This is a repository copy of Determination of amygdalin in apple seeds, fresh apples and processed apple juices. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/81239/ Version: Accepted Version Article: Bolarinwa, IF, Orfila, C and Morgan, MRA (2015) Determination of amygdalin in apple seeds, fresh apples and processed apple juices. Food Chemistry, 170. 437 - 442. ISSN 0308-8146 https://doi.org/10.1016/j.foodchem.2014.08.083 Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version - refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher’s website. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ 1 Determination of amygdalin in apple seeds, fresh apples and the 2 processed apple juices 3 Islamiyat F. Bolarinwaa,b,c, Caroline Orfilaa, Michael R.A. Morgana 4 aSchool of Food Science & Nutrition, University of Leeds, Leeds LS2 9JT, United Kingdom 5 bDepartment of Food Science & Engineering, Ladoke Akintola University of Technology, PMB 4000, Ogbomoso, Nigeria 6 cCorresponding author : email [email protected], [email protected], phone no: +2348022526700 7 8 9 10 Abstract 11 Cyanogenic glycosides are natural plant toxicants. Action by endogenous plant enzymes 12 can release hydrogen cyanide causing potential toxicity issues for animals including 13 humans. We have quantified amygdalin in seeds from different apple varieties, determined 14 the effects of processing on the amygdalin content of apple juice and quantified amygdalin 15 in commercially-available apple juices. Amygdalin contents of seeds from fifteen varieties of 16 apples ranged from 1 mg g-1 to 4 mg g-1. The amygdalin content of commercially-available 17 apple juice was low, ranging from 0.01 to 0.04 mg ml-1 for pressed apple juice and 0.001 to 18 0.007 mg ml-1 for long-life apple juice. Processing led to juice with low amygdalin content, 19 ranging from 0.01 mg ml-1 to 0.08 mg ml-1. The results presented show that the amygdalin 20 contents of commercially-available apple juices are unlikely to present health problems to 21 consumers. 22 23 Keywords: Cyanogenic glycosides, amygdalin, apples, apple juice, pasteurization. 24 25 26 27 22 28 Introduction 29 Apple (Malus domestica) is a member of the Rosaceae family that also includes 30 apricots, cherry, peaches, pear and plum and is the most widely consumed fruit in the UK. 31 World apple production in 2011/12 was estimated to be 65.23 million metric tons, out of 32 which 12.2 million metric tons was used for the production of apple juice (Negro & Lojo, 33 2011). Although apple contains compounds which may confer significant health benefits to 34 humans, apple seeds contain amygdalin (Fig.1), a potentially toxigenic compound. 35 Commercial apple juice is usually made from a blend of apples to produce an 36 acceptable juice in terms of flavour. Apples are soaked in water to remove soil and other 37 foreign material. The cleaned apples are then inspected, and damaged or decayed fruit 38 should be removed to avoid patulin contamination from the final product. The sorted 39 apples are ground into mash or pulp for extraction, crushing or cutting the apples to 40 appropriate consistency. The mashed apples are pressed by applying pressure to obtain 41 the juice. In some cases, enzymatic mash treatment is used to improve the pressability of 42 the mash and increase juice yield, achieved by adding a pectinase enzyme such as 43 pectinol specifically developed for apple mash pre-treatment. The enzyme acts mainly by 44 breaking down the structure of the cell wall, thus freeing the juice. 45 Cyanogenic glycosides, including amygdalin, are naturally-occurring plant toxins. 46 They are widely distributed in the plant kingdom, being present in more than 2500 species 47 (Ganjewala, Kumar, Asha, & Ambika, 2010). Cyanogenic glycosides are stored in vacuoles 48 within plant cells. When tissues are disrupted, for example by crushing, the cyanogenic 49 glycosides come into contact with endogeneous enzymes (-glucosidases and - 22 50 hydroxynitrile lyases) resulting in the release of hydrogen cyanide (Zagrobelny, Bak, 51 Rasmussen, Jørgensen, Naumann, & Møller, 2004). In plants, consequently, cyanogenic 52 glycosides serve as important chemical defence compounds against herbivores 53 (Zagrobelny et al., 2004; Ganjewala et al., 2010). In humans, consumption of cyanogenic 54 plants can cause sub-acute cyanide poisoning with symptoms including anxiety, headache, 55 dizziness and confusion. Acute poisoning results in decreased consciousness, 56 hypotension, paralysis, coma and even death. Acute cyanide poisoning has been reported 57 from the ingestion of apricot kernels (Sahin, 2011), almonds (Sanchez-Verlaan, 2011) and 58 cassava (Akintonwa & Tunwashe, 1992). 