Effect of Storage on Cloud Stability of Cloudy Apple Juice

/InternationalFood Science and Technology/content/14/1/105The online version of this article can be found at:DOI: 10.1177/10820132080901762008 14: 105Food Science and Technology International Guang-Yuan Zhao, Wei Zong and Guang-Jie AnEffect of Storage on Cloud Stability of Cloudy Apple JuicePublished by: On behalf of:Consejo Superior de Investigaciones Científicas (Spanish Council for Scientific Research) can be found at:Food Science and Technology International Additional services and information for/cgi/alerts Email Alerts:/subscriptions Subscriptions: /journalsReprints.nav Reprints:/journalsPermissions.nav Permissions:/content/14/1/105.refs.html Citations:What is This?- Apr 22, 2008Version of Record >>Effect of Storage on Cloud Stability of Cloudy Apple Juice Guang-Yuan Zhao,*Wei Zong and Guang-Jie AnSchool of Food and Biological Engineering,Zhengzhou University of Light Industry5Dongfeng Road,Zhengzhou,Henan450002,ChinaCloud stability of particles in cloudy apple juice during its storage for120days at4,22and40 C is conducted.The juices are added with or without ascorbic acid.The cloud stability of juices during storage is studied by optical microscopy,electron microscopy and size distribution analysis of suspending particles combined with a polyphenols component analysis by high performance liquid chromatography.At the beginning of storage,the turbidity of juice added with or without ascorbic acid decreased quickly because particles of larger size in juices aggregated and deposited,and since there are less larger particles at the upper portion of the bottles,the depositing speed of particles decreases.The loss of total phenolic compounds,epicatechin,chlorogenic acid and tannin increases with the increase of temperature and storage time.The polyphenols degraded quickly during the initial period of storage and then it slowed down.The addition of ascorbic acid to the juice can inhibit the oxidation and polymerisation of phenolic compounds to avoid the formation of new small insoluble particles during the storage,and the efficacy of inhibiting between adding0.024%or0.006%(w/w)ascorbic acid is not remarkably different.Key Words:cloudy apple juice,turbidity,viscosity,polyphenols,cloud stabilityINTRODUCTIONOwing to its higher sensory and nutritional properties, the naturally cloudy apple juice has a considerable market potential(Beveridge,1997).The main quality criteria in cloudy fruit juices are the stability and inten-sity of cloud particles which provide turbidity,flavour and aroma(Genovese et al.1997;Baker and Cameron, 1999).The cloud stability of particles in cloudy juice was affected by the size of particles,the viscosity of juice and the electrostatic repulsion between the particles (Genovese and Lozano,2006).The cloud of naturally cloudy apple juice consists of negatively charged,partly demethoxylated pectin wrap-ping around a core of positively charged protein and the cloud was formed when the juice was extracted from apples.As the protein and polyphenols could form a protein–polyphenols complex,therefore,the main components responsible for the cloud are proteins, pectin and polyphenols.In cloudy apple juice,the interaction between proteins and polyphenols compounds can have an effect on cloud stability by forming a protein–polyphenols complex to increase the size of particles(Hoon and Johan,1987; Siebert et al.,1996a,b).The o-dihydroxyphenolic groups in polyphenols and keto-imide groups in polypeptides are thought to be the primary sites for interaction (McMurrough et al.1999).In addition,a more specific hydrophobic bonding has been proposed between the aromatic rings of polyphenols and the hydrophobic proline residues in proteins(McMurrough et al.,1999). Non-tannin flavans,such as catechin and epicatechin may associate on the protein surface,increasing the hydrophobic surface area which promotes flocculation and precipitation.The condensation of oxidised phenolic compounds also contributes to the growth of particles in cloudy apple juice.Especially procyanidins oxidised by poly-phenol oxidase(PPO)form tiny insoluble droplets which coalesce into