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Measurement of lipoxygenase in Australian white wheat flour: the effect of lipoxygenase on the quality properties of white salted noodles

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Measurement of lipoxygenase in Australian white wheat flour: the effect of lipoxygenase on the quality properties of white salted noodles
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   Journal of the Science of Food and Agriculture J Sci Food Agric  86 :1670–1678 (2006) Measurement of lipoxygenase in Australian white wheat flour: the effect oflipoxygenase on the quality properties of whitesalted noodles Larisa Cato, ∗  Andrew L Halmos and Darryl M Small School of Applied Sciences, RMIT University, GPO Box 2476V, Melbourne, VIC 3001, Australia Abstract: The enzyme lipoxygenase has a number of functions in breadmaking. Although white salted noodles area staple food in various countries, the significance and potential of lipoxygenase in noodlemaking are less wellunderstood. In these products a bright, uniform appearance is particularly important and so the aim of the presentresearch has been to study the effect of endogenous and exogenous lipoxygenase upon discolouration of whitesalted noodles as well as on the textural and structural attributes. Similar lipoxygenase levels were recorded in theflours studied and no significant losses of activity were found during noodle manufacture and subsequent storage.Less discolouration occurred in treated noodle sheets compared with control samples. Discolouration happenedto a lesser extent when samples were cooked immediately after preparation or drying for both treated and controlnoodles. Whiter noodle sheets were obtained when a soybean lipoxygenase was added to the formulation. Texturaland structural properties of white salted noodles were not adversely affected by enzyme addition, giving firm,elastic and non-sticky products. It is concluded that the incorporation of the lipoxygenase preparation offersprospects for colour enhancement of white salted noodles. © 2006 Society of Chemical Industry Keywords:  Asian noodles; white salted noodles; lipoxygenase; textural characteristics; noodle colour INTRODUCTION Although rice is the staple cereal throughout Asia,wheat is also an important commodity as the rawingredient for many different types of noodles, whichare a staple food of the region. Over 12% of globalwheat production is utilised for noodle manufacture,and the consumption of wheat in Asian countriescontinues to expand. Furthermore, a significantproportion ( ∼ 39%) of all Australian bread wheatexports is used for noodle production. 1 White salted noodles (WSN) are one of the pri-mary types of Asian noodles. 2 , 3 Although WSN aretypically made using only flour, water and salt, thereis considerable variation in them, due to the char-acteristics of the raw materials used, product shapeas well as processing methods. This partially reflectsvarying regional preferences. 4 The quality of WSNhas various aspects: appearance, eating quality, tasteand cooking properties. 5 , 6 Colour and brightness areimportant, while eating quality is the primary criterionfor consumer acceptance. Although preferences varyfrom region to region, generally softness, tendernessand some feeling of elasticity are desirable 7 and thesedepend upon both the protein and starch componentsof the flour. 8 The enzyme lipoxygenase (LOX, EC 1.13.11.12)has a variety of effects on wheat flour dough. It hasbeen reported that LOX increases mixing toleranceand generally enhances dough rheological properties. 9 However, the enzyme is most commonly thought of asableachingagent.Thisactionisbelievedtoinvolvetheoxidation of pigments and unsaturated fatty acids byoxygen. Fatty acid radicals produced during the inter-mediatestepsofsubstrateperoxidationareresponsiblefor oxidative degradation of pigments including  β -carotene, xanthophylls and chlorophylls. 