R Cingrape ◦ Candscrew ◦ 2009 Institute of Food Technologists doi: 10.1111/j.1750-3841.2009.01221.x Further reproduction without permission is prohibited C While monomeric and some oligomeric procyanidins have been Extrusion is one of the most common industrial processes mace is in monomoric and polymeric formsbrings and about whether any extrusion change in the distribution of these polyphenols. found to be absorbed (Shoji anddins others are 2006), poorly polymeric absorbed procyani- (Donovan andothers others 2003). 2002; Gonthier Processing and techniques that can convert/degrade the procyanidin content in cocoa and chocolate1999). (Adamson and others used to make snacks,all and digestibility and its bioavailability main ofously goal food performs, among is nutrients. others, It to mixing, simultane- cutting,expanding, enhance crushing, drying, the pressing, and over- sterilizingers functions 1996). (McDougall In and particular, oth- itat provides high shearing effects temperature concomitantly anding pressure the (Kokini 1993), food thereby matrixenzymes modify- that to cannot release be theamount acted of associated procyanidins upon nutrients. present by in Sinceform fruits, the a not including digestive readily substantial grapes, available is for intigate absorption, a how it much is of important to the inves- procyanidins present in its seed and po- RIOR C in pomace) and screw speed (100, 150, and 200 rpm in both) were tested using ◦ Vol. 74, Nr. 6, 2009 R.L. P — AND , Introduction OWARD ,L.R.H H: Health, Nutrition, and Food JOURNAL OF FOOD SCIENCE HANAL Keywords: anthocyanins, catechin, epicatechin, extrusion, grape pomace, grape seed, LC/MS, procyanidins, rocyanidins (PAs) or condensed tannins are the oligomeric and polymeric polyhydroxy flavan-3-ol units that are ubiquitous Practical Applications: Extrusion processing cangrape be seed used and pomace. to Procyanidins increase in procyanidin grapemolecular by-products monomer weight have compounds, and many which health dimer are benefits, contents poorly butogy in most absorbed. are Extrusion to present processing increase as levels appears large of to the be bioactive a low promising molecular technol- weight procyanidins. tannins ABSTRACT: Grape juicepounds processing that may by-products, afford grape protection against seed chronicconditions and disease. to This pomace enhance study the are was undertaken contents a to ofoligomers rich identify monomers and optimal source and polymers extrusion of in dimers grape at procyanidins, seed the com- and expense pomace. of Extrusion large variables, molecular temperature weight (160, procyanidin 170, and 180 mixtures of grape seed ascontent well of 45%. as Samples pomace of with grapetrusion, seed decorticated and and white pomace total sorghum were anthocyanins analyzed flour forphase were at procyanidin determined high-performance a composition in before liquid ratio and pomace. chromatography of after Additionally,both were 30 ex- chromatograms : grape compared 70 by-products from for increased and diol the the moisture and biologicallyature separation important normal and of monomer screw and procyanidins. dimers speeds. Extrusion considerably Highest of across monomer all temper- content resulted when extruded at a temperature of 170 seed, and 160, 170, 180, and 190 creases in monomer and dimerby 27% contents to were 54%, apparently or enhanced thecessing extraction result reduced facilitated total of by anthocyanins disruption reduced in of polymer pomace the by contents, food 18% matrix which to during declined 53%. extrusion. Extrusion pro- speed of 200 rpm, which were 120% and 80% higher than the unextruded grape seed and pomace, respectively. In- 174 Buelga and Scalbert 2000;ers Wang 2001; and Anderson others and 2000;ical others Joshi absorbance and 2004). capacity oth- Moreover, (ORAC) the was oxygen found rad- to correlate with the in plants and constituteral phenolics the after 2nd lignin (Porter most 1994).foods Their abundant bears existence importance group in not common only of because theysible natu- are partially for respon- organoleptic characteristicsand cocoa of (Sarni-Manchado foods and others like 1999),their grapes, but reported also wines, antioxidant because capacity of andin possible human protective effects health (Facino and1999; others Zhu 1999; and Yamakoshi others and 1999; others Broadhurst and others 2000; Santos- tion Center, Little Rock, AR 72202,(E-mail: U.S.A. [email protected]). Direct inquiries to author Howard H MS 