Physicochemical and nutritional characteristics of blueberry juice after high pressure processingFrancisco J.Barba,Maria J.Esteve,Ana Frigola ⁎Nutrition and Food Chemistry,Universitat de Valencia,Avda.Vicent Andrés Estellés,s/n.46100Burjassot,Spaina b s t r a c ta r t i c l e i n f o Article history:Received 13October 2010Accepted 26February 2011Available online xxxx Keywords:High pressure processing Physicochemical properties Bioactive compounds ColorBlueberry juiceThis study was carried out to investigate the impact of high pressure processing (HPP)at different pressure (200,400and 600MPa)and treatment times (5,9and 15min)on ascorbic acid,total phenolics,anthocyanin stability and total antioxidant capacity,were also studied at different physicochemical parameters such as pH,°Brix and color.HPP treatments resulted in more than 92%vitamin C retention at all treatment intensities.On the other hand,total phenolic content in the juice was increased,mainly after HPP at 200MPa for all treatment times.The total and monomeric anthocyanin were similar or higher than the value estimated for the fresh juice being maximum at 400MPa/15min (16%increase).Antioxidant capacity values were not statistically different for treatments at 200MPa for 5–15min in comparison with fresh juice,however for 400MPa/15min and 600MPa for all times (8–16%reduction),the lowest values were observed for total antioxidant capacity determined with TEAC method.No signi ficant changes were observed in pH and °Brix.Color changes (a*,b*,L*and ΔE)were not visually noticeable for pressurized beverage for all pressures and times.©2011Elsevier Ltd.All rights reserved.1.IntroductionBlueberry juices are becoming more attractive to consumers due to their high levels of antioxidants,which promote human health.Blueberry juice with higher amounts of anthocyanins and other antioxidant phytochemicals such as vitamins C and E among other bene ficial substances,is healthful to humans (Nindo,Tang,Powers,&Singh,2005).Vitamin retention studies to assess the effects of food processing on the nutritional value of foods are of great importance to food technologists and consumers.Ascorbic acid has been used as quality indicator in fruits and vegetables (Esteve,Farré,&Frígola,1996;Giannakourou &Taoukis,2003)because this is a temperature-sensitive bioactive compound,which is also acting as a valid criterion for other organoleptic or nutritional components (Blasco,Esteve,Frigola,&Rodrigo,2004;Esteve,Farre,&Frígola,1995).Anthocyanins are the major polyphenolics present in blueberry juice,which are responsible for the red,violet,purple,and blue color of this fruit (Kong,Chia,Goh,Chia,&Brouillard,2003;Prior,2003),and exhibit a wide range of antioxidant protection and therapeutic bene fits including the integrity of genomic DNA,potent cardioprotective,neuroprotective,antiin flam-matory,and anticarcinogenic properties.Among berry fruits,blueberry has the highest antioxidant capacity,which is mainly related to the anthocyanin content (Juranic &Zizak,2005;Kalt,Forney,Martin,&Prior,1999).Thermal blanching above 80°C remains the most widely adopted technology for preservation of fruit juices,however at these process conditions,blueberry products typically lose their natural flavor and some of their nutritional value (Rossi,Giussani,Morelli,Lo Scalzo,Nani &Torregiani,2003).Consumer demand for minimally processed foods,has led to interest in non thermal technologies such as HPP (Hendrickx &Knorr,2002).The application of HPP allows microbial and enzyme inactivation in order to enhance safety and shelf-life of perishable foods,while reducing thermal effects on nutritional and quality parameters (Butz,Fernández García,Lindauer,Dieterich,Bognár &Tauscher,2003;Knorr,1993).A commercial application of HPP consists of subjecting the packaged or unpacked food in water at pressures of 100–900MPa for 1–20min at room