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Riboflavin in Asian noodles: The impact of processing, storage and the efficacy of fortification of three product styles

Riboflavin in Asian noodles: The impact of processing, storage and the efficacy of fortification of three product styles
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  Riboflavin in Asian noodles: The impact of processing, storageand the efficacy of fortification of three product styles Lan T.T. Bui * , Darryl M. Small 1 School of Life Sciences and Technology, Victoria University, Werribee Campus, P.O. Box 14428 MCMC, Victoria 8001, Australia a r t i c l e i n f o  Article history: Received 15 September 2008Received in revised form 5 October 2008Accepted 12 November 2008 Keywords: RiboflavinAsian noodlesFortificationMicronutrientsVitamin retention a b s t r a c t Asian noodle products are a staple food in many countries, representing the end-use of approximatelyone eighth of all wheat produced globally. Relatively little has been published on the contribution of Asian noodles to dietary intakes of essential nutrients including the B group vitamins. Therefore, theaimof this study has been to investigate the factors influencing the retention of riboflavin in these prod-ucts. The three most common styles of Asian noodles (white salted, yellow alkaline and instant) havebeen prepared under controlled laboratory conditions and riboflavin contents measured at each stageof processing. The stability varied markedly between the different styles with losses occurring at eachstep. Declines rangingfrom27%to38%of theinitialriboflavinlevelswerefoundinfortifiednoodles priorto cooking. During boiling at the optimum temperature, further decreases occurred resulting in overalllosses of 52–74% of the amounts incorporated into the various formulations. The presence of alkalinesalts as ingredients appeared to be the primary factor influencing the extent of losses in the three styles.White salted and instant Asian noodles are effective vehicles for enhancing dietary intakes of riboflavin.   2008 Elsevier Ltd. All rights reserved. 1. Introduction Asian noodles have been consumed for thousands of years (Fu,2008) and continue to be a staple food in many countries aroundthe world. With globalisation they have more recently beenadopted into the cuisines of Western countries. Asian noodles, tra-ditional pasta and European noodle products probably share acommon srcin (Huang, 1996; Kill, 2001). Overtime, differencesin formulation and processing have developed, and today the pri-maryingredientofAsiannoodlesisrefinedflourmilledfrombreadwheat ( Triticum aestivum , also known as commonwheat), whereasfor pasta it is semolina from durum wheat ( Triticum durum ). Asiannoodle formulations and processes vary widely, providing a rangeof colour and textural attributes in order to satisfy the preferencesof a diverse range of consumers in Asia (Collado &Corke, 2004; Fu,2008; Hou, 2001). These products are increasingly consumedglob-ally( JapanConvenienceFoodsIndustryAssociation. InstantRamenFacts, 2008), and the production of Asian noodles accounts formore than twelve percent of all wheat produced each year (FAO,2005; Nagao, 1995).Wheat flour products are generally regarded as good sources of various essential nutrients including the B group of vitamins(Guthrie, 1989). Whilst these have been researched in pasta prod-ucts (Dexter, Matsuo, & Morgan, 1982; Douglass & Matthews,1982; Ranhotra, Gelroth, & Novak, 1986), there is relatively littleinformation on the contribution of Asian noodles to dietary intake,the stability of the nutrients during processing, and the suitabilityoftheproductsforfortification.Onthebasisofthegeneralinstabil-ity of vitamins, it can be hypothesised that endogenous and addedvitamins might be subjected to substantial losses during process-ing as well as subsequent cooking prior to consumption.Recently, we have reported on thiamine and folates in Asiannoodles (Bui & Small, 2007a, 2007b, 2007c, 2007d, 2007e, 2008),showing that thiamine losses during processing are substantialand vary for the three most common styles (white salted, yellowalkalineandinstant). ProductpHappearstobeanimportantfactorwith overall losses of up to 97% occurring in the most alkalinetypes of noodles (Bui & Small, 2007a). On the other hand, folatelosses are approximately 40% for each of the three styles (Bui &Small, 2007e).In order to extend our understanding of the contribution of Asian noodles to dietary intakes of B group vitamins, the purposeof the study reported here has been to investigate the retentionof riboflavin during processing and storage. In particular, the po-tential of these products to provide a vehicle for effective fortifica-tion has been evaluated. 