20090188 Submitted 3/3/2009, Accepted 4/27/2009.Howard are Authors Khanal with and Dept. of72703, Food U.S.A. Science, Author Univ. of Prior Arkansas, is Fayetteville, AR with USDA-ARS, Arkansas Children’s Nutri- P and Total Anthocyanin Content ofPomace Grape as AffectedR.C. by K Extrusion Processing JFS Procyanidin Content of Grape Seed and Pomace, H: Health, Nutrition, & Food iladgon nacfe rne.Bt aeil eete mixed mate- then extraneous were materials any Both grinder. exclude coffee a to in sieve ground were and 6 seeds rial nr Grape grinder. an coffee through a screened in was grinding seeds before and dried skins, stems, freeze of consisting pomace The U.S.A.). (Fayetteville, was program Ark., Enology Sunbelt) Arkansas (variety of Univ. the pomace from obtained grape and U.S.A.), Wash., (Prosser, apepreparation Sample (ACY) anthocyanin total as pomace. well grape as of content pomace of grape composition and procyanidin seed on grape effects their determine to ex- studied were these speed, In screw grape and benefits. temperature, variables, and health extrusion periments, pro- seed associated grape the the improve in enhance and contents to pomace, dimer potential and same monomer the cyanidin by has extrusion increased hypoth- to that us substantially led esize studies These be 2009). others and could (Khanal extrusion monomers oligomers that lower-level Our demonstrated 2003). pomace and blueberry others on and work (Awika pro- recent polymers MW higher the reduced including but cyanidins, procyanidins, significantly MW sorghum pre- lower of A enhanced extrusion oligomers. that MW demonstrated lower study in other vious useful and prove monomers may the procyanidins increasing (MW) weight molecular higher . . . extrusion and procyanidins Grape iue1--Rpeettv PCcrmtgaso nxrddadetue rp edo omladdo phases. diol and normal on seed grape extruded and unextruded of chromatograms HPLC --- Representative 1 Figure rp ed(ait isig a bandfo FruitSmart from obtained was Riesling) (variety seed Grape 100 150 200 250 300 100 200 300 400 500 600 700 800 LU 50 LU 0 1020304050600 102030405060700 FLD1 A, Ex=230,Em=321(RCK6&7\08260805.D) FLD1 A, Ex=230,Em=321, TT (RCK6\04010803.D) 1 2 1 aeil n Methods and Materials nxrddgaese ndo phase diol on seed grape Unextruded 2 3 nxrddgrapeseedon normal phase Unextruded 3 4 4 5 5 6 6 7 7 8 Polymer 9 10 Polymer mi mi 1000 1200 1400 1600 200 400 600 800 100 200 300 400 500 600 0 LU LU a edn apoiaey10gfeig a mlydduring employed man- was continuous g/feeding) A extrusion. 100 speeds. (approximately screw feeding rpm 150 ual and 100 at extrudates xrso a are u ttmeaue f10 7,10 and 180, 170, 160, of temperatures at 2009). 190 out others carried and was (Khanal previously Extrusion described been ex- have the about truder Details Germany). ex- Karlsruhe, System Haake, PolyLab Haake (Thermo screw truder twin a using batches 3 in extruded rdts oee,n aaaerpre o rp edetue at extruded seed grape for 190 reported are ex- data of no quality However, the trudates. enhanced and extruder the of clogging minimized eprtr nzn a h ot fteetue)wsstat set was extruder) the of mouth the (at 90 A zone in temperature xrso processing Extrusion were samples All 2009). at others stored and using been (Khanal for have previously Reasons content 6. moisture described 45% at and set to sorghum was content white mixture moisture decorticated total kitchen the (a the bring basis of weight to Speed dry added 45%. a was on Water 70 kg/batch). : Har- 30 2 of Benton of ratio K45SS, a a at Model in U.S.A.) Classic Mich., U.S.A.) bor, Aid Tex., (Kitchen Station, mixture College Quality kitchen Univ., Cereal Rooney, A&M L. Texas Dr. Laboratory, of courtesy ATx436xRTx631 Univ. Hybrid A&M (Texas sorghum white decorticated milled hammer with 0 1020304050600 102030405060700 FLD1 A, Ex=230,Em=321(RCK6&7\08260806.D) FLD1 A, Ex=230,Em=321, TT (RCK6\04010824.D) itr fgaese ihdcriae ht ogu was sorghum white decorticated with seed grape of mixture A ◦ sopsdt h te oe.Tetmeauegradient temperature The zones. 3 other the to opposed as C ◦ ◦ ic tddntpoueapeibeqaiyadqatt of quantity and quality appreciable produce not did it since C ihsrwsed f10 5,ad20rm oee,the However, rpm. 200 and 150, 100, of speeds screw with C 1 o.7,N.6 2009 6, Nr. 74, Vol. − 20 2 ◦ 1 ni ute aoaoyanalyses. laboratory further until C 3 2 3 Extruded grape seed on normal phase normal seed on grape Extruded xrddgaese ilphase Extruded grape seed diol 4 4 — 5 ORA FFO SCIENCE FOOD OF JOURNAL 5 6 6 7 8 910 Polymer Polymer H mi mi 175