a very dispersed population of larger droplets(Beveridge,1999a).Condensation of oxidised phenolic compounds caused the formation of a haze in the clarified apple juice during the storage period (Beveridge et al.,1997).Electrostatic repulsion provided by the negative charges present in the partly demethy-lated pectin by galacturonic acid residues(Beveridge, 1999b)also contributed to the stability of suspensions of hydrated cloud particles.However,if methoxy content*To whom correspondence should be sent(e-mail:guangyuan-zhao@).Received23February2007;revised11April2007. Food Sci Tech Int2008;14(1):105–113ßSAGE Publications2008Los Angeles,London,New Delhi and Singapore ISSN:1082-0132DOI:10.1177/1082013208090176of pectic compounds dropped below a critical level because of the action of pectin methylesterase(PME)in apples,the system is not stable(Castaldo et al.,1989; King,1990;Genovese et al.,1997).The low methoxy pectic compounds form insoluble pectates with divalent ions,such as Ca2þand cause the growth of particles (Javeri and Wicker,1991;Baker and Cameron,1999). In addition,the low methoxy pectic-compounds become substrates of polygalacturonases(Marangoni et al., 1995).These enzymes catalyse the depolymerisation of pectic compounds and may cause the reduction of juice viscosity which is effective on cloud stability(Stoforos and Reid,1992;Tantchev et al.1993).Genovese et al.(1997)reported that steam heating during crushing could not only inactivate the PPO in apple to maintain the juice’s natural color and sensory properties but also produced natural juices with good cloud stability.In addition,they found that particles in freshly pressed cloudy apple juice were3m m but after 15min of centrifugation at4200Âg,almost all particles had a diameter of1m m,and the cloud could be consi-dered stable.Unfortunately,when the haze formation and factors affecting their formation in the system were investigated, a lot of studies(Siebert et al.,1996a;Beveridge, 1999a,b)were focused on the clarified apple juice or modelled systems and there were only a few studies which focused on the cloud stability of cloudy apple juice during its storage.The objective of the present work was to investigate the change of turbidity,viscosity and the content of polyphenols of cloudy apple juice with and without ascorbic acid added during its storage,and analyse the influence of ascorbic acid on the cloud and stability of the juice.MATERIALS AND METHODSMaterialsApples(Fuji variety)were purchased from the market.Polyphenols standards[(þ)catechin,chlorogenic acid, epicatechin,rutin,phlorizin and quercetin]and 5-(hydroxymethyl)furaldehyde(5-HMF)were pur-chased from Sigma Chemical Co.All other reagents used were of analytical grade.MethodsJuice Processing and StoringA special apparatus designed by the working group was used to crush apple,which had a cooling coat outside and a cooling coil inside to cool purees as quickly as possible.Once apples with peel(10kg) were crushed,the purees were heated by introducing hot steam at95 C for80s.It was then centrifuged after cooling.The soluble solid content(SS)of purees was 10.3 Brix.The residue was discarded and the juice was centrifuged again(3000rpm for20min).Then the juice was collected and0.006or0.024%(w/w)ascorbic acid (VC)was added,degassed and filled into150mL glass bottles.After being covered,the bottles were heated in 100 C boiling water for8min and then cooled.Control was the same batch,but without ascorbic acid added. The juice and the control were stored at4,22,and40 C for120days and samples were taken out at intervals to be assayed.Turbidity MeasurementsDuring the storage,aliquots(5–7mL)of the stored juice were drawn from the upper portion of the bottles at different intervals.The turbidity of the juice was determined in a1cm path cuvette cell as absorbance at 660nm,Abs660(Randall et al.,1997;Yemenicioglu et al., 2000)with a spectrophotometer(Model FGC-S,Nice Science Instrument Ltd,Shanghai).Viscosity MeasurementsDuring the storage,aliquots(6–8mL)of the stored juice were drawn from the upper portion of the bottles at different intervals and the viscosity was determined. The viscosity of the juice