0308-8146/$ - see front matter    2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2008.11.048 *  Corresponding author. Present address: DSTO-Scottsdale, 74 George St.,Scottsdale, Tasmania 7260, Australia. Tel.: +613 6352 6606; fax: +613 6352 3044. E-mail addresses: (L.T.T. Bui), (D.M. Small). 1 Present address: RMIT-Melbourne, Australia. Food Chemistry 114 (2009) 1477–1483 Contents lists available at ScienceDirect Food Chemistry journal homepage:  2. Materials and methods  2.1. Samples The reference sample (code VMA 399) was supplied by AACCInternational (American Association of Cereal Chemists Interna-tional, St Paul, Minnesota, USA), and P-farina extra-white flourwas from Allied Defiance, New South Wales, Australia.  2.2. Noodle preparation White salted, yellow alkaline and instant noodles were pre-pared in the laboratory using methods based on the proceduresof  Moss, Gore, and Murray (1987), as recently described (Bui & Small, 2008). For the purpose of this study, the three styles of noo-dles were prepared from the same flour, P-Farina and having thesamestrandsize(1.6mm).Carewastakenatallstagestominimiseexposureof noodlestolight. Sampleswerefortifiedwithriboflavin(Sigma of noodels Chemical Co., USA, R-4500) at a level of 25% of Australian recommended daily intake values (RDI) per serving(corresponding to 0.43mg/35g dried noodles).  2.3. Noodle storage Threereplicatebatchesofbothwhitesaltedandyellowalkalinenoodles were prepared for the study of storage conditions. Sub-sampleswerestoredatdifferentconditionsfor24h:roomtemper-ature, refrigeration temperature and drying at 40  C. Similarly,fresh yellow alkaline noodles were also stored for up to 96h inthe dehydrator at 40  C.An additional three replicate batches of white salted, yellowalkalineandinstantnoodleswerepreparedforthestudyofribofla-vin losses during processing.  2.4. Cooking of noodles The noodle samples were cooked by placing a small amount(approximately 30g) in a saucepan of gently boiling water(600ml). After every minute, a strand of noodle was removed,placed in cold water, and then squeezed between two microscopeslides. The noodle was determined to be fully cooked if a uniformcolour was obtained, indicating that the uncooked core had disap-peared. It is noted that for this study, these noodles were preparedto have the same strand size in order to reduce the variability of cooking time. Cooking water was discarded as this is what con-sumers are most likely to do.  2.5. General analysis procedures Approved procedures of AACC International were used for theanalysis of moisture contents of all noodle samples by the air ovenmethod (AACC, 2005) and pH values of samples (AACC, 2005).  2.6. Extraction and preparation of samples for riboflavin analysis All chemicals used in this study were of analytical grade or of the highest purity available, unless otherwise specified. MilliQ water was used for all extraction and analysis procedures. All pro-cedures were carried out in subdued light, and the procedure of Brubacher and others (Brubacher, Muller-Mulot, & Southgate,1985) was adopted with modifications. Flour and noodle samples(approximately6–7g)aswellasaAACCreferencesample(approx-imately 0.5–1g) were weighed into 250-ml wide-necked conicalflasks. Approximately, 30ml of H 2 SO 4  (0.01M) was added, andthe samples were homogenised for between 20s and 2min usingan Ultra-Turrax (T 25, Janke and Kunkel, Staufen, Germany) seton medium speed. The time used for each particular sample wasbased upon that required for the achievement of a homogeneousextract, which flowed freely. For cooked noodles, short times weresufficient. In the case of dried noodles, both ground and ungroundsamples were analysed and the recovery of riboflavin compared.Within each batch of samples prepared for analysis, aliquots of standard test solution I (10ml) were also prepared in duplicate,and sufficient H 2 SO 4  (0.01M) was added to give the same volumeas that used for the noodle samples. The standard flasks were thensubjected to the same steps used for samples. Homogenates andstandards were autoclaved for 15min at 121  C. After cooling toroom temperature, the pH of the mixture was adjusted to pH4.5±0.1 by adding 6ml of sodium acetate solution.  