H: Health, Nutrition, & Food mi mi m Luna silica μ Polymer Polymer m filter. Separations based on 6 μ 5 5 4 4 m, Phenomenex, Torrence, Calif., U.S.A.) μ 3 Extruded grape pomace on normal phase 3 2 Extruded grape pomace on diol phase 4.6, 5 4.6 mm) (Phenomenex) at a column temperature × × 1 2 1 C.Themobilephaseconsistedof(A)82:14:2:2methylene ◦ The HPLC was connected to a Bruker Esquire-LC ion trap mass FLD1 A, Ex=230, Em=321, TT (RCK7.1\04210823.D) TT A, Ex=230, Em=321, FLD1 0 10203040506070 FLD1 A, Ex=230, Em=321 (RCK6&7\08260804.D) A, Ex=230, FLD1 column (250 of 37 chloride:methanol : acetic acid : water, (B) 96acid: : water. 2 : 2 The methanol : 70-min acetic B gradient linear; was 20 025.6% to to to 87.7% 50 20 B min min linear, 55 0% 11.7% to 65 to to min 11.7% 25.6% 87.7% B B isocratic, and linear; 65 50 to to 55 min tracts were passed through adegree 0.45- of polymerization werecolumn conducted (250 using aas Develosil described Diol previously (Kelm and2009). others 2006; A Khanal flow and rate others ofMobile phase 1 consisted mL/min of (A) was 98 maintained :95 2 : for acetonitrile 3 : : a acetic 2 75-min acid methanol : and run. water3 (B) : acetic min acid. 7% The B 75-min isocratic, gradient 337.6% was to 0 60 to to min 100% 7% linear, to 63 37.6%7% B to linear, B 70 60 to min linear 63 100% min followedProcyanidin B peaks isocratic, by were 70 monitored 10-min to by re-equilibration 75 fluorescenceexcitation detection min at of with 230 nm the and emission column. at 321 nm. spectrometer (Bruker Daltonics, Billerica,the Mass., procyanidin peaks U.S.A.) with to their molecular verify the weights. HPLC After chromatograms studying from both normalrations of and both diol grape phase seed sepa- andtitation grape pomace, of it was individual felt that procyanidinsphase quan- was while probably identification of better the withvidual linkages procyanidins diol present was within better the withdata the indi- normal obtained phase. from As a massphase result, separation, spectrometer which were was those carried out from using normal a 5- 0 0 102030405060 50 400 350 300 250 200 150 100 mi 80 60 40 20 LU 180 160 140 120 100 LU mi Polymer Polymer 9 10) purified from 8 < 7 Vol. 74, Nr. 6, 2009 6 6 — 5 C for 8 h and procyanidin con- 5 ◦ 4 4 3 Unextruded grape pomace on normal phase 3 2 Unextruded grape pomace on diol phase 1 2 1 0.05) in an experiment with 3 varieties of grape seeds ≤ JOURNAL OF FOOD SCIENCE P FLD1 A, Ex=230, Em=321, TT (RCK7.1\04210801.D) TT A, Ex=230, Em=321, FLD1 0 10203040506070 FLD1 A, Ex=230, Em=321 (RCK6&7\08260803.D) FLD1 0 102030405060 0 50 LU 300 250 200 150 100 90 80 70 60 50 40 30 20 LU 100 Procyanidins were analyzed using an Agilent 1100 HPLC system Identification and quantification of the procyanidin peaks on Procyanidins were extracted and isolated as previously de- 36.1 containing no procyanidins with DP 176 Figure 2 Representative --- phases. HPLC chromatograms of unextruded andH extruded grape pomace on normal and diol High-performance liquid chromatography (HPLC) and HPLC-electrospray/ionization-mass spectrometry (HPLC-ESI-MS) Standards Grape procyanidins and extrusion . . . Extraction and analysis of procyanidins centrations are expressed on dry weight (DW) basis. consisting of a quaternary pump,pler, a a solvent degasser, thermostat an column autosam- a compartment, fluorescence a detector, and diode-array ChemStation detector, manipulation for (Agilent data Technologies, collection Palo and Alto, Calif., U.S.A.). Ex- the HPLC chromatogram was based onpared procyandin from standards pre- purified cocoamonomers (Gu through and decamers. A others polymeric 2003a) fraction> (average that DP of contained scribed (Khanal and otherswhich samples 2009) were with soaked overnight aextraction in since simple extraction it solvent modification enhanced before the in extractioncantly of ( procyanidins signifi- (unpublished data). Moisture contentmined of in a the forced samples air oven was at deter- 103 blueberry was used as polymeracterization standard. of Fractionation both and oligomer char- and and blueberries polymer were described standards previously from (Gu and cocoa others 2002). H: Health, Nutrition, & Food aebe ecie rvosy(hnladohr 2009). others and (Khanal previously spectrometer described mass been the have of at conditions set operational was the range about scan Details The kV. 3.5 was voltage lary ehd(isiadWosa 01.Caii -lcsd (c3g) 3-glucoside Cyanidin 2001). Wrolstad and (Giusti method ngaepomace grape in anthocyanins total of Determination 350 of temperature gas drying a with respectively, nitrogen The pump. spec- HPLC mass series the pressureandflowrateonthenebulizerwere50psiand10L/min, 1100 to HP prior auxiliary just an the by apparatus in trometer mL/min HPLC tee a the 0.06 via of of added stream rate was eluant which flow acetate, A ammonium for 2001). used others was and acetate (Prior ammonium methanol mmol/L in 10 post-column using enhanced procyanidins of was Ionization spectrometer. mass the into introduced nm. out 650 at carried wavelength was reference a detection with compared UV nm nm. 280 at at 321 emission maintained and and was 230 excitation rate were detection, Flow wavelengths fluorescence run. For next mL/min. re-equilibration the of 0.8 before min 10 column by followed the linear of B 0% to 87.7% min 70 . . . extrusion and procyanidins Grape oto)o rp ed he eesec ftmeauea 6,10 n 180 over and change 170, (% 160, composition at procyanidin temperature on of extrusion each during levels speed Three screw seed. and grape temperature of of control) --- Effect 3 Figure 5 (- 150 oa nhcaiswr eemnduigtep differential pH the using determined were anthocyanins Total h ltn tem( Lmn rmteHL paau was apparatus HPLC the from mL/min) (1 stream eluting The % change