was determined with an Ostwald viscosimeter.Size Distribution of SuspendingParticles MeasurementsDuring the storage,aliquots(20–30mL)of the stored juice were drawn from the upper portion of the bottles at different intervals and the size distribution of particles were determined.The size distribution of suspending particles was measured with the Mastersizer(Model 2000,Malvern,England).The mean diameter is the sum of all diameters divided by the number of samples. Transmission Electron MicroscopyAliquots(0.2mL)of the stored juice were taken from the upper portion of the bottles after120days storage and1drop was placed on the grid and allowed to stand for5min.Excess sample was touched off to a filter paper and the remaining sample was stained with1drop of saturated uranyl acetate for1min and excess stain was touched off to a filter paper.The stained grids were allowed to air dry at least15min before examination in a H-700transmission electron microscope(TEM).106G.-Y.Z HAO ET AL.Scanning Electron MicroscopyAliquots(5mL)of the stored juice were drawn from the upper portion of the bottles after120days storage. The juice was fixed with2.5%glutaraldehyde at4 C and then washed for some time using0.1M phosphate buffer.The samples were fixed with1%osmium tetra-sulphide and washed for some time using0.1M phosphate buffer.The cleaned samples were dehydrated with alcohol of30,50,70,90,and100%in turn and then put in isoamyl acetate solution,critical-point-dried. The dried lobes were sputtered with gold for5min and were examined with the QUANTA-200scanning elec-tron microscope(SEM,FEI Company).Phenolic Compounds AssayDuring the storage,aliquots(10mL)of the stored juice were drawn from the upper portion of the bottles at different intervals and were mixed with15mL methanol for5min.The extract was filtered through filter paper and the solution was analysed for phenolic compounds content.Total phenolic compounds were analysed according to the method described by Ough and Amerine(1988).Tannin was analysed using the vanillin-HCl assay (Broadhurst and Jones,1978).The standard curve was established using(þ)catechin.Measurements were made at500nm using a spectrophotometer(Model FGC-S, Nice Science Instrument Ltd,Shanghai)and are expressed as catechin equivalents.HPLC Analysis of Specific PolyphenolsPhenolic composition of the juice from the upper portion of the bottles at different intervals was deter-mined using a modified procedure of the HPLC tech-nique described by Bengoechea et al.(1997).For this study,only the samples stored at22 C were studied. The juice(15mL)was homogenised in15mL of methanol/hydrochloric acid(1000:1,v/v)with a blender (1min).Methanol was evaporated under vacuum,and the residue was re-dissolved in25mL of water/ethanol (80:20,v/v)and extracted three times with ethyl acetate (25mL).The organic fractions were combined,dried for30min with anhydrous sodium sulphate,filtered through a filter,and evaporated to dryness in a rotary evaporator,always keeping the bath temperature under 35 C.The residue was re-dissolved in2mL of methanol/water(50:50)and filtered through0.45m m filter.Aliquots(10m L)of the extract were directly injected onto a Phenomenex Jupiter C-18column (100Â4.6mm)for HPLC analysis using solvent A and B as an eluting buffer.Solvent A was water/acetic acid (98:2v/v),and solvent B was water/acetonitrile/acetic acid(78:20:2v/v/v).The concentration of solvent A decreased from100to20%over a47min period,using a flow rate of1.0mL/min;then it rapidly changed to10% in2min,and was kept under these conditions for 60min.The polyphenols were monitored with a diode array UV detector at280nm.A solution containing 2.5-HMF,catechin,chlorogenic acid,epicatechin,rutin, phlorizin and quercetin was used as standard. The standard curve was established using epicatechin, chlorogenic acid and5-HMF respectively for determin-ing the polyphenolic content of them in the juice. Relative Degree of Polymerisation Assayfor ProanthocyanidinsThe relative degree of polymerisation assay for proanthocyanidins was analysed according to the method described by Butler(1982).Sample preparation was made as described in the procedure of