temperature,which results in the inactivation of vegetative microorganisms and enzymes while maintaining physicochemical and nutritive properties,allowing the storage of the product at 4–6°C (Cheftel,1995).Over the last 20years,HPP has been investigated and several commercial products,including fruit juices,i.e.mandarin,grapefruit,apple,orange,carrot juices and broccoli –apple juice mixture treated by HPP are currently available on market (Buzrul,Alpas,Largeteau,&Demazeau,2008;Hayakawa,Kanno,Yoshiyama,&Fujio,1994).In addition,at this stage of development of HPP technology,evaluating the in fluence of process variables on the stability of bioactive compounds as well as antioxidant capacity and physicochemical parameters of blueberry juice is a key factor in de fining treatment conditions to avoid the loss of these important properties of foods and to obtain a food beverage with high bene fits for the health of the consumer.Thus,the operating pressure,and holding times at the pressure set point wereFood Research International xxx (2011)xxx –xxx⁎Corresponding author.Tel.:+34963544955;fax:+34963544954.E-mail address:ana.frigola@uv.es (A.Frigola).FRIN-03571;No of Pages 50963-9969/$–see front matter ©2011Elsevier Ltd.All rights reserved.doi:10.1016/j.foodres.2011.02.038Contents lists available at ScienceDirectFood Research Internationalj o u r n a l h o me p a g e :w w w.e l sev i e r.c o m /l oc a te /fo o dre schanged over a wide range,with the aim of optimizing the processing condition,in order to obtain a beverage with high content in bioactive compounds and optimal physicochemical characteristics,and in any case with an assured microbiological stability of the processed juice. 2.Materials and methods2.1.SamplesBlueberries(Vaccinium myrtillus,harvested in Poland)were pur-chased at Berlin's wholesale market(Berlin,Germany).The fruits were washed,drained and chopped.Then,the squeezed blueberry juice was centrifuged at4000×g for15min and the supernatant wasfiltered using a steel sieve with a mesh of2mm.2.2.ChemicalsTrolox®(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid),ABTS(2,2-azinobis(3-ethylbenzothiazoline6-sulphonate)), potassium hydroxide,sodium and disodium phosphate,acetonitrile (special grade),magnesium hydroxide carbonate and tetrabutyl ammoniumhydrogen sulfate were obtained from Fluka(Steinheim, Germany)L(+)-ascorbic acid,ethanol,methanol metaphosphoric acid and sodium chloride(special grade)were obtained from Merck (Darmstadt,Germany).2.3.Microbiological assayThe number of surviving cells,N,after each test was determined, after a proper dilution of the treated samples in distilled water,by plate count method.The count of microbial colonies,grown on PCA(Plate Count Agar,Oxoid)slants at32°C for72h,was expressed in cfu/mL (colony forming units per milliliter of sample).The survival fraction, S=N/N0and the level of inactivation,log(S)were evaluated for each test.2.4.HPP systemThe samples,inserted in PE-LD bottles,were placed in polyethylene bagsfilled with water and were heat-sealed(MULTIVAC Thermosealer) before being placed in the HPP unit(U4000Unipress,Poland).The pressurization liquid was a mixture of distilled water and1,2-propanediol(50:50v/v).The pressure level,pressurization time,and temperature were controlled automatically.The treatment time stated in this study does not include come-up and come-down times.The samples were pressurized at200,400,and600MPa for specific times in a range from5to15min at a maximum temperature of42°C(initial temperature was25°C).All the treatments were applied in triplicate, with three bottles per replicate.Immediately after pressurization the samples were transferred to an ice/water bath,packed,and then stored under refrigeration(4±1°C)until needed for analysis.2.5.Ascorbic acidAscorbic acid was determined according to Rückemann(Rückemann, 1980).All reagents were of analytical grade.The eluent contained2.5g of tetrabutyl ammoniumhydrogen sulfate in945mL distilled water and 55mL methanol.A standard ascorbic acid solution of50mg ascorbic acid in6%metaphosphoric acid was prepared with appropriate dilutions. 