-80 -64 -48 -32 -16 % change % change

-80 -60 -40 -20 % change  20 0 -60.0 -45.0 -30.0 -15.0 100 0 ) n 0 (- 200 and -), 20 40 60 80 0.0 0 6 7 180 170 160 160 170180 6 7 180 170 160 6 7 180 170 160 Temperature, °C  )rmwr sdfretuiggaese naPlHaeExtruder. PolyHaake a in seed grape extruding for used were rpm -) Decamer Heptamer Monomer Tetramer

% change % change % change -20 -80 -64 -48 -32 -16

% change 20 40 60 80 -50 -40 -30 -20 -10 10 -75 -60 -45 -30 -15 0 0 m 0 0 / ◦ z .Tecapil- The C. 5 o2200. to 150 6 7 180 170 160 6 7 180 170 160 6 7 180 170 160 6 7 180 170 160 Temperature, °C LD etwt infcnedcae at declared significance difference with significant test least (LSD) using separated same were the Means using the approach. analyzed determine were to ACY Total necessary conditions. was extrusion con- process best included stepwise also a Such that fixed data. model only trol randomized the completely as a used in was speed factor screw and combi- temperature polymer, of and nation When oligomers control. major in unextruded observed for was obtained interaction data the experiment. include each not in did replicates It 3 with respectively, seed, grape 4 and of fac- arrangement fixed factorial as a included in were tempera- tors interaction constituent), their determining and the speed, screw as ture, – absorbed be can with that compared was First, contents a experiments. using the monomer control both in unextruded in increase process lowest 3-step the a in U.S.A.) N.C., ttsia analysis Statistical equiva- c3g of mg DW. was as kg 449.2 expressed per of were lents Results MW standard. and the 26900 as of used coefficient extinction molar a with ( P ≤ ttsia nlsswscridoti M SSIs.Ic,Cary, Inc., Inst. (SAS JMP in out carried was analysis Statistical .5 a bevd(sn ooe h rmr procyanidin primary the – monomer (using observed was 0.05) Polymer o.7,N.6 2009 6, Nr. 74, Vol. Pentamer Octamer Dimer

% change % change % change -80 -64 -48 -32 -16 % change -80 -64 -48 -32 -16 -35 -20 -30 -25 -20 -15 -10 0 10 25 40 -5 -5 0 0 — ◦ n ce pe f10(- 100 of speed screw and C t ORA FFO SCIENCE FOOD OF JOURNAL ts.We infcn difference significant a When -test. 6 7 180 170 160 6 7 180 170 160 6 7 180 170 160 × 3and3 P Temperature, °C ≤ 0.05. × o rp pomace grape for 3 Trimer Total Nonamer Hexamer H  177 -),

H: Health, Nutrition, & Food

iue ntesm ounwt ifrn uesrpsaesaitclydfeet( different statistically are superscripts different with column same the in Figures 0.05). P

≤

C

n h n atsrwsedi rpm. in speed screw part 2nd the and C in temperature represents treatment the of part First

B ◦

rcaii opsto speetda ereo oyeiain(P,i hc P en ooe,D2dmr n oo.Alprocyanidins All on. so and dimer, DP2 monomer, means DP1 which in (DP), polymerization of degree as presented is composition Procyanidin P0aegopda n n ecie ntetx spolymer. as text the in described and one as grouped are DP10

> A

428 7271 180/200 31 1287 122 1300 39 1886 211 2718 74 2204 861 26058 177 6646 56 437 98 1071 16 693 14 546

± ± ± ± ± ± ± ± ± ± ± ±

de cde ab a a ab a b bcd cd bc c

268 5901 180/150 57 1246 92g 870 39 1198 152 1675 95 1473 156 19995 444 5248 43 372 148 1057 99 455 69 502

± ± ± ± ± ± ± ± ± ± ± ±

e f bc de cd b cd cd cd c c

84 7152 180/100 38 1432 40 1047 89 1431 67 1986 68 1787 350 21532 278 4147 33 305 79 1092 191 653 83 500