2.7. Enzymatic digestion for extraction of riboflavin Clara-diastase solution (Fluka Chemie Gmbh, Germany, 10%,5ml) was added to each of the extracts and standards, and wasmixed thoroughly. The mixtures were then incubated for 90minat 45  C. After cooling to room temperature, the mixtures wereacidified by H 2 SO 4  (2.5M, 4ml). The contents of each flask werequantitatively transferred to a 100ml volumetric flask and madeup to the mark with water. The resulting solutions were filteredthroughpleatedfilterpaper(number1), andtheinitial 10ml offil-trate was rejected. The extracts were then further filtered through0.2 l mmembranesbeforeinjectionintoaHPLC.Samplesfromrep-licate trials were analysed in triplicate.  2.8. HPLC analysis of riboflavin contents Sample test solutions were analysed using a Varian system(model 9012, Varian, Melbourne, Australia). Isocratic elution wasperformed on an analytical column, Spherisorb ODS 2, C-18 col-umn, length 250   4.6mm, 5 l m pore size, with a matching guardcartridge. The mobile phase was based upon MeOH–water (15:85)and the water incorporated 1-hexansulphonic acid (0.005M), gla-cial acetic acid (2.4%) and triethylamine (0.5%). Analyses were car-ried out at 30  C with a column heater used to control thetemperature, and the flow rate of the mobile phase was 1.0ml/min. The injector loop used had a volume of 25 l l, and the columneluate was monitored with a spectrophotometric detector (model9050) set at 268nm. The total run time required for each analysiswas less than 35min.ThechromatogramswererecordedusingtheStarChromatogra-phy (Star WS 5.31) software package, which provided retentiontimesforeachpeakalongwithpeakareas.Forquantitationofribo-flavincontents,samplesofstandardtestsolutionsIandIIwerealsoinjected with each batch of sample extracts analysed. In addition,the calibration and stability of the detector were monitored atthebeginningof eachsetof samplesandduringthecourseof anal-yses.ThiswasdonebyinjectingasampleofstandardtestsolutionIafter every set of five analyses of noodle or flour samples.  2.9. Calculation of riboflavin data Riboflavin contents of sample test solutions were calculated bydirectcomparisonwiththepeakareavaluesobtainedfortheexter-nal standard. At the beginning of each series of analyses, at leastthree injections were included for each of standard test solution IandalsoforstandardtestsolutionII. Boththesehadbeenpreparedand processed in parallel with the unknown samples in a singlebatch. The peak areas of standard test solution II corresponded to2ng riboflavin per 10 l l. The peak areas of the sample test solu-tions were compared with those of standard test solution I. Theweight of the sample portion srcinally taken, the volume used 1478  L.T.T. Bui, D.M. Small/Food Chemistry 114 (2009) 1477–1483  for the analysis and the dilution was also taken into account. Theequation used to calculate the riboflavin content in samples is asfollows: Riboflavin content ð mg = 100g Þ ¼  F  p    C  s   1000  F  s    S  w   100 where  F  p  is the peak area of riboflavin in sample test solution,  F  s  isthe peak area of riboflavin in standard test solution I,  C  s  is theamount of riboflavin in 10ml volume of standard test solution I(20 l g),  S  w  is the amount of sample srcinally weighed (expressedin g), 1000 * is the conversion factor so that result is expressed inunits of mg of riboflavin, and 100 is the conversion factor so thatthe result is expressed per 100g of sample.  2.10. Calculation for dry weight basis In order to facilitate the direct comparison of results, the calcu-latedresultswerealsoadjustedtoadryweightbasisbythefollow-ing formula: Riboflavin content ð Corrected to dry weight basis Þ¼  Riboflavin content ð as is Þ   100100  Actual moisture of sample  2.11. Statistical analysis Statistical analysis was performed using SPSS software version14.0 (SPSS, Chicago, Illinois). Comparison of means was conductedusingone-wayanalysisofvariance(ANOVA)withPostHocTukey’stest at  p  <0.05. 