polyphenolic compounds assay.The relative degree of polymerisation for proanthocyanidins was defined as the ratio of absor-bance in the vanillin assay(using glacial acetic acid as the solvent)to the absorbance in the anthocyanidins formation assay.Statistical AnalysisData were analysed using the Statistical Analysis System(SAS Institute,Cary,NC,USA)by the analysis of variance(ANOVA).Experiments were replicated three times and data were subjected to analysis of variance and Duncan’s multiple range test(P0.05). RESULTS AND DISCUSSIONTurbidity and ViscosityDuring the storage period of120days,some bottles were taken out of the cultured box and some juice was drawn from the upper portion of the bottles and assayed directly or assayed after being centrifuged.Since the turbidity of the juice with0.024and0.006%(w/w) ascorbic acid was not significantly different(P50.05), only the turbidity of the juice with0.006%(w/w) ascorbic acid is displayed in Figure 1.The centri-fugation conditions(4200Âg,15min)were according to Genovese et al.(1997)and some unstable particles with a larger size were removed from the juice.Without considering the juice stored at40 C,the turbidity of juices decreased with prolonged storage time,and it decreased quickly during the initial period of storage and then slowed down.At the beginning of storage,the turbidity of juices decreased quickly because the particles of larger size in the juices collided, aggregated and settled,so the larger particles at the upper portion of the bottle decreased,and theEffect of Storage on Cloud Stability of Cloudy Apple Juice107depositing speed of particles diminished.On the other hand,the formation of some new particle small enough in size also contributed to slow down the speed.However,within the juices stored at 40 C,the degree of oxidation and polymerisation of phenolic compounds was the most acute compared with other juices stored below 40 C.As a result,more insoluble pigments were formed in the juice stored at 40 C and juice turbidity reached the highest value after 120days storage.The turbidity of centrifuged juices increased during the initial period of storage,then decreased and then remained constant.Owing to the fact that some larger particles were removed by centrifuging,one could not find out a decrease in turbidity of the juice.But as time went on,with the oxidation and polymerisation of phenolic compounds,some new small size particlesformed and the turbidity of the juice increased.The new small size particles collided,aggregated and increased their size.Hence they could be removed by centrifuging (4200Âg for 15min)and the turbidity decreased.Especially at the end of the storage of 120days,the turbidity of the juice stored at 40and 4 C was higher compared with that of the juice stored at 22 C.As the viscosity of the juice stored at 4 C was the highest (Figure 2),the growth and deposition of particles in it was slower.Though the viscosity of the juice stored at 40 C was the lowest (Figure 2),the oxidation and polymerisation of phenolic compounds in it was most acute and more small pigments were formed,so the turbidity of it was the highest.Since it was found that soluble pectin increased the juice viscosity significantly (Genovese and Lozano,2000)and that adding VC could not affect the content of soluble pectin during the storage (results not shown),the viscosity of juice with or without VC added stored at the same temperature was not significantly different (P 50.05).As to every time point,especially at the end of the storage of 120days,the turbidity of the juice with added VC was greater than that of the control.Since VC could protect phenolic compounds from being oxidised and polymerised,less pigments were formed in the juice with added VC than in the control and this led to a lower turbidity of the juice with added VC.When particles in cloudy apple juice were investigated using an electron microscope,negative staining was determined to be the best method of particle visualisa-tion between embedding and sectioning,shadow casting and negative staining techniques.Cloud particles in negatively stained preparations were composed of