± ± ± ± ± ± ± ± ± ± ± ±

cde efg def b bc ab b e d cd bc c

236 7508 170/200 132 1501 42 1788 60 1753 174 2049 77 1996 414 25447 203 5964 74 413 149 967 59 775 54 736

± ± ± ± ± ± ± ± ± ± ± ±

bcd a abc b ab a a bc bcd d b ab

245 6377 170/150 104 1382 99 1381 152 1551 205 1829 60 1646 813 21640 276 4601 35 478 100 915 120 711 81 769

± ± ± ± ± ± ± ± ± ± ± ±

cde bcd cde bc cd c b de bcd d bc a

280 6554 170/100 78 917 47 914 17 1430 100 1701 125 1707 604 20407 296 4844 49 396 107 1076 7 461 52 408

± ± ± ± ± ± ± ± ± ± ± ±

f fg def bc cd bc b de bcd cd c c

145 5460 160/200 28 1543 161 1175 42 1309 61 1476 62 1252 568 21095 169 5325 50 431 13 1659 120 883 77 580

± ± ± ± ± ± ± ± ± ± ± ±

bc def ef c e d b cd bcd ab ab bc

310 7223 160/150 128 1721 61 1544 3 1622 105 2013 73 1805 689 25533 526 6170 99 560 286 1279 53 837 32 759

± ± ± ± ± ± ± ± ± ± ± ±

b abc bcd b bc ab a bc ab bcd b a

240 6172 160/100 72 1538 75 1393 89 1729 179 2091 103 1830 808 24974 309 6490 40 542 135 1461 73 912 14 817

± ± ± ± ± ± ± ± ± ± ± ±

bcd bcd abc b bc cd a b abc bc ab a

187 4228 Control 120 2008 128 1653 203 1977 156 1964 76 1369 1808 26446 625 8590 95 714 171 1982 128 1160 75 802

± ± ± ± ± ± ± ± ± ± ± ±

a ab a b e eC a a a a a a

P P P P P P P P P P0PlmrTotal Polymer DP10 DP9 DP8 DP7 DP6 DP5 DP4 DP3 DP2 DP1 Treatment