3. Results and discussion  3.1. Preliminary considerations and evaluation of procedures Inestablishingproceduresfor thestudy of riboflavinstabilityinnoodles, a known amount of the vitamin was added into the prod-uct formulationfollowingdissolutionin the distilled water used inpreparing the noodle dough. It should be noted that the riboflavinsolution was prepared separately from the alkaline salt (kansui)solution so that the riboflavin remained at neutral pH until allthe ingredients were combined, immediately prior to commence-ment of mixing. This approach was applied in the preparation of both yellow alkaline and instant noodles in the laboratory.Consideration was also given to the levels that might be added.In fortification, it is a common practice to add an amount, whichcorresponds to one quarter of the RDI for the specific nutrientper serving of food. In current Australian regulations (Food Stan-dards Australian New Zealand, 2008) fortification with riboflavinis not mandatory. Where it is permitted, claims of addition referto 0.43mg of riboflavin per serving with a serving correspondingto 35g of dried noodles as purchased. Therefore, this amountwas used as the basis for the level of addition into the noodleformulations.A further preliminary evaluation was undertaken using driedfortified noodles. Extraction of the products could be achievedusing either ground samples or unground noodles through highspeed maceration with the Ultra-Turrax. One possible concernwith mechanical grinding of noodles could be overheating, whichmight have an impact upon vitamin levels. The problem wouldnot occur, when an acidic solution was used for maceration of pre-cooked noodles. A direct comparison of the results obtainedby these two alternate procedures is provided in Table 1. This data show that no significant difference was obtained between the twoapproaches. As the Ultra-Turrax offered a more convenient meth-od, this was used for all dried samples in this study.The riboflavin values then obtained for a series of separatebatchesof driednoodlesarepresentedinTable2. Theresultsshowthat there was a considerable difference in the riboflavin contentsfor the different styles of noodles. There was also some variationfromdaytodayfor eachanalysis. Thevariabilitywas greaterasre-flectedinthecoefficientofvariabilityvaluesfortheyellowalkalinenoodles, whichhad the higher levels of alkaline salt in the ingredi-ents. Thecoefficientsfor thesenoodlescanbe compared withpub-lished repeatability values. In the standard method of theInternational Association for Cereal Science and Technology(1990), it was noted that coefficients of up to 13% might be foundforvitamindeterminations.Therefore,basedonthedatainTable2,itwasconcludedthatthelevelofrepeatabilityiswithinreasonableand normal levels. Hence, the procedure was used for all furtherstudies in this project.In using this method, it is noted that particular care was takentoapplyuniformtechniques, particularlyfortheextractionstep. Inaddition, for all analyses, the riboflavin reference standard wasanalysed along with samples. The purpose of this was to assessthereproducibilityof thepeak areagivenbytheinstrument. Theseresultsagainindicategoodrepeatability, andshowthat theperfor-mance of the HPLC in conjunction with the extraction methodadopted for this study was good.  3.2. The influence of noodle processing on riboflavin losses Laboratory scale processing methods were selected and set upto allow control of individual steps during studies of the threestyles of Asian noodles. The procedures were selected to reflectthe typical current commercial practice for each of the three stylesof noodles and the methods were those that were previously de-scribed (Bui & Small, 2008). After fortification, the riboflavin con-tents of white salted, yellow alkaline and instant noodles at eachstage of processing were analysed and are shown in Fig. 1. It isnoted that the optimal cooking times determined for the threestyle noodles were 2.5, 3.0 and 1.5min for white salted, yellowalkaline and instant noodles, respectively.Relatively, little loss occurred for white salted noodles at eachstepfromflourtodoughtothedriednoodles.However,almosthalf of the amount of total riboflavin was lost during cooking of thedried prepared noodles. In the case of yellow alkaline noodles,there was clearly loss of riboflavinat each stage of noodle process-ing. Large losses occurred during the preparation before cooking,and there was also a significant loss upon cooking with retentionof less than one fourth of the amount of riboflavin srcinally pres-ent in the ingredients.Theresultsoftheriboflavinanalysisofsamplestakenduringin-stant noodle preparation (Fig. 1) indicate that relatively little ribo-flavinwaslostatthedoughpreparationstage,withsomelossuponsteaming and greater loss during the frying step. Besides thermaldecomposition, losses of riboflavin may have taken place due tothewatersolubilityofthevitamin.Morethanhalfofthetotallevelof riboflavin was lost during cooking of the instant noodles.  