400μL of blueberry juice was diluted with1600μL of6%metaphosphoric acid.The samples prepared in this way werefiltered throughfilters with a pore size of0.22μm and injected in the chromatograph(HPLC column, pump,variable wavelength monitor,Knauer GmbH,Berlin,Germany). Elution is obtained at aflow rate of1mL/min and eluate absorbance is measured at251nm.Retention was expressed as mg/100g.2.6.Total phenolic compoundsTotal phenolic content was measured using the Folin–Ciocalteu method(Singleton&Rossi,1965).Results were expressed as milligrams of gallic acid equivalents per gram of fresh weight.2.7.Total monomeric,polymeric anthocyanins,monomeric indexTotal anthocyanins,monomeric and polymeric anthocyanins,and monomeric index were determined using a modified method of Mazza, Fukumoto,Delaquis,Girard,and Ewert(1999).A10-fold diluted blueberry juice of100μL was mixed with1700μL of distilled water and200μL of5%(v/v)HCl.The sample was held at room temperature for20min before measuring the absorbance at520nm in a10mm cuvette.This reading corresponds to the total anthocyanins content after considering the relevant dilution(A ta).Separately,100μL of juice aliquot,800μL of5%(w/v)SO2,1700μL of distilled water and200μL of 5%(v/v)HCl were mixed,and absorbance was measured at520nm (A SO2)after20min at room temperature.From this reading,polymeric anthocyanins were obtained,and monomeric anthocyanins were obtained from the difference between total anthocyanin(A ta)and polymeric anthocyanins(A SO2).From these measurements monomeric index absorbance units were calculated as follows:monomeric index=A ta−A SO2=A SO2:Calculations of total anthocyanins were based on malvidin-3-glucosid(molar absorptivity28,000for blueberry).All spectrophoto-metric analyses were performed using a UV–visible spectrophotometer Lambda20(Perkin-Elmer,Überlingen,Germany).2.8.Antioxidant capacityThe Trolox Equivalent Antioxidant Capacity(TEAC)test was adapted from Re,Pellegrini,Proteggente,Pannala,Yang and Rice-Evans(1999). This method is based on the capacity of antioxidants to quench the radical cation2,2-azino-bis(3-ethylbenzothiazoline6-sulphonate) (ABTS),which has a characteristic long-wavelength absorption spec-trum showing a maximal peak at734nm.The ABTS radical cation is formed by the interaction of ABTS(7mM)with K2S2O8(2.45mM).2.9.Physicochemical parameterspH and°Brix(total soluble solid content(g/100g))were measured in accord to IFU methods(2001).Conductivity was measured at20°C using the Conductivity Meter WTW LF323(WTW,Germany).Color measurements were done in a Hunter Lab Labscan spectrophotometer (CR-200,Minolta,Japan)and the Hunter color parameters L*:lightness (0=black,100=white),a*(−a*=greenness,+a*=redness)and b* (−b*=blueness,+b*=yellowness)were used.Tests for each sample were conducted in triplicate and the values were averaged.These values were then used to calculate hue degree(h0=arctangent[b*/a*]), chroma[C=(a*2+b*2)1/2],which is the intensity or color saturation, andΔE,total differences of color,[ΔE=((ΔL*)2+(Δa*)2+(Δb*)2)1/2] Calvo(2004).2.10.Statistical analysisSignificant differences between the results were calculated by analyses of variance(ANOVA)and the possible interactions between the parameters.An LSD test was applied to indicate the samples between which there were differences.A multiple regression analysis was performed for each parameter to study the influence of pressure and time of treatment.Also the correlations between a pair of variables were studied.All statistical analyses were performed using2 F.J.Barba et al./Food Research International xxx(2011)xxx–xxxStatgraphics Plus 5.0(Statistical Graphics