both1.35(A)(B)1.301.251.201.151.101.051.000.950.500.480.460.440.420.40204060Storage time (day)A b s o r b a n c eA b s o r b a n c e80100120204060Storage time (day)80100120Figure 1.Effect of storage time on the turbidity of cloudy apple juice without (A)and with centrifuging (B).Temperature:(g )4 C,(f )22 C (control)(m )22 C;(n )40 C,(^)40 C (Control).Turbidity of juice was determined with an absorbance of 660nm (Abs 660)described as Abs 660with a spectrophot-ometer.1.261.241.221.201.181.161.14204060Storage time (day)V i s c o s i t y (m P a .s )80100120Figure 2.Effect of storage time on the viscosity of cloudy apple juice.Temperature:(g )4 C,(f )4 C (control)(m )22 C;(n )22 C (Control),(^)40 C (p )40 C (Control).Since the viscosity of juice with 0.024%and 0.006%(w/w)VC added was not significantly different (P 50.05),only the turbidity of juice with 0.006%(w/w)VC added is displayed.108G.-Y.Z HAO ET AL .positively and negatively stained particles making up an aggregate network(Figure3).The halo and surrounding negative stain give the appearance of particles.It was obvious that the surroun-ding negatively stained material around the halo in the control was darker and more intense compared with that of the juice with0.006%(w/w)or0.024%(w/w)VC added and it was composed of pectin and new small particles formed by the oxidation and polymerisation of phenolic compounds.Using scanning electron microscope,it was found that the cloudy juice looked like a network and some par-ticles were shuttled in it(Figure4).It was also found that there were more small particles shuttling in the network in control than in the juice with0.006%(w/w) or0.024%(w/w)VC added,and the particles size in the latter two juices was not remarkably different.These particles in control were thought to be the result of the oxidation and polymerisation of phenolic compounds without the protection of VC and the efficacy of protection between adding0.024%VC and0.006% (w/w)VC was not remarkably different.It was also found that the size of particles in the bottom of the juice bottle was smaller than it in the upper portion (Figure4).Size Distribution of Suspending ParticlesAt the beginning of the storage of the juice with 0.006%ascorbic acid added,there were no particles of 0.105–0.158m m,but at the end of storage,the volume percentage of those particles was14.03%(Table1)and this meant some new particles with a smaller size were formed during storage.The same results were also found for juice without and with0.024%ascorbic acid added (Table1).Genovese and Lozano(2000)found cloudy apple juice without ascorbic acid added resulted in a suspension of irregular shape particles ranging from0.25 to5m m,but they did not study the changing of size distribution of suspending particles during the storage of cloudy apple juice.The mean size of particles in the juice with0.006% ascorbic acid was 1.443m m at the beginning and 0.514m m at the end of storage(Table1).The change of size distribution of suspending particles could be explained as follows:First,the volume percentage of particles in the juice with greater size decreased during the storage of120days.The volume percentage of par-ticles of0.550–2.188m m was49.64%,36.49%after 15days and decreased to32.43%at the end of storage. The volume percentage of particles of2.512–19.953m m was16.17%,11.28%after15days and decreased to 0.01%at the end of storage(Table1).Second,the volume percentage of particles in the juice with smaller size increased during the storage of120days.The volume percentage of particles of0.182–0.240m m was 3.46%initially and up to23.81%at the end of storage. Third,some new and smaller particles which did not exist in the juice initially were formed during the storage (Table1).There were no particles of0.105–0.120m m and 0.138–0.158m m in the juice at the beginning of storage, but the volume percentage of particles of0.105–0.120m m and0.138–0.158m m increased to 2.43and 11.60%respectively.The same phenomena were also found in the juice without and with0.024%ascorbic acid(Table1).Hence,it is concluded from the behavior of particles in the juice during the storage that,first, particles greater in size(named particles1)collided, aggregated