10 , 12 Whilstthesignificanceoftheenzymeforbreadandpastaqual-ity has been researched in considerable detail, 9 , 11 , 13 less is known of its role, influence and potential innoodle products 13 , 14 . Accordingly, the objective of this study has been to examine the effect of an exoge-nous LOX preparation from soybean on textural andcolour properties of WSN. MATERIALS AND METHODS Australian commercial white wheat flours, UltraWhite (UW) and P-Farina (PF), were from AlliedMills (Melbourne, Australia). Enzyme LOX (TypeV; L6632; suspension in 2.3molL  − 1 ( NH 4 ) 2 SO 4 solution,pH ∼ 6;purifiedby2 × crystallisation)wasof soybeansrcin,obtainedfromSigma-Aldrich(Sydney,Australia). LOX units, defined by the manufacturer,cause an increase in  A 234  of 0 . 001 min − 1 at pH ∗ Correspondence to: Larisa Cato, AWB Ltd., 260 Princess Hwy, Werribee, VIC 3030, AustraliaE-mail: lcato@awb.com.auContract/grant sponsor: Grains Research & Development Corporation (GRDC), Canberra, Australia(  Received 2 June 2005; revised version received 15 November 2005; accepted 30 November 2005  )Published online 12 July 2006 ;  DOI: 10.1002/jsfa.2539 ©  2006 Society of Chemical Industry.  J Sci Food Agric  0022–5142/2006/$30.00  Lipoxygenase effects in white salted noodles 9 and 25 ◦ C when linoleic acid is the substrate in3mL volume (1cm light path). One  A 234  unit isequivalent to the oxidation of 0 . 12 µ mol of linoleicacid. Linoleic acid (minimum 99%, sealed ampoule)was from Sigma-Aldrich. Chemical analysis Moisture content of flour and noodles was determinedby oven drying at 130 ◦ C until constant weight wasreached. 15 Ash content of flour and noodles wasdetermined by dry combustion in a furnace for 24h at590 ◦ C. 15 Protein content (N × 5 . 7) was determinedusing the Kjeldahl method. 15 pH values of flourand noodle samples were measured by the AACCmethod. 15 Total starch was determined using theMegazyme Total Starch Kit (Megazyme InternationalIreland Ltd, Bray, Ireland). 15 This method allowsthe measurement of total starch in most processedor unprocessed cereal products. Here the starchhydrolysis proceeds in two phases: first, starch ispartially hydrolysed and totally solubilised; second,the starch dextrins are quantitatively hydrolysed toglucose by amyloglucosidase. 16 Each batch of raw and dried noodles was cookedto optimum time, which had been established in apreliminary cooking test. Portions of 10g were cookedin 300mL of distilled water and then rinsed withwater at ambient temperature. 15 Noodle strands werepressedbetweentwo glassplatesandtheoptimumwasrecorded as the time when the gelatinised zone hadreached the centre of the strands. For WSN this was2.30min.Cooked weight and cooking loss were determinedby methods modified from Refs 17 and 18. Noodles(10g)werecookedin300mLofdistilledwatertotheiroptimum cooking time, rinsed with distilled water, leftto drain for2minatroom temperature,reweighed andresults reported as the increase on cooking (%). Theresidual cooking water was dried in an oven at 110 ◦ Cand results reported as the proportion of initial weightlost (%) during cooking.All results have been adjusted to a 14% moisturebasis and are presented as mean ± standard deviation. Preparation of WSN Noodles were prepared in the laboratory by methodsmodified from Refs 8, 19 and 20. Optimum waterabsorption was determined based on the appearanceof the dough and dough sheet as well as handlingproperties of the sheet during the noodlemakingprocess. Flour (200g) was mixed with water (68mL)containing 8g of NaCl using a Kenwood mixer fittedwith a K-beater. The crumbly dough was repeatedlypassed through a pair of rolls (type MOD 150,Imperia, Torino, Italy), with a final gap of 2mm,and subsequently cut into strips. LOX preparationwas dissolved in phosphate buffer (0.1molL  − 1 , pH7.5) prior to addition to the formulation. Raw noodleswereassessedimmediatelyfollowingpreparation(timezero) and following 24h of storage at either ambienttemperature ( ∼ 25 ◦ C) or 4 ◦ C. Samples were stored inpolyethylene bags to prevent moisture loss. Noodleswere also cooked and then assessed immediately andfollowing storage. Batches of raw noodles were alsodried in a convection oven at 40 ◦ C for 30h. LOX extraction from flour LOX was extracted with phosphate buffer(0.1molL  − 1 , pH 7.5). Flour or noodle samples(2g) were mixed with phosphate buffer (10mL) andagitated on ice for 30min. The