Corporation,Inc.,Rockville,MD,USA).3.Results and discussionDifferent pressures (200,400,and 600MPa)were applied at different treatment times (5up to 15min).All treatments were compared to the untreated blueberry juice.The samples of blueberry juice,characterized by an initial microbial concentration of approximately 104cfu/mL,were treated by high hydrostatic pressure.Despite the initial high microbial load,the microbiological assays performed on the treated samples show that the microbial load after the HPP cycles is always less than 10cfu/mL.This result highlights the ef ficacy of the HPP,whatever pressure and holding time are set for the experiment and con firm the experimental data reported in literature for acidic fruit juices (Alpas,Kalchayanaud,Bozoglu,&Ray,2000;Donsì,Ferrari,Di Matteo,&Bruno,1998).According to the experimental results achieved,the residual microbial load and therefore the microbiological criteria do not give any useful indications on the optimal operating condition.All the combinations of pressure and holding time should be taken into account in the following experiments.Moreover,the chemical –physical parameters provide suf ficient information for the choice of the optimal process conditions.pH and °Brix values in the untreated beverage were (2.95±0.00)and (7.40±0.00)respectively.No statistically signi ficant changes were observed between the treatments (data not shown).The impact of HPP on ascorbic acid,total phenolics,total anthocya-nins,antioxidant capacity,and color is shown in Tables 1,2.The ascorbic acid content in unprocessed and processed blueberry juice ranged from 14.6to 15.9mg/100g showing no signi ficant differences.These values are in the range of those previously reported by Prior et al.(1998).Samples treated at 200MPa for 5–15min preserved all the ascorbic acid content,however,levels of ascorbic acid in samples treated at 400and600MPa for 5–15min were signi ficantly lower than in fresh samples (p b 0.05).Results indicate that pressurization at 400and 600MPa min preserved 92%of the ascorbic acid in the samples.One explanation of these differences between 200MPa and 400–600MPa could be a pressure-induced enzyme activation during processing at 400and 600MPa.Nagy (1980)reported two main reasons of vitamin C reduction which are:oxidative reactions by enzymes such as cyto-chrome oxidase,ascorbic acid oxidase and peroxidase found in fruits,aerobic and nonenzymatic anaerobic reactions.HPP was reported to affect the hydrophobic and electrostatic bonds of proteins,therefore affecting their secondary,tertiary and quaternary structures.Such conformational changes can enhance enzyme activity by uncovering active sites and consequently facilitate catalytic conversion.In this way,Del Pozo-Insfran,Del Follo-Martínez,Talcott,and Brenes (2007)found a 3-fold and 2.5fold increase in polyphenol oxidase activity in muscadine grape juice after pressurization at 400and 550MPa,respectively.Tiwari,O'Donnell,and Cullen (2009)also have found an implication of other enzymes such as peroxidase and β-glucosidase in degradation of other bioactive compounds in berry juices.In this study,regression analysis performed showed a signi ficative relation of pressure and time on ascorbic acid concentration (Fig.1):y =16.07–0.003*P +0.013*t,where y is the concentration of ascorbic acid (mg/100mL),P is the pressure applied (MPa)and t is the time (min)(R 2=80.4,error standard=0.27).Ascorbic acid retention levels obtained for 400and 600MPa were in accordance with those found by Barba,Esteve,and Frigola (2010),they obtained 91%ascorbic retention or higher after HPP (100–400MPa/2–9min)in a vegetable beverage and Patras,Brunton,Da Pieve,Butler and Downey (2009a,b)found ascorbic acid retention levels higher than 91%for strawberry purees processed at 400and 600MPa and tomato purees treated at 400MPa.Yen and Lin (1996)reported retentions of 89%of the initial