and deposited in the bottom of the bottle. Second,particles of a smaller size(named particles2) collided and aggregated to form particles1.Third,new particles of the smallest size(named particle3)collided, and aggregated to form particles2.Fourth,a lot of(A)(B)(C)Figure3.Ultrastructure of cloudy apple juice with0.006%(w/w)VC added(A),with0.024%(w/w)VC added(B) and without VC added(C)stored at22 C for120days observed by TEM(Â25500).Effect of Storage on Cloud Stability of Cloudy Apple Juice109particle 3formed as the storage time went on.The behaviors described above were considered contin-uous,but were divided into four parts only to be understood easily.Hence,the changes in turbidity (Figure 1)could be described as that,since behavior 1was the main behavior for particles in the juice,the turbidity of the juice decreased quickly at the initial storage period.As time went on,as behavior 2became acute and behavior 1slowed down,the decline of turbidity of the juice became constant.Behavior 4became more acute with prolonged storage time.According to the polyphenols contents (described below),it was thought that behavior 4was the result of the oxidation and polymerisation of phenolic compounds.Ascorbic acid protects against the oxida-tion and polymerisation of phenolic compounds,there-fore turbidity in the control juices was greater than that in juices added with ascorbicacid.Figure 4.Ultrastructure of cloudy apple juice of the upper portion of the bottle with 0.006%(w/w)VC added (A),the upper portion of the bottle with 0.024%(w/w)VC added (B),the upper portion of the bottle without VC added (C),the bottom of the bottle with 0.006%(w/w)VC added (D),the bottom of the bottle with 0.024%(w/w)VC added (E)and the bottom of the bottle without VC added (F)stored at 22 C for 120days observed by SEM.110G.-Y.Z HAO ET AL .At the beginning of storage,the volume percentage of particles of0.138–0.158m m in control juice was2.12%, however these particles did not exist in the juice with 0.006and0.024%ascorbic acid,since these particles were formed by the oxidation and polymerisation of phenolic compounds which could be partly protected by adding ascorbic acid during processing.During the storage period,the oxidation and polymerisation of phenolic compounds did not stop and new small particles were formed.At the end of storage,the volume percentage(4.01%)of new small particles in the control juice was more than in samples added with 0.006and0.024%ascorbic acid(2.43and 2.31% respectively).This result was proved by scanning electron microscopy(Figure4).At the end of the storage of120days,there were no particles of0.105m m and the volume percentage of particles of0.105m m increased to0.09and0.56%for juice with0.006%ascorbic acid,stored at22and40 C respectively(Table2).This result showed that some new small particles were formed with the increased storage temperature and it was considered that increased temperature of storage had a positive effect on the oxidation and polymerisation of phenolic compounds.More pigments were formed and the turbidity increased as the temperature increased(Figure1).Changes in PolyphenolsDuring the storage experiment of juices with and without VC added at4,22and40 C for120days,the loss of total polyphenols,epicatechin,tannin and chlorogenic acid became more important by increasing temperature and prolonged storage time(Table3and Figure5).Since the polyphenol oxidase(PPO)in cloudy apple juice was inactivated(results not shown),the decrease of polyphenols was caused by non-enzymatic reaction.In order to investigate the effect of VC on the polyphenols during the storage period,a parallel assay was made without ascorbic acid(control)during the storage experiment and was found that,total polyphe-nols,tannin and epicatechin retained after storage in juice with0.006and0.024%ascorbic acid added were not significantly different,but they were significantly higher(P0.05)than those retained in the juice without preservative addition at the same storage temperature (Table3and Figure5).At the initial time of storage, the relative degree of polymerisation of proanthocyani-dins in the juice was1.3and it increased