B

al - feto xrso eprtr n ce pe nprocyanidin on speed screw and temperature extrusion of Effect --- 1 Table E)o rp seed. grape of SEM) mean DW, (mg/kg composition ± A Vol. 74, Nr. 6, 2009 — )-catechin and which one was + Results and Discussion JOURNAL OF FOOD SCIENCE Closer examination of the chromatograms obtained from nor- One of our objectives was to study if extrusion affects the Procyanidins in grape and wine have been studied in-depth. Retention times of external standards as well as the mass spec- )-epicatechin. Although peak height in the chromatogram alone 178 − H Grape procyanidins and extrusion . . . Analysis of grape seed and grape pomace procyanidins cannot determine thedividual quantitative procyanidins, increase an (or obvioussome decrease) difference of in in the the in- components, peakmers such size could as be for observed monomers, betweenucts dimers, extruded and for and poly- both unextruded prod- grapein seed both normal and and grapemade diol pomace. to phase This determine chromatograms. was the Noclear apparent DP attempt in of was the polymers. HPLCfirmed Although chromatogram, the it compound presence may mass of notpomace. spectra 2 be hexamers con- in both grape seed and grape mal and diol phasesult separations about suggested which method a to employ rather during interestingpeaks HPLC separation. obtained re- While from diol phase werenormal more pronounced phase and clean, separatedcyanidins compounds better. Normal within phase did not the provide aspentamers individual clear through of decamers pro- peaks for as did thedicated diol that quantification phase. would These probably data be in- carrieddiol out than better normal by phase. However, identification of thedetermination linkages of and the number and typevidual of procyanidins compounds would within be indi- carriedseparation. We out are better currently comparing by the normal normalmethods phase and for diol phase thefood quantification products. of Regardless of procyanidins thepeaks in separation were a method observed used in number no extrudedpomace. of new products of either grape seed or changes in thegrape linkages seed present or grape in pomace.had procyanidin exclusively All B-type of oligomers linkages. the This from is oligomersfindings in suggesting we agreement most with investigated of previous the foods, includingclusively grapes, B-type contain procyanidins ex- (Gu and others 2003b). Extrusionno had effect on the linkagesobserved with present either in diol the phase procyanidinsseparations. (data not as shown) could or be normal phase Grape seeds contain partly galloylated procyanidinsepicatechin, with and catechin, epicatechin gallate atunits a (Prieur ratio and of others 1994). 1 Using : MALDI-TOF 3 mass :try, Krueger 1 spectrome- and being others (2000) sub- detected procyanidin dimers through undecamers in grape seed with 0DP to of 7 gallic up acid substitutions. to The 28 has been tentatively determined in a grape seed ( tra obtained fromthe HPLC-ESI-MS procyanidin monomers analyses through decamers were and polymers. usedever, How- no to mass identify spectral dataorescence are response provided curves beyond obtained heptamers.phase Flu- from HPLC separations both for normal unextrudedand and and grape extruded diol pomace grape are seed As presented can in Figure be 1separation observed, and demonstrated the 2, the fluorescence respectively. presencemore peak of trimers, from 2 tetramers, normal monomers andtra pentamers. and phase (data Compound 3 mass not or spec- of shown) (epi)catechin confirmed units thatpresence that these or had oligomers absence exclusively consisted above of B-type was A- linkages. not or The determined.could Since B-type not the identify linkages which 2 one in monomers was hexamers co-eluted, ( we and H: Health, Nutrition, & Food Fgr )o h xrddaduetue rp edclearly seed grape unextruded and extruded the chromatogram of representative A 1) contents. dimer (Figure for rpm 200 and 2% oe oprdt rp seed. grape to proportionately compared was lower pomace (21%) grape poly- of Conversely, DW) seed. mg/kg (905 grape content higher mer than had oligomers pomace lower-level grape of though, levels compared Proportionately lower seed. much grape also were to pomace and grape dimer Similarly, in seed. contents grape trimer in present in amount the DW) was of it 38% mg/kg seed, only grape present in (1609 than amount higher content the proportionately was monomer of pomace grape DW) Although the seed. of grape 0.43% in (or seed, 16% grape only than was 2) (Table which contents procyanidin total consid- lower had pomace erably of grape 7% hand, other and the 5% On respectively. contributed DW, Holt 2005) the 2002; others others and and Tsang (Baba 2002; been others intestine and have the in which absorbed trimers, be and which to 33% shown Dimers of approximately mg/kg). and basis; (8590 DW) DW polymer mg/kg on the (4228 2.64% monomer content was the was procyanidin seed 16% total grape unextruded The the 1). in (Table monomers the cluding rue soeaddsrbdi h eta oye.OioesD7truhD1 eentdtce eywl nmn ae n hrfr o presented. not therefore and cases many in well very detected not were DP10 through DP7 Oligomers polymer. as text the in described and one as grouped opsto n content and composition procyanidin on extrusion of Effect pomace grape and seed grape of content and composition Procyanidin epicatechin- and 3- epigallocatechin, skin grape epicatechin, galloylation in catechin, Procyanidins of 2003). contain degree others and the (Hayasaka with eight to MS up ESI using extract procyanidin . . . extrusion and procyanidins Grape C 3144 B 3092 A 2590 3278 190/200 3092 190/150 2981 190/100 3587 180/200 2880 180/150 3269 180/100 2752 170/200 2691 170/150 2839 170/100 1609 160/200 160/150 160/100 Control Treatment procyanidin on speed screw and temperature extrusion of --- Effect 2 Table nxrddcnrli rp ed h etrslswr obtained the were over 170 results 80% at best as extruded The much when seed. as grape by in