content of ascorbic acid in strawberry coulis and in strawberry nectar after treatment at 400MPa/20°C/30min.Phenols appeared to be relatively resistant to HPP,even though they were signi ficantly increased (13–27%)after 200MPa for 5–Table 1Values (mean ±standard deviation of three replicates per sample)of ascorbic acid,total phenolics,total anthocyanins (TACN)and TEAC in HPP blueberry juice (200,400,600MPa for 5,9and 15min.).Treatment Time (min)Ascorbic acid (mg/100g)Total phenolics (mg/g)TACN (mg/g)TEAC (μmol/g)Untreated 015.9±0.1a 2.45±0.13a 2.25±0.03a 32.2±1.4ab HPP515.8±0.2a 3.13±0.05b 2.34±0.23a 32.6±0.2a 200MPa 915.9±0.1a 2.89±0.05bcd 2.34±0.04a 31.9±1.7ab 1515.9±0.1a 2.79±0.18cde 2.40±0.18ab 28.4±2.9bcd HPP515.0±0.1bc 2.64±0.05ade 2.30±0.06a 31.6±0.1ab 400MPa 915.1±0.4b 2.45±0.08a 2.29±0.04a 32.3±1.3a 1514.7±0.1bc 3.03±0.05bc 2.60±0.14b 29.6±3.9abcd HPP514.6±0.2c 2.53±0.12ae 2.45±0.06ab 26.3±0.4d 600MPa914.7±0.2bc 2.57±0.01ae 2.24±0.12a 30.2±0.4abc 1514.8±0.1bc2.59±0.11ae2.28±0.05a26.9±0.3cda –eDifferent letters indicate signi ficant statistical differences in function of the applied treatment.Table 2Values of a*,b*,chroma and L*(mean ±standard deviation of three replicates per sample)of HPP blueberry juice (200,400,600MPa for 5,9and 15min).Treatment Time (min)a*b*L*Chroma ΔEUntreated 00.36±0.04ab−6.80±0.03a32.66±0.23a6.81±0.03a0aHPP50.33±0.13a −8.12±0.05b 32.09±0.20b 8.12±0.04b 1.44±0.13b 200MPa 90.38±0.01ab −7.93±0.16c 31.78±0.03cd 7.94±0.16c 1.43±0.12b 150.28±0.06a −7.57±0.02d 31.70±0.05cde 7.58±0.02d 1.23±0.04cde HPP50.33±0.03a −6.54±0.11d 31.32±0.10f 6.55±0.11e 1.37±0.08bc 400MPa 90.37±0.03ab −6.66±0.00d 31.41±0.20ef 6.67±0.00e 1.26±0.20bcde 150.31±0.07a −7.39±0.02e 31.49±0.24def 7.40±0.02f 1.33±0.22bcd HPP50.36±0.01ab −6.92±0.08a 31.53±0.04cdef 6.93±0.08a 1.14±0.04de 600MPa90.34±0.09a −6.63±0.05d 31.86±0.09bc 6.64±0.05e 0.83±0.09f 150.46±0.10b−6.59±0.05d31.58±0.07cde6.61±0.06e1.11±0.07ea –lDifferent letters indicate signi ficant statistical differences in function of the applied treatment.Chroma (a*2+b*2)1/2,ΔE=((ΔL*)2+(Δa*)2+(Δb*)2)1/2.Pressure (MPa)Time (min)A s c o r b i c a c i d (m g /100m L )100200300400500600036912151414.414.815.215.61616.4Fig.1.Ascorbic acid variation in blueberry juice as a function of pressure and treatment time.3F.J.Barba et al./Food Research International xxx (2011)xxx –xxx15min and24%after400MPa for15min.This increase in total phenolic content may be related to an increased extractability of some of the antioxidant components following high pressure processing. Corrales,Toepfl,Butz,Knorr and Tauscher(2008)reported an increase in total phenolic content of grape by-products following HPP.The total anthocyanin content is similar or higher than the value estimated for the fresh juice.This result indicates that in this particular range of processing conditions the high pressure treatment mainly modifies the mechanism of anthocyanin degradation by affecting themolecules involved in the kinetics of reaction,such as enzymes.Ferrari, Maresca and Ciccarone(2010)observed similar results when they studied pomegranate juice using pressures higher than400MPa and temperatures higher than45°C.Monomeric index,which reflects a relation between monomeric and polymeric anthocyanins was not modified significantly in comparison with fresh juice.TEAC values for unprocessed blueberry juice was32.2±1.4μmol/g. In general,treatments at200MPa for5–15min obtained similar TEAC values than fresh juice,however for400MPa/15min and600MPa for all times,the lowest values for TEAC were observed,these results were in accord to those found for ascorbic acid.This could be due to the highly oxidative conditions that resulted from enzyme activation during pressurization.Our results confirmed the hypothesis formulated by Talcott,Brenes,Pires and Del Pozo-Insfran(2003)when they studied muscadine grape juice,they suggested the involvement