Table 1 Comparison of grinding dry noodles and Ultra-Turrax homogeniser for extraction of riboflavin from dried fortified noodle samples. Sample Ground Ultra-Turrax homogeniserWhite salted noodles 1.038±0.003 1.033±0.047Yellow alkaline noodles 0.969±0.026 0.993±0.017 Notes:  (a) For each noodle style six sub-samples were analysed. (b) Riboflavin dataare presented as mean±sd, and are expressed in units of mg/100g on a dry weightbasis. L.T.T. Bui, D.M. Small/Food Chemistry 114 (2009) 1477–1483  1479  Thecumulativelossesofriboflavinateachstageduringthepro-cessing of the three different styles of Asian noodles are presentedin Table 3. Again, the data indicated that the presence of alkalinesalt at high levels in the ingredients of yellow alkaline noodles af-fected the stability of riboflavin. Losses of up to 74% of riboflavinwere found in yellow alkaline noodles after cooking, compared toapproximately a 53% loss in white salted and instant noodles.The results of white salted, yellow alkaline and instant noodles,as a final product before cooking, showed a 27% loss of riboflavinin white salted, a 36% loss in yellow alkaline noodles comparedto a total loss of 38% in instant noodles. In order to clarify the im-pact of formulation, the pH of the dried noodle samples was mea-sured with results of pH 5.85, 10.6 and 7.4 for white salted, yellowalkaline and instant noodles, respectively. These reflect the incor-poration of 0%, 1% and 0.1% of alkaline salt (sodium carbonate)(Bui & Small, 2008) in the respective formulations, and these con-firm the relatively wide range of pH of Asian noodles and theresults found for a variety of commercial products reported re-cently (Bui & Small, 2007b, 2007c, 2007d). The greater loss maybe due to the low level of alkaline salt in the ingredients of instantnoodles resulting in a neutral pH, in addition to the steaming andfrying steps during processing.The lowest retention of riboflavin was found during cooking of the yellow alkaline noodles compared with the white salted and  Table 2 The repeatability of analyses for riboflavin in dried white salted, yellow alkaline and instant noodles. White salted noodles Yellow alkaline noodles Instant noodlesTime span of analyses 6 weeks 6 weeks 2 weeksNumber of batches of noodles analysed 5 5 3Number of analyses 17 11 8Mean riboflavin content 1.13 0.91 0.94Standard deviation 0.062 0.076 0.032Coefficient of variability (%) 5.49 8.39 3.37 Notes:  (a) Riboflavin data are expressed as mg/100g on a dry weight basis. (b) Each analysis was carried out on an individual extract. Flour Vitamin addedto formulationTotal vitaminlevel iningredientsDough Dried noodles Cookednoodles Processing conditions    R   i   b  o   f   l  a  v   i  n   (  m  g   /   1   0   0  g   ) White salted noodlesYellow alkaline noodles Flour Vitamin added toformulationTotal vitaminlevel iningredientsDough SteamednoodlesFriednoodlesCookednoodles Processing conditions    R   i   b  o   f   l  a  v   i  n   (  m  g   /   1   0   0  g   ) AB Fig. 1.  Riboflavin contents in (A) white salted and yellow alkaline noodles and (B) instant noodles in different processing conditions. Data expressed as mg/100g on a dryweight basis.1480  L.T.T. Bui, D.M. Small/Food Chemistry 114 (2009) 1477–1483  instant noodles. This might be due to the presence of alkaline saltlimiting the liberation of riboflavin into the cooking water. The ex-tent of vitamin loss appears to be dependent on the temperature,the time of the treatment and the contribution of the ingredientsin the formulation.Whilst there is relatively little published data to compare theresults for Asian noodles, a summary is presented in Table 4. Mostof the products analysed, including the egg noodles, have durum-based ingredients. The only data relating to processing losses re-ported(Watanabe&Ciacco, 1990) arelower thantheresultsfoundhere for Asian noodles.The influence of the cooking of pasta on riboflavin is very vari-able although the relative losses are typically higher than thosefound for the three styles of Asian noodles (Table 3). The loss cal-culated for the Japanese soba product from US food compositiondata (US Department of Agriculture, Agricultural Research Service,2007) is similar to that found in this study. However, the potentiallosses which might occur during the storage of products are notclear. Accordingly, the impact of varying storage conditions wasconsidered in further experiments on Asian noodles prepared inthe laboratory.  