contents control dimer unextruded as well as monomer oti h osiun nt htaecnrbtdb ohteskins the seeds. both the by and contributed may are pomace that units grape constituent seeds, the some contain and and pulp, 2003), skins, others contains and (Monagas pomace skins grape of that similar as have composition wine from extracted the procyanidins in or units Since study. seed constituent current their grape identify to either attempt we in did the nor procyanidins determine pomace to of attempt polymerization not of did We degree 2003b). higher others sub- and galloylated a (Gu of have units proportion skins lower and grape polymerization seed, of grape degree in procyanidins to parison rp pomace. grape rcaii opsto speetda ereo oyeiain(P,i hc P en ooe,D2dmr n oo.Alprocyanidins All on. so and dimer, in DP2 temperature monomer, represents means treatment DP1 the which of in part (DP), First polymerization of degree as presented is composition Procyanidin iue ntesm ounwt ifrn uesrpsaesaitclydfeet( different statistically are superscripts different with column same the in Figures O xrso nacd( enhanced Extrusion in- procyanidins, total of amounts considerable had seed Grape glaea osiuieuis(oqe n tes19) ncom- In 1996). others and (Souquet units constitutive as -gallate B ± ± ± ± ± ± ± ± ± ± ± ± ± P P P P P P oye Total Polymer DP6 DP5 DP4 DP3 DP2 DP1 42 127 69 30 160 142 33 99 96 64 108 111 87 ◦ n 0 p o ooe,ad180 and monomer, for rpm 200 and C bc g b a cdef b efg hC P bcd bcd cde fg defg ≤ .5 h ilgclyimportant biologically the 0.05) 736 653 524 673 688 634 724 647 732 581 538 504 329 ± ± ± ± ± ± ± ± ± ± ± ± ± 40 37 6 11 33 20 30 61 35 30 18 23 24 d a abc ab ab bc ab abc a cd d d e ◦ n h n atsrwsedi rpm. in speed screw part 2nd the and C 570 516 398 566 535 530 629 581 593 548 499 395 384 ± ± ± ± ± ± ± ± ± ± ± ± ± 13 28 24 21 14 14 30 68 33 12 26 4 34 d abc bc d abc bc bc a abc ab abc c d 342 328 233 363 342 361 404 328 346 305 379 331 348 ◦ C ± ± ± ± ± ± ± ± ± ± ± ± ± P fgaese t190 at seed Extrusion grape trimer. the of of for effect variable speeds, was speed screw were screw and and contents temperature temperature dimer all Although at results. increased same primarily the produce not did s10 ntecs fmnmra 170 at monomer of case the much in as by 120% speeds as screw and temperature all across pomace grape monomer in increase highest the contents. with exactly correspond not did hl teswudrdc t iia ogaese,hgetde- at highest occurred pomace seed, grape grape extruded 190 of to contents Similar polymer it. in crease reduce would others enhance would the while conditions at extrusion certain occurred increase when Transition tetramer highest seed. of the level grape in with resulted contents that monomer condition in same the is which et curdars l eprtrsadsrwsed ihthe with 190 speeds at screw occurring decrease and highest temperatures all across con- occurred polymer tents in reduction significant A conditions. extrusion the of etlnal,btdigs thge eprtrso 8 n 190 and 180 of temperatures higher at so doing but linearly, tent et eeete eue rntafce yetuin except extrusion, by affected not or 180 reduced at either con- Trimer were oligomers counterparts. oligomer higher-level tents lower some their into of polymers conversion and the by probably con- dimer tent, and monomer in increase substantial the demonstrates oae hi fet r rsne nasre fgah in graphs 170 of to 160 in- series from grape was creased temperature a as extrusion when seed, increased well grape in contents With pomace). monomer as (grape presented 4 Figure and seed are seed) (grape 3 grape Figure effects with their observed pomace, was extrusion ing ex- the here. in po- reported unextruded not amounts therefore, in and small present mace were very they though in even present products truded or detected not were ( decamers either through significantly heptamers conditions, reduced extrusion all were across polymers pentamers, and While contents. hexamers, monomer in increase highest the with conditions. h xrdrbre t160 at inside barrel time extruder residence the the Increasing temperature. increasing with h nraei ooe otnswssgiiaty( significantly was 170 to contents compared monomer smaller because here in presented increase rpm are 200 data the at no extrusion though extrudates, Even quality speed. produced screw but of 150, rpm or 200 100 either at at only extrudates quality produce not did and had ( < 30 34 16 13 15 38 22 46 20 8 57 21 23 P bc 0.05). xrso nacdmnmr ie,adtie otnsof contents trimer and dimer, monomer, enhanced Extrusion ic neato ewe eprtr n ce pe dur- speed screw and temperature between interaction Since ≤ ab ab c ab ab ab a ab ab ab ab ab ◦ .5.Alohroioeswr eue ( reduced were oligomers other All 0.05). n 0 p,acniinta i o orsodexactly correspond not did that condition a rpm, 100 and C ◦ 238 224 170 238 190 242 303 241 266 209 256 263 364 n 0 p hc eutdi infcn increase significant a in resulted which rpm 200 and C o.7,N.6 2009 6, Nr. 74, Vol. ± ± ± ± ± ± ± ± ± ± ± ± ± 19 22 28 12 25 16 11 27 28 16 47 4 40 bcd bcde cde e bcde de bcde ab bcde bc cde bcd a A opsto m/gD,mean DW, (mg/kg composition ◦ a ahrdfiutwt h xrdrwe extruder the with difficult rather was C 172 226 153 258 256 221 274 220 213 285 280 348 365 ◦ ◦ ,btn ute nraewsobserved was increase further no but C, ◦ n 0 p rsm te extrusion other some or rpm 200 and C nacdgaese ooe con- monomer seed grape enhanced C — ± ± ± ± ± ± ± ± ± ± ± ± ± ORA FFO SCIENCE FOOD OF JOURNAL 41 3 19 44 12 4 18 39 23 16 25 43 5 cd cd a d d c c bc cd cd bc bc ab ◦ n 0 p,acniinthat condition a rpm, 100 and C 589 517 370 473 503 500 531 503 495 505 471 577 905 ◦ n 0 p Tbe2), (Table rpm 200 and C ± ± ± ± ± ± ± ± ± ± ± ± ± 6 30 16 5 25 26 18 17 21 15 50 18 34 P b d cd e d d bcd d d d d bc a ≤ .5 ne most under 0.05) > 5791 5556 4469 5850 5605 5468 6453 5401 5914 5184 5115 5256 4304 P0are DP10 ± P P E)of SEM) ± ± ± ± ± ± ± ± ± ± ± ± ± ≤ ≤ 173 165 38 237 165 23 150 314 185 126 208 70 148 H 0.05) 0.05) 179 e bcd d ◦ bc bcd bc bcd a cd b d d e C