of residual polyphenol oxidase in the degradation of ascorbic acid and antioxidant capacity takes place during HPP.Another contributing factor may be the decrease in volume that takes place during pressurization,which may facilitate oxidative reactions in accordance to Le Chatelier principle (Butz&Tauscher,2002).In relation with our results,other authors obtained that HPP at 600MPa/60°C/30min only slightly affects antioxidant capacity(deter-mined as TEAC value)of apple juice(Fernández García,Butz&Tauscher, 2000)and Sánchez-Moreno,Plaza,De Ancos and Cano(2006)indicated that total scavenging activity(DPPH)in aqueous and organic fractions of tomato purée was unaffected by a HPP treatment of400MPa/25°C/ 15min.Dede,Alpas and Bayindirli(2007)observed also a significant decrease in antioxidant capacity in tomato and carrot juice after HPP (250MPa/15min/35°C)and Oey,Van Loey and Hendrickx(2004) reported a decrease in TEAC number for the antioxidant capacity of orange juice during the high pressure treatments.However,Butz et al. (2003)observed that TEAC did not significantly change after the application of600–800MPa of pressure to several fruit and vegetable products.Patras et al.(2009a,b)reported that HPP either increase or does not affect antioxidant activity of liquid foods.Instrumental color parameters of unprocessed and HPP blueberry juice are shown in Table2.No significant changes were observed in a* values(0.36±0.04)after different HPP.L*values significantly(p b0.05) decreased(31.32±0.10to32.09±0.20)in relation to the unprocessed blueberry juice(32.66±0.23).The multifactor analysis of variance showed the existence of interaction(p b0.01)between pressure and time of treatment,when applied pressure was200MPa.Beverage luminosity(L*)decreased when the treatment time was longer,while an increase in L*was observed when pressure was higher,although when pressure was higher than9min a decrease in L*value was observed when pressure was600MPa.Patras et al.(2009a)found similar results in tomato puree and Saldo,Suarez-Jacobo,Gervilla, Guamis and Roig-Sagues(2009)also reported a decrease in L*value in HPP apple juice at100MPa/4°C and200MPa/4–20°C.Depending on the value ofΔE,the color difference between the treated and untreated samples can be estimated such as not noticeable (0–0.5),slightly noticeable(0.5–1.5),noticeable(1.5–3.0),well visible (3.0–6.0)and great(6.0–12.0)(Cserhalmi,Sass-Kiss,Tóth-Markus& Lechner,2006).In this study no noticeable changes were observed (ΔE b1.5)in color in comparison to the unprocessed juice.These results were in accordance with those found by Barba et al.(2010)in a vegetable beverage after applying HPP and Patras et al.(2009b),who only found a small effect of HPP on the color in strawberry and blackberry purees(ΔE b3,after HPP(500and600MPa/15min).A discriminant analysis was performed,establishing as a classifi-cation variable the treatment applied:untreated,and HPP(200,400 and600MPa).Three statistically significant(p b0.05)discriminant functions were obtained(Table3).4.ConclusionsOptimizing pressure–time conditions after HPP can be considered as a factor of great interest because it is possible to achieve microbial inactivation by applying high pressure at much lower temperatures and shorter treatment times than what is commonly used in thermal preservation,which would allow a better retention of bioactive compounds and physicochemical characteristics.In any case,more studies on the combined effect of pressure and time are required to elucidate the effects of high pressure process parameters on bioactive compounds and color in foods,and further studies dealing with the effects of HPP in blueberry juice during storage are needed.In any case, we can conclude that HPP has good prospects for use in the food industry as an alternative to thermal pasteurization. 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