3.3. The changes in riboflavin contents of noodles during short termstorage For the study of storage conditions, white salted and yellowalkaline noodles were prepared in the laboratory. Fresh preparednoodles were placed in sealed plastic bags and were covered withaluminiumfoiltoexcludelight,priortostorageatroomandrefrig-eration temperatures. For drying, freshly prepared noodles wereplaced loosely in a tray and were dried at 40  C. The results(Fig. 2) indicated that minor losses occurred at each stage duringprocessing of white salted noodles. Storage of noodles at roomand refrigeration temperatures did not affect the retention of ribo-flavin significantly. However, for this type of noodle, some losseswere found after drying the fresh noodles at 40  C for 24h.For yellow alkaline noodles, the amount of riboflavin lost washigherwhennoodlesweredriedat40  Cfor24hcomparedtonoo-dles stored fresh at room temperature. There was no significantdifference (  p  >0.05) in riboflavin levels, when fresh noodles werestored at room or refrigerated temperature. However, there wasa greater decrease in the riboflavin levels for yellow alkaline noo-dlesthanforwhitesaltednoodles.Thismightbeduetothestorageof fresh noodles at a condition of high moisture content (33g/100g) and high pH level (pH 10) as riboflavin is known to be rela-tively unstable under alkaline conditions (Gregory, 2008).  3.4. The riboflavin contents of noodles during storage for up to four days Theprecedingexperimentsshowedthat morethanone-thirdof total riboflavinin the ingredients was lost during the processing of driedyellowalkalinenoodles.Therefore,afurtherstudyonthelossof riboflavin in yellow alkaline noodles was designed. For thisexperiment, the noodles were prepared in the laboratory, and thefresh noodles were divided into two; one was stored in a sealed  Table 3 Relative and cumulative losses of riboflavin at each stage during processing of laboratory noodles. Noodle style Processing stage Riboflavin loss Cumulative riboflavin lossWhite salted Flour  ?  dough 9±3 9±3Dough  ?  dried noodles 18±2 27±2Dried noodles  ?  cooked noodles 26±1 53±1Yellow alkaline Flour  ?  dough 12±3 12±3Dough  ?  dried noodles 24±3 36±3Dried noodle  ?  cooked noodles 38±2 74±2Instant Flour  ?  dough 11±2 11.3Dough  ?  steamed noodles 3±2 14±2Steamed  ?  fried noodles 24±2 38±2Fried noodles  ?  cooked noodles 13±3 52±3 Note:  Losses are expressed as% values in relation to the total amount of riboflavin in the ingredient formulation (from flour plus that added during fortification).  Table 4 A summary of relevant literature values for riboflavin losses during processing and cooking of pasta, noodles and related products. Riboflavin content Riboflavin loss (%) ReferencesSpaghetti Watanabe & Ciacco (1990)Loss during processing – 4Loss during cooking – 51Noodles, eggs enriched 0.53 50.0 US Department of Agriculture, Agricultural Research Service (2007)Noodles, eggs, spinach enriched 0.52±0.01 25.2Noodles, Japanese, soba 0.14±0.00 31.0Spaghetti, enriched 0.50±0.01 41.8Spaghetti, protein fortified 0.52±0.02 23.6Spaghetti 0.36–0.55 36.4–52.8 Ranhotra, Gelroth, Novak, & Bock (1983)Noodles 0.34–0.59 35.3–56.4Macaroni 0.27–0.33 7.4–45.5Spaghetti 0.37–0.50 44 (40–56) Ranhotra, Gelroth, Novak, & Matthews (1985)Noodles 0.37–0.65 46 (41–49)Macaroni 0.32–0.45 37 (37–41)Couscous (commercial) 0.054–0.091 31.7–43.1 Rahmani & Muller (1996)Bulgur 0.084±0.02 28.6 Kadakal, Ekinci, & Yapar (2007) Notes:  (a) Riboflavin contents are for dried noodles (as purchased at retail), and are expressed in units of mg/100g on an as is basis. (b) Loss values are the proportion of riboflavin measured in the dried noodle sample, which was lost during cooking and are expressed as a percentage. (c) Loss values have been calculated from data in thesrcinal references after first adjusting values to a dry matter basis. (d) Loss values are expressed as means or ranges or both in some cases. (e) Where precision data werepresented and the results are reported here as mean±sd. (f) Different analytical procedures were applied in these studies. L.T.T. Bui, D.M. Small/Food Chemistry 114 (2009) 1477–1483  1481
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