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Cyanogenesis in Macadamia and Direct Analysis of Hydrogen Cyanide in Macadamia Flowers, Leaves, Husks, and Nuts Using Selected Ion Flow Tube–Mass Spectrometry

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Cyanogenesis in Macadamia and Direct Analysis of Hydrogen Cyanide in Macadamia Flowers, Leaves, Husks, and Nuts Using Selected Ion Flow Tube–Mass Spectrometry foods Article Cyanogenesis in Macadamia and Direct Analysis of Hydrogen Cyanide in Macadamia Flowers, Leaves, Husks, and Nuts Using Selected Ion Flow Tube–Mass Spectrometry Hardy Z. Castada 1,* , Jinyi Liu 2, Sheryl Ann Barringer 1 and Xuesong Huang 2,* 1 Department of Food Science and Technology, The Ohio State University, 2015 Fyffe Road, Columbus, OH 43210, USA; [email protected] 2 Department of Food Science and Engineering, Jinan University, Guangzhou 510632, China; [email protected] * Correspondence: [email protected] (H.Z.C.); [email protected] (X.H.); Tel.: +1-614-247-1945 (H.Z.C.) Received: 16 January 2020; Accepted: 7 February 2020; Published: 11 February 2020 Abstract: Macadamia has increasing commercial importance in the food, cosmetics, and pharmaceutical industries. However, the toxic compound hydrogen cyanide (HCN) released from the hydrolysis of cyanogenic compounds in Macadamia causes a safety risk. In this study, optimum conditions for the maximum release of HCN from Macadamia were evaluated. Direct headspace analysis of HCN above Macadamia plant parts (flower, leaves, nuts, and husks) was carried out using selected ion flow tube–mass spectrometry (SIFT-MS). The cyanogenic glycoside dhurrin and total cyanide in the extracts were analyzed using HPLC-MS and UV–vis spectrophotometer, respectively. HCN released in the headspace was at a maximum when Macadamia samples were treated with pH 7 buffer solution and heated at 50 ◦C for 60 min. Correspondingly, treatment of Macadamia samples under these conditions resulted in 93–100% removal of dhurrin and 81–91% removal of total cyanide in the sample extracts. Hydrolysis of cyanogenic glucosides followed a first-order reaction with respect to HCN production where cyanogenesis is principally induced by pH changes initiating enzymatic hydrolysis rather than thermally induced reactions. The effective processing of different Macadamia plant parts is important and beneficial for the safe production and utilization of Macadamia-based products. Keywords: Macadamia; hydrogen cyanide; cyanogenic glycosides; headspace analysis; SIFT-MS 1. Introduction Macadamia-based commercial products have rapidly increased in recent years. In addition to Macadamia nuts, Macadamia flowers, husks, leaves, and shells are now widely used as a source of functional foods, beverages, and raw materials in cosmetics, feed, and other applications. Abundant antioxidant substances, such as polyphenols, can be extracted from Macadamia skin and husks for utilization in the food and pharmaceutical industries [1–3]. Bioactive constituents in Macadamia are believed to provide health benefits such as improved blood lipid profiles, decreased inflammation, oxidative stress, and reduced cardiovascular disease risk factors [4,5]. Species within the genus Macadamia, however, contain cyanogenic glycoside compounds, which are secondary metabolites that release hydrogen cyanide through cyanogenesis [6,7]. Cyanogenic glycosides are widely distributed in plants and are usually O-β-glycosidic derivatives of α-hydroxynitriles originating from the five hydrophobic protein amino acids tyrosine, phenylalanine, valine, leucine, and isoleucine [8,9]. Biological cyanogenesis requires unstable cyanohydrin or a stable cyanogen and degradative enzymes [10,11]. In plants, cyanogenesis mostly occurs via Foods 2020, 9, 174; doi:10.3390/foods9020174 www.mdpi.com/journal/foods Foods 2020, 9, x FOR PEER REVIEW 2 of 15 Foods 2020, 9, 174 2 of 15 cyanogen and degradative enzymes [10,11]. In plants, cyanogenesis mostly occurs via hydrolysis of cyanogenic glycosides that is enzymatically catalyzed by one or more glycosidases and α- hydrolysis of cyanogenic glycosides that is enzymatically catalyzed by one or more glycosidases and hydroxynitrile lyases. These substrates and enzymes are localized in separate vacuoles intact within α-hydroxynitrile lyases. These substrates and enzymes are localized in separate vacuoles intact within the subcellular or tissue level of the plant so that under normal physiological conditions, hydrolysis the subcellular or tissue level of the plant so that under normal physiological conditions, hydrolysis does not occur and autotoxicity is prevented [12,13]. However, tissue disruption allows mixing of does not occur and autotoxicity is prevented [12,13]. However, tissue disruption allows mixing of substrate and enzymes initiating cyanogenesis and subsequently, hydrolysis [6,7]. Figure 1 shows a substrate and enzymes initiating cyanogenesis and subsequently, hydrolysis [6,7]. Figure1 shows two-step enzymatic hydrolysis of