proteins and estimated that roughly 10% of the spots in a Coomassie-stained gel derive from in vivo deamidation at particular asparaginyl residues. Several of the deamidated protein forms, e.g. tropomodulin-2, V-type proton ATPase subunit B, and protein disulfide-isomerase A3 were also found in 2D gels of proteins extracted from rat hippocampus. All identified deamidation sites contained a glycine residue on the carboxyl side of the asparaginyl residue. Strikingly, a second glycine residue at the +3 position was found in the majority of the deamidated peptides. We propose that the NGxG motif confers exceptional susceptibility to in vivo asparagine deamidation.

No evidence of increased asparagine levels in the bone marrow of patients with acute lymphoblastic leukemia during asparaginase therapy.

BACKGROUND: Mesenchymal cells (MSCs) in bone marrow (BM) may produce asparagine and form protective niches for leukemic cells. In vitro, this led to high levels of asparagine and conferred asparaginase resistance to acute lymphoblastic leukemia (ALL) cells. The aim of this study was to investigate whether MSCs or other cells in BM indeed produce such significant amounts of asparagine in vivo as to result in clinical asparaginase resistance. PROCEDURE: Twenty-six patients with newly diagnosed ALL were enrolled. All children received induction chemotherapy according to the Dutch Childhood Oncology Group (DCOG) ALL-10 protocol. Asparaginase was administered from days 12-33. Asparaginase, asparagine, aspartic acid, glutamine, and glutamic acid levels were measured in BM and blood at diagnosis, days 15, 33, and 79. RESULTS: Median asparaginase trough levels were not significantly different at days 15 and 33. Only at diagnosis, asparagine level was significantly higher in BM than in blood (P = 0.001). Asparagine levels were all below the lower limit of quantification in BM and blood at days 15 and 33. However, aspartic acid level in BM was significantly higher than in blood (P < 0.001) at diagnosis, and also at days 15, 33, and 79. CONCLUSIONS: We demonstrate higher aspartic acid levels in BM compared to blood; however, no increased asparagine levels were seen during induction therapy containing asparaginase in BM when compared to blood. Therefore, increased asparagine synthesis by MSCs is of relevance for resistance to asparaginase of leukemic cells in vitro, but it is questionable whether this leads to asparaginase resistance in childhood ALL patients.

Acrylamide reduction in potato chips by selection of potato variety grown in Iran and processing conditions.

BACKGROUND: Acrylamide as a possible carcinogen is known to form in heated carbohydrate-rich food such as potato chips. In this study, the effect of three potato varieties (Agria, Sante and Savalan) and two blanching conditions (75 °C for 9 min and 83 °C for 2.5 min) on the concentration of precursors and acrylamide reduction in potato chips was investigated. RESULTS: Results revealed that potato variety and blanching time-temperature were important parameters for acrylamide formation in potato chips. Acrylamide content in Sante variety potatoes, which contained the highest amount of reducing sugars, was found to be the highest (8825 µg kg(-1)). However, Savalan, containing the highest asparagine concentration, showed the lowest amount of acrylamide due to its lower reducing sugar content. Blanching reduced acrylamide formation; it was more efficient at 75 °C for 9 min, with an average reduction of 74%. The effect of three frying temperatures (170, 180 and 190 °C) on acrylamide formation was also studied just for the Agria potato variety. Increasing frying temperature led to a significant increase in acrylamide formation. CONCLUSION: Potato variety and processing conditions were important parameters for acrylamide formation in potato chips. The combination of a suitable variety and appropriate processing conditions could considerably reduce acrylamide content.

Identifying the origin of acrylamide in Peruvian panela production to inform strategies for its reduction.

Maximum levels of acrylamide have been set by the European Commission (EU) 2017/2158 for several food products due to its carcinogenic properties. Although not regulated yet, European buyers are requesting maximum levels of 0.8 mg kg-1 in artisanal panela (raw cane sugar) from northern Peru. Panela in this area is produced by 600 small holder farmers and exportation guarantees a respectable price in an area with a high index of poverty. The objective here was to determine the cause of high acrylamide concentrations in panela to inform cost effective minimisation strategies. We monitored panela production from field to final product to understand the scale of the problem, identify the cause of acrylamide formation, as well as the effect of storage on its concentration. We also determined the utility of rapid kits for asparagine quantification. Our results indicate that high acrylamide levels are a widespread problem (85% of samples analysed) and there was a correlation between acrylamide and asparagine of R2 = 0.58 (p < 0.001), but not with any post-harvest processing variable. We estimate that with a concentration of asparagine of <0.1 g l-1 in sugarcane juice, the threshold set by buyers for acrylamide can be met. Potential solutions to reduce asparagine include varietal selection, improved agronomic practices and the use of asparaginase during panela production. However, any proposed measure should be applicable in the context of the rural Peru. Additionally, we confirm the utility of rapid and low-cost kits for measuring asparagine. This pioneering study provides a baseline for effective management for acrylamide minimization in panela.