to2.5,1.9and 1.8respectively for the control,juice with0.006and 0.024%added ascorbic acid after storage for120days at22 C.Hence,it was concluded that ascorbic acid could protect phenolic compounds against being oxi-dised and polymerised,and could also avoid the non-enzymatic browning caused by the oxidation and polymerisation of phenolic compounds.Table1.The effect of time of storage at22 C on size distribution of suspending particle in juice.Juice1:juice with0.024ascorbic acid.Juice2:juice with0.006ascorbic acid.Volume percentage of particles in juice0day15days120daysParticle size(m m)Juice1Juice2Control Juice2Control Juice1Juice2Control0.105–0.1200000.03 1.46 2.31 2.43 4.010.138–0.15800 2.12 4.309.3611.5411.6013.560.182–0.240 3.99 3.4612.2917.019.0623.1423.8123.750.275–0.47931.9930.6930.2330.9124.0630.8829.7227.410.550–2.18850.5649.6441.2936.4937.6932.5032.4331.252.512–19.95316.4416.1714.0711.288.400.000.010.00Mean size(m m) 1.520 1.443 1.533 1.294 1.1230.5450.5140.504Table2.The effect of storage temperature on sizedistribution of suspending particle in juice with0.006%ascorbic acid.Volume percentage of particles injuice stored for120daysParticle size(m m)At4 C At22 C At40 C0.1050.000.090.560.120 1.47 2.34 3.360.138 4.16 4.91 5.950.158 5.88 6.697.380.1827.247.787.860.105–0.18218.7521.8125.110.2097.888.117.600.2408.017.92 6.940.209–0.47947.9745.7539.650.550–0.72413.4313.2014.360.832–0.9558.508.739.931.096–1.259 6.58 6.497.101.445–2.188 4.76 4.013.852.512–3.8020.010.010.000.550–3.80233.2832.4435.24Mean size(m m)0.5330.5140.521 Effect of Storage on Cloud Stability of Cloudy Apple Juice111Table3.Effect of storage time on the content of phenolic compounds in juice with different contentsof ascorbic acid added.Time (days)Temperature and ascorbicacid added(%)Total phenolic compounds(mg gallic acid/L)Tannin(mg catechin/L)Chlorogenicacid(mg/L)Epicatechin(mg/L)ProanthocyanidinRelative degree ofpolymerisations0Control465.9Æ8.6a255.3Æ4.1a101.3Æ2.1a77.6Æ1.6a 1.30.006489.1Æ9.0b269.6Æ3.9b105.7Æ2.2a82.1Æ2.0b 1.30.024515.2Æ8.0c272.4Æ4.0b102.2Æ2.3a82.9Æ1.8b 1.3 54 C Control454.6Æ8.1250.1Æ4.8100.1Æ2.277.4Æ1.9 1.50.006472.8Æ8.3261.8Æ5.0104.7Æ2.081.9Æ2.0 1.40.024493.1Æ7.5262.3Æ4.7102.9Æ2.182.1Æ1.8 1.322 C Control442.0Æ8.1250.9Æ5.199.4Æ1.974.9Æ1.5 1.40.006460.2Æ7.9253.1Æ4.7103.4Æ2.179.2Æ1.6 1.40.024483.7Æ6.1250.8Æ4.895.3Æ2.280.2Æ1.9 1.440 C Control412.5Æ6.0186.0Æ3.989.7Æ2.065.4Æ1.7 1.40.006432.5Æ8.5235.2Æ4.6101.9Æ2.272.2Æ2.1 1.50.024440.9Æ6.9226.1Æ4.192.6Æ2.069.0Æ1.9 1.5 154 C Control450.2Æ7.8246.8Æ4.799.0Æ2.176.3Æ1.4 1.50.006451.4Æ8.4252.6Æ4.7104.0Æ2.481.2Æ1.8 1.50.024490.5Æ6.5253.8Æ3.9102.3Æ2.380.9Æ2.0 1.422 C Control424.6Æ8.1222.8Æ4.296.2Æ2.171.6Æ1.9 1.50.006442.2Æ8.2239.0Æ4.5102.5Æ2.077.6Æ1.2 1.40.024470.4Æ7.7233.8Æ4.393.3Æ2.586.8Æ1.1 1.440 C Control357.8Æ7.2150.2Æ4.387.6Æ2.762.1Æ1.3 1.70.006428.1Æ8.2180.2Æ4.1100.0Æ2.369.3Æ1.4 1.60.024431.8Æ6.8173.3Æ3.974.9Æ2.176.3Æ1.4 1.5 454 C Control427.8Æ6.4225.4Æ3.395.1Æ1.966.0Æ1.0 1.60.006438.9Æ8.4233.5Æ4.3101.0Æ2.270.8Æ1.8 1.50.024457.7Æ7.5242.3Æ3.6100.2Æ2.172.6Æ1.2 1.522 C Control387.9Æ7.6192.8Æ4.192.0Æ2.465.0Æ1.7 1.60.006397.8Æ8.0205.3Æ3.893.8Æ1.965.7Æ1.5 1.50.024441Æ6.8203.5Æ3.793.0Æ2.264.4Æ1.4 1.540 C Control341.6Æ7.4124.1Æ3.188.3Æ2.356.8Æ1.1 1.80.006356.0Æ7.6150.9Æ3.992.6Æ2.262.0Æ2.0 1.60.024385.6Æ5.8144.3Æ3.189.0Æ2.159.5Æ1.3 1.6 754 C Control400.5Æ7.6214.1Æ2.5 1.60.006437.1Æ7.8227.0Æ5.1–– 1.40.024458.5Æ6.1227.5Æ3.9–– 1.522 C Control385.3Æ8.0162.3Æ4.2–– 1.70.006401.4Æ8.1172.6Æ4.2–– 1.60.024415.2Æ5.2171.8Æ3.1–– 1.740 C Control285.4Æ6.594.8Æ2.3–– 1.70.006363.9Æ7.3111.2Æ4.1–– 2.80.024377.1Æ6.2110.1Æ2.7–– 3.0 1204 C Control386.5Æ5.9d201.1Æ3.7c92.2Æ3.0b66.5Æ1.8c 1.60.006443.5Æ8.1e218.3Æ4.1d94.9Æ3.0b72.3Æ1.7d 1.50.024451.4Æ5.8e222.2Æ3.9d95.8Æ2.8b75.8Æ3.1d 1.522 C Control333.5Æ7.9f151.9Æ3.5e78.1Æ1.8c39.5Æ1.1e 2.50.006363.9Æ6.5g169.2Æ3.9f79.4Æ2.1c44.9Æ1.3f 1.90.024375.9Æ6.2dg164.1Æ4.1f74.3Æ3.1c42.7Æ1.9f 1.840 C Control263.5Æ7.1h78.3Æ3.1g68.6Æ4.1d30.6Æ1.3g 4.40.006337.0Æ5.7i101.8Æ3.8h78.1Æ1.8c37.7Æ1.7eh 3.40.024344.9Æ6.598.7Æ3.3h72.0Æ2.0e35.4Æ1.8h 3.3 Data bearing different letters within the same temperature same fraction and column were significantly different(P50.05).112G.-Y.Z HAO ET AL.。