dhurrin (4-hydroxymandelonitrile-β-D-glucoside), a cyanogenic a two-step enzymatic hydrolysis of dhurrin (4-hydroxymandelonitrile-β-d-glucoside), a cyanogenic glycoside found in Macadamia. A more complex biosynthetic pathway for dhurrin hydrolysis is glycoside found in Macadamia. A more complex biosynthetic pathway for dhurrin hydrolysis is available in the literature [14,15]. In Figure 1, hydrolysis is initially catalyzed by endogenous available in the literature [14,15]. In Figure1, hydrolysis is initially catalyzed by endogenous dhurrinase (a β-D-glucoside glucohydrolase) producing D-glucose and an unstable intermediate, p- dhurrinase (a β-D-glucoside glucohydrolase) producing D-glucose and an unstable intermediate, hydroxy-(S)-mandelonitrile. The nitrile group is rapidly cleaved off of this intermediate, producing p-hydroxy-(S)-mandelonitrile. The nitrile group is rapidly cleaved off of this intermediate, producing hydrogen cyanide and p-hydroxybenzaldehyde, which is catalyzed enzymatically by α- hydrogen cyanide and p-hydroxybenzaldehyde, which is catalyzed enzymatically by α-hydroxynitrile hydroxynitrile lyase [16,17]. lyase [16,17]. Figure 1. Enzymatic hydrolysis of dhurrin [17,18]. Figure 1. Enzymatic hydrolysis of dhurrin [17,18]. Acute and chronic toxicities of hydrogen cyanide from plant-derived food have been reported [16,19,20]. IngestionAcute of and0.5–3.5 chronic mg cyanidetoxicities/kg of body hydrogen weight cyani resultsde from in acute plant-derived toxicity. Sublethal food have doses been could reported lead [16,19,20].to headache, Ingestion hyperventilation, of 0.5–3.5 mg vomiting, cyanide/kg weakness, body weight abdominal results cramps, in acute and toxicity. partial Sublethal circulatory doses and couldrespiratory lead systemsto headache, failure. hyperv Moreover,entilation, cyanide vomiting, can inhibit weakness, cellular respiration,abdominal whichcramps, could and result partial in circulatoryfatal poisoning and [respiratory7,16,21]. The systems concentration failure. Moreover of cyanogenic, cyanide glycosides, can inhibit such cellular as dhurrin respiration, and proteacin, which couldvaries result among in plantfatal poisoning species of [7,16,21].Macadamia The(i.e., concentrationM. ternifolia of, M. cyanogenic integrifolia glycosides,, and M. tetraphylla such as dhurrin)[6,22]. andThese proteacin, compounds varies are alsoamong unevenly plant species distributed of Macadamia within the (i.e., different M. ternifolia parts of, aM. plant integrifolia (i.e., nuts,, and seeds, M. tetraphyllaand roots),) [6,22]. and their These concentrations compounds changeare also atunevenly different dist developmentalributed within stages the different from seed parts germination of a plant (i.e.,to plant nuts, maturation seeds, and [ 23roots),,24]. and their concentrations change at different developmental stages from seed Ingermination this study, to di ffplanterent maturation conditions causing[23,24]. the hydrolysis of cyanogenic compounds in Macadamia that subsequentlyIn this study, produce different hydrogen conditions cyanide causing gas werethe evaluated.hydrolysis Mostof cyanogenic characterization compounds studies in Macadamia thatand othersubsequently plants only produce involved hydrogen analysis cyani of cyanogenicde gas were glycosides evaluated. usingMost time-consumingcharacterization studiesassays coupled in Macadamia with HPLC and other analysis plants [6,25 only,26]. involved Furthermore, analysis typical of cyanogenic analysis of releasableglycosides cyanide using time- uses consumingtedious assays assays and coupled subsequent with spectrophotometry HPLC analysis [6,25,26]. or LC- Furthermore, or GC-MS analysis typical [14 analysis,27,28]. Inof releasable this study, cyanidehydrogen uses cyanide tedious was assays measured and subsequent directly above spectr theophotometry headspace of or the LC- di fforerent GC-MS parts analysis of the Macadamia[14,27,28]. Inplant this including study, hydrogen the flowers, cyanide leaves, was husks, measured and nutsdirectly using above selected the headspace ion flow tube–mass of the different spectrometry parts of (SIFT-MS).the Macadamia To ourplant knowledge, including the this flowers, is the first leaves, study husks, to measure and nuts hydrogen using selected cyanide ion inflow real tube–mass time and spectrometrydirectly above (SIFT-MS). the headspace To our of Macadamia knowledge,samples this is using the first SIFT-MS. study The to measure rapid and hydrogen real-time cyanide analysis in of realhydrogen time and cyanide directly is particularly above the headspace important inof theMacadamia processing samples of the using various SIFT-MS. parts of TheMacadamia rapid andthat real- are timeknown analysis to contain of hydrogen cyanogenic cyanide glycosides is particularly and can subsequentlyimportant in hydrolyzethe processing and undergoof the various cyanogenesis. parts of MacadamiaThe optimum that conditions are known
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