mixture was thenmaceratedusing an Ultra Turrax (Stauffen,Germany)(2 × 15s) and centrifuged immediately at 3000 ×  g  for 20min at 4 ◦ C. Assays for LOX activity werecompleted within 30min of extraction. LOX assay  The activity of the enzyme LOX was determinedby a method modified from Refs 14 and 21–26.LOX activity was determined spectrophotometricallyat 25 ◦ C. The substrate used was prepared as follows.Tween 20 (0.5mL) was dissolved in borate buffer(50mmolL  − 1 , pH 9, 10mL); vigorous mixing wasavoided to prevent bubble formation in the solution.Linoleic acid (0.5mL) was added dropwise and themixture was stirred thoroughly to disperse the acidinto a fine emulsion. To this, NaOH (1molL  − 1 ,1.3mL) was added and the mixture was agitateduntil a clear solution was obtained. Finally, boratebuffer (50mmolL  − 1 , pH 9, 90mL) was added,the total volume was made up to 200mL withdistilled water and the pH was adjusted to 7 withHCl. The resulting solution was approximately 7 . 5 × 10 − 3 molL  − 1 in linoleic acid. 22 The reaction mixturecomprised sodium acetate buffer (50mmolL  − 1 , pH5.5, 950 µ L), linoleic acid substrate (30 µ L) andenzyme extract (20 µ L). LOX activity was expressedas  µ mol hydroperoxide formed min − 1 g − 1 using anextinction value of 2 . 5 × 10 4 Lmol − 1 cm − 1 . 23 Colour measurement of WSN ThecolourofnoodleswasdeterminedusingaMinoltaCR310 Chroma Meter (Osaka, Japan). 27 For rawnoodle sheets, measurements were made immediatelyafter preparation (time zero), then every hour for theperiod up to 9h, and at 24, 48 and 72h followingstorage at either 25 or 4 ◦ C. The colour of cookednoodles was measured immediatelyafter fresh noodleshad been cooked to the optimum. For this, noodleswere packed between two glass plates and readingswere taken. The colour of noodle sheets dried underpreviously defined conditions was also measured. Textural profiles of WSN WSN were tested with a TA-XT2 texture analyserequipped with Texture Expert Exceed software(Stable Micro Systems Ltd, London, UK). 28 Forall measurements the TA-XT2 was equipped with a5kg load cell. Two attachments were evaluated. Firsta cylinder probe (P/45) was used and compression  J Sci Food Agric  86 :1670–1678 (2006)  1671 DOI: 10.1002/jsfa  L Cato, AL Halmos, DM Small force (N s) was measured. For each test, two strandswere placed side by side, as close to each otheras possible and ensuring that the strand test regionwas positioned centrally under the probe. For probecalibration a 15mm return trigger path was used(Stable Micro Systems Ltd). The following settingswere used: mode – measure force in compression;option – return to start; pre-test speed 2mm s − 1 ; testspeed2mms − 1 ;post-testspeed2mms − 1 ;strain75%;trigger type – auto, 10g; data acquisition rate 200pps.Analyses were performed on two separate batches of noodles for each treatment, with 20 subsamples beingcompressed for each batch.The secondattachmentused wasaflatcutting blade(7cm × 11 . 5cm × 0 . 3cm) measuring maximum cut-tingforce(Ns).Againtwobatcheswereanalysed,with15 cuttings for each. Settings were: mode – measuremaximum cutting force; option – return to start; pre-test speed 0.5mm s − 1 ; test speed 0.17mm s − 1 ;post-test speed 10mm s − 1 ; distance 4.5mm; triggertype – button; data acquisition rate 400pps; test set-up – five strands of noodles adjacent to one anothercentrally under the knife blade, with the axis of theproduct strands at right angles to the blade. 28 The results from the cylinder probe were designatedas noodle hardness and force required to compressnoodles to 75%, and for the cutting blade as noodlefirmness,whichisthemaximumcuttingforcerequiredto penetrate strands, expressed as the work (N s)required to shear one strand of noodle. Structural characteristics of WSN Structural properties of WSN were examined using aPhilips XL (Eindhoven, the Netherlands) 30 scanningelectron microscope (SEM) with the followingsettings: temperature 23 ◦ C, pressure 0.5Torr, spotsize 5, voltage 30kV. Cooked noodles were freeze-dried immediately after cooking and stored at 4 ◦ Cin polyethylene bags to prevent moisture gain untilanalysed. Raw noodle samples were examined assoon as