59 Cyanogenic glycosides are present in economically important food plants such as 60 apple, almond, various beans, cereals, cassava, taro and sorghum (Jones, 1998; Donald, 61 2009). Processing techniques such as pounding, crushing, grinding, soaking, fermentation, 62 boiling and drying have been used over the years to reduce the cyanide contents of foods. 63 Processing allows contact between cyanogenic glycosides and endogeneous enzymes 64 which results in the hydrolytic breakdown of cyanogenic glycosides to hydrogen cyanide. 65 Because the boiling point of hydrogen cyanide is 26 oC, it easily volatilizes during food 66 processing (Montagnac, Davis, & Tanumihardjo, 2009). Quantification of cyanogenic 67 glycosides in plants is carried out either indirectly (by determining the amount of hydrogen 68 cyanide released after hydrolysis) or directly (by determining the intact form). The indirect 69 method is the most commonly used analytical method and usually involves enzymatic 70 hydrolysis followed by colorimetric determination of total cyanide (Bradbury, Egan, and 71 Lynch, 1991; Santamour, 1998). Methods for determination of intact cyanogenic glycosides 72 include liquid chromatography with refractive index detection (Sornyotha, Kyu, & 22 73 Ratanakhanokchai, 2007), gas chromatography/mass spectrometry (Chassagne, Crouzet, 74 Bayonove, & Baumes, 1996), and HPLC with UV detection (Bolarinwa, Orfila & Morgan, 75 2014). 76 Although humans, generally, do not consume apple seeds, apple juice is generally 77 produced from whole apples including the seeds. Apple seeds disintegrate during juice 78 production and contaminate the juice. While there are extensive studies on the antioxidant 79 composition of apple juice (Spanos, Wrolstad, & Heatherbell, 1990; Miller, Diplock, & Rice- 80 Evans, 1995), microbial safety and preservation of apple juice (Evrendilek, Jin, Ruhlman, 81 Qui, Zhang, & Richter, 2000), there has been no study on the amygdalin content of apple 82 juice, and there is limited information on the amygdalin contents of apple seeds 83 (Holzbecher, Moss, & Ellenberger, 1984; Haque & Bradbury, 2002). 84 85 2. Materials and Methods 86 2.1. Reagents and standards 87 Amygdalin, ethanol, diethyl ether, and HPLC-grade methanol were all purchased 88 from Sigma-Aldrich (Dorset, UK). Water was prepared using a Millipore Milli-Q purification 89 system. All other reagents were of analytical grade. 90 2.2. Apples 91 Fifteen varieties of apples (Braeburn, Bramley, Cox, Elstar, Empire, Egremont 92 Russet, Fuji, Golden Delicious, Granny Smith, Jazz, Pink Lady, Red Delicious, Royal Gala, 93 Rubens and Spartan) were purchased from local supermarkets in Leeds (UK). The apples 94 were stored at 4 oC immediately after purchase prior to processing. 22 95 2.2.1. Extraction of amygdalin from apple seeds 96 Apples were each cut into four equal parts and apple seeds were separated from 97 other tissues with a knife and extracted immediately. Apple seeds (2 g) were disintegrated 98 with a mortar and pestle, and 1 g was weighed into a round-bottom flask (500 ml). Ethanol 99 (50 ml) was added, and the mixture was boiled under reflux for 100 min. The extract was 100 filtered (Whatman No. 1 filter paper) and transferred into plastic polypropylene tubes (50 101 ml). Ethanol was completely evaporated from the filtrate with a rotary evaporator (low BP, 102 35 oC, 7 mbar). Diethyl ether (10 ml) was added to the dried sample and the mixture was 103 vortexed (1 min) at room temperature (20o ± 2 oC) to precipitate amygdalin. The diethyl 104 ether was allowed to evaporate overnight and the extracted amygdalin was dissolved in 105 water (5 ml) and prepared for HPLC analysis (2.5). 106 107 2.2.2. Apple juice extraction 108 2.2.2.1. Apple juice from whole apple 109 Four samples of apple juice were produced from four apple cultivars (Braeburn, 110 Egremont Russet, Golden Delicious and Royal Gala). Whole apples (10) were each 111 washed, cut into 4 pieces and pressed in a commercial juice extractor (Kenwood JE 600). 112 The extracted juice from each apple variety was divided into