prepared, using environmental SEM (ESEM)with the following settings: temperature 4 ◦ C, pressure6Torr, spot size 5, voltage 30kV. RESULTS AND DISCUSSIONFlour properties In a preliminary evaluation a number of commercialflours were characterised. Based on the proteincontents, two were selected for preparation of WSN. These two flours (PF and UW) had similarcharacteristics (Table 1) in terms of moisture, proteinand ash contents as well as LOX activity and colourparameters. Levels of LOX in flour and stability during noodleprocessing The flours analysed contained relatively low levelsof LOX (Table 1), similar to data reported forIndian wheat by Rani  et al  . 23 The endogenous enzyme Table 1.  Properties of flours used for noodle preparation a Parameter PF UWMoisture (%) 11 . 29 ± 0 . 25 11 . 29 ± 0 . 25Protein (%) 9 . 96 ± 0 . 22 10 . 36 ± 0 . 20 Ash (%) 0 . 69 ± 0 . 07 0 . 60 ± 0 . 08 Total starch (%) 71 . 3 ± 3 . 9 75 . 0 ± 4 . 3LOX activity b 30 . 2 ± 0 . 3 36 . 0 ± 0 . 4 L ∗ 92 . 96 ± 0 . 08 92 . 63 ± 0 . 02  a ∗ − 0 . 12 ± 0 . 01 0 . 1 ± 0 . 03  b ∗ 5 . 32 ± 0 . 05 5 . 13 ± 0 . 06 a  Abbreviations: PF, P-Farina flour; UW, Ultra White flour; LOX,lipoxygenase. b Unit for LOX:  µ mol min − 1 g − 1 (see ‘Materials and methods’). Table 2.  Activity of LOX in control and supplemented WSN storedunder different conditions a LOX activity b  Treatment UW PF Controls Day 1 34.9a 29.3ab24h RT 33.4ab 30.6ac24h FR 32.6b 31.6cDried 30.3 28.6b LOX 1 . 85 × 10 3 U per batch added  c Day 1 38.3cd 39.4de24h RT 38.6ce 40.1dfg24h FR 40.9 42.6efhDried 39.8de 40.4gh LOX 3 . 70 × 10 3 U per batch added  c Day 1 45.5fg 46.8i24h RT 44.8fh 47.2ij24h FR 44.3ghi 47.6jk Dried 43.6i 46.6k  a  Abbreviations: LOX, lipoxygenase; WSN, white salted noodles; UW,Ultra White flour; PF, P-Farina flour; RT, storage at room temperature;FR, storage at 4 ◦ C. Means followed by the same letter within a columnare not statistically different (  P  <  0 . 05). b Unit for LOX:  µ mol min − 1 g − 1 (see ‘Materials and methods’). c LOX added was from Sigma-Aldrich; 1 U causes an increase in  A 234 of 0 . 001 min − 1 at pH 9 and 25 ◦ C with linoleic acid as substrate in3mL volume (1cm light path). remained active during noodle preparation, with nolosses under any of the storage conditions studied(Table 2, controls). This probably results from thepresence of only salt as an ingredient that mightinfluence enzyme stability. Accordingly, the pH of the WSN samples was measured and found to be 6.1which is slightly lower than the optimum pH of LOX.Although some statistically significant decreases wereobserved (Table 2) these were less than fifteen percentof the initial levels of activity. Impact of LOX on the texture of WSN For this study a highly purified enzyme preparationwas selected. Despite the application of recrystallisa-tion, is it is acknowledged that other enzymes mayhave been present at low levels of activity to influ-ence product attributes. It could be speculated that 1672  J Sci Food Agric  86 :1670–1678 (2006)DOI: 10.1002/jsfa  Lipoxygenase effects in white salted noodles 020406080100120140160control 3.71.85control 3.71.85    M  a  x .  c  o  m  p  r  e  s  s   i  o  n   f  o  r  c  e   (   N   ) 05101520253035404550 LOX x 10 3 (U/batch)    M  a  x .  c  o  m  p  r  e  s  s   i  o  n   f  o  r  c  e   (   N   ) Figure 1.  Effect of different levels of lipoxygenase (LOX) addition on noodle hardness of fresh raw (top) and fresh cooked (bottom) white saltednoodles (made from Ultra White flour) as measured using the P/45 flat cylinder probe. Error bars represent standard deviation values. these might include peroxidases and lipases. Hsiehand McDonald 24 reported peroxidase activity in apurified LOX from durum wheat endosperm. Thisindicates a direct link of peroxidase to colour prop-erties of wheat end products. In the current study,when WSN prepared from a formulation incorporat-ing the enzyme preparation were evaluated, similarresults were obtained for the two flours. Thereforedata for only one (UW) are presented in this paper.Two different levels of LOX addition were assessed.The soybean preparation was dissolved in 5mL of extraction buffer (50mmolL  − 1 sodium phosphate,pH 7.5), then 1 . 85 × 10 3 units (U) and 3 . 70 × 10 3 Uwere added per batch of noodles. In terms of visualappearance and handling, relatively little differencewas seen between controls and treatments. Hardnessof WSN was measured with the flat cylinder probe(P/45), and the incorporation of enzyme resulted inno differences from the controls in either the raw orcooked form (Fig. 1). Firmness of WSN was assessedusing the cutting attachment blade of the TA-XT2.Here a tendency of firmer WSN was seen upon theaddition of LOX (Fig. 2). Addition of 1 . 85 × 10 3 Uper batch seemed to result in slightly firmer noodlesthan addition of 3 . 70 × 10 3 U, with both being firmercomparedwiththecontrolsamples.Thiswasobservedfor both raw and cooked WSN.Both levels of enzyme addition were evaluated afternoodles have been stored under various conditions,and results of these measurements are presentedin Tables 3 and 4. Again, through the storage ateither room temperature or 4 ◦ C or in the case of dried noodles, only minor differences were recordedbetween controls and WSN treated with LOX. Atendencyofslightlyfirmerandhardernoodleswasseenupon the storage of noodles at 4 ◦ C compared withthe storage at room temperature ( ∼ 25 ◦ C), and alsodried noodles showed the same tendency (Tables 3and 4); however, most of the time the differences werenot statistically significant. Therefore the differencesseen between the two levels of LOX used were notstatistically significant. Effects of LOX on cooking yield Noodles were cooked for the optimum period andresults presented as yield loss and water uptake(Table 5). Fresh WSN had lower cooked weight thandried noodles. The slightly higher losses in freshnoodles upon higher levels of LOX addition werenot statistically different from the controls. Relationship between LOX and colour propertiesof WSN Discolouration ( L ∗ value, whiteness) of noodlesheets was observed for all samples includingcontrols (Figs 3(a) and 3(b)), although there was nodevelopment of speckiness in any case. Formulations  J Sci Food Agric  86 :1670–1678 (2006)  1673 DOI: 10.1002/jsfa  L Cato, AL Halmos, DM Small Table 3.  Effects of LOX (1 . 85 × 10 3 U per batch) on the textural properties measured by TA-XT2 for WSN made from UW a Hardness (P/45) Firmness (blade) Treatment Total energy tocompress (N s)Maximum compressionforce (N) Total energy towork (N s)Maximum cuttingforce (N)Raw Control 41.6a 130a 11.8a 2.42aLOX 34.7b 114 8.51 2.74aRT control 43.3ac 134ab 13.6b 2.55aLOX RT 46.6ac 141 12.5ab 2.48aFR control 44.5bac 136ab 12.1ab 2.4aLOX FR 39.8abc 128ab 11.8a 2.14aCooked Control 8.8a 33.3a 3.54a 0.70aLOX 10.2ab 35.1a 2.56 1.20RT control 9.2ab 34.8a 3.54a 0.70aLOX RT 13.2c 43.3 3.13a 0.68aFR control 10.0ab 37.0a 4.24b 0.78aLOX FR 13.5c 45.9 4.12ab 0.65aDried b Control 11.9a 39.5a 6.01 0.99aLOX 10.3a 35.3a 3.87 1.31a a  Abbreviations: LOX, lipoxygenase; WSN, white salted noodles; UW, Ultra White flour; RT, storage at room temperature; FR, storage at 4 ◦ C. Meansfollowed by the same letter within a column and treatment (raw, cooked or dried) are not statistically different (  P  <  0 . 05). b Measurements taken after cooking to optimum. 00.511.522.533.5control 3.71.85control 3.71.85    M  a  x .  c  u   t   t   i  n  g   f  o  r  c  e   (   N   ) 00.511.522.5 LOX x 10 3  (U/batch)    M  a  x .  c  u   t   t   i  n  g   f  o  r  c  e   (   N   ) Figure 2.  Effect of different levels of lipoxygenase (LOX) addition on noodle firmness of fresh raw (top) and fresh cooked (bottom) white saltednoodles (made from Ultra White flour) as measured using the flat blade attachment. Error bars represent standard deviation values. incorporating LOX darkened less, and at a slowerrate when the level of addition was 3 . 70 × 10 3 U.Darkening occurred at a faster rate and was observedearlier in noodles stored at 25 ◦ C as comparedwith those stored at 4 ◦ C. Under these conditions,control samples were visually white and off-whiterespectively at time zero. After 72h the productcolour was an unattractive beige. On the other hand, 1674  J Sci Food Agric  86 :1670–1678 (2006)DOI: 10.1002/jsfa
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