H: Health, Nutrition, & Food 0.05) in grape pomace (as ≤ P C and 3 levels of screw speed at ◦ much as 49%), it wasseed. either It reduced substantiates or one remained of(Khanal similar the in and propositions grape we others made 2009)and previously lower-level that oligomer contents part may of haveextraction come the facilitated from enhanced increase by in disruptionshearing monomer of effects the concomitantly pomace at(Kokini matrix high 1993). due temperature Since and to pomace pressure containsand pulp leaves as (in well addition asseed, to some and some stems seeds) extrusion that provides shearingpossible have that effect more it may fiber on actually than the help releaseextraction material, more from procyaindins it pomace during than is seed. procyanidin contents significantly ( C, which was ◦ Vol. 74, Nr. 6, 2009 C and 200 rpm for grape po- — ◦ -) rpm were used for extruding grape pomace in a PolyHaake Extruder.  -), and 200 (-  C with increasing residence time inside the extruder ◦ -), 150 (- JOURNAL OF FOOD SCIENCE  One striking difference about the effect of extrusion between As stated previously, monomers, dimers, trimers, and even 180 100 (- barrel, their enhancement declined linearly with the increasingidence res- time at higher temperatures of 180 and 190 Figure 4 Effect --- control) of of temperature grape and pomace. Four screw levels speed of during temperatureH extrusion at 160, on 170, procyanidin 180, composition and (% 190 change over Grape procyanidins and extrusion . . . mace, which was true onlyseed. for While monomers the through monomer tetramers in160 increased the to linearly case 170 from of grape similar to grape seed. Similarly, enhancementcontent in in grape total pomace procyanidin followed similar pattern as monomers. grape seed and grapecontents pomace was of observed the on 2 total materials. procyandin While extrusion enhanced the total tetramers increased the most at 170 H: Health, Nutrition, & Food encnrbtdb nacdetato n o uhthrough much how have and conversion. may extraction increase enhanced it the by of made contributed much been has how studies exactly determine extrusion to poly- for difficult purified oligomers higher-level of or lower-level amounts mers and sufficient monomers dur- of and into Unavailability lost 2004) converted oligomers. oligomers get others higher-level may and and extrusion ing polymers Bourvellec all Le not 2001; therefore, others and interactions polysaccha- (Renard hydrophobic large cell-wall and/or bonding to that hydrogen irreversibly through demonstrated rides bind have may pomace procyanidins apple More-MW oligomers. with lower-level accounted and studies was monomer over, extrusion in increase during lower the lost and by for monomer polymer to all converted not been have oligomers, may polymer amount monomers, certain the a increased of While oligomers. the smaller to other extrac- some enhanced contributed and dimers, as actually well have as the may conversion in tion that reduction con- possible procyani- and extrusion is the pomace, of it all grape ditions, contents across of polymers total case and increased oligomers the higher-level in the least oth- of at and light dins, (Khanal In previously 2009). contents ers oligomer lower-level and extruder and extrusion matrix by food the procyanidins the on MW that type. dependent lower suggest be the may studies in processing others These increase and 2009). of (Gu others studies pomace extent and previous blueberry Khanal to and compared 2008; sorghum higher tannin contents were high polymer study in in current decrease the and in contents monomer in crease eotd vna oe eprtrs(150 blue- temperatures not (data both lower extruder with screw at single contents observed even a monomer using also reported) in pomace have grape increase We and 40% berry both. to in higher- 15% in polymers a decrease and concomitant to other a oligomers 100 the with level than in 2003) trimers more others and and and dimers, (Awika 2008) monomers, others extractable and of 400% (Gu sorghum experiment tannin high one in 65% in enhanced to up extrusion by tetramers Indeed, extruder through larger monomer conditions. a extrusion with reproduced similar be under to its extruder, expected scale be pilot could small a results is Haake Although (Khanal 2009). contents others polymer and po- the decreased blueberry concomitantly in and contents mace trimer and dimer, monomer, increased rp rcaiisadetuin... . . extrusion and procyanidins Grape iue5--Ttlatoynncnet m/gD)in treatment the of DW) part tem- in 1st temperature various The represents (mg/kg at speeds. extrusion screw after and contents perature and before anthocyanin pomace grape --- Total 5 Figure ce pe nrpm. in speed screw ehv rpsd3psiiiisfrteehne monomer enhanced the for possibilities 3 proposed have We ehv eetyrpre htetuinsgiiaty( significantly extrusion that reported recently have We o n h n atrepresents part 2nd the and C ◦ ) ohtein- the Both C). P ≤ 0.05) inwr olwdb esr vlaint eemn consumer determine products. to formula- the of evaluation additional acceptability sensory require by will followed and work flavor tion bland a benefits have health trudates contents perceived dimer into and translated monomer be in could extrac- increases enhanced such or whether conversion monomers and to the tion due in been increases have may such dimers of and much how and deter- to whether important mine very is It studies. previous than anthocyanins of retention greater monomers in resulted of oligomers concentrations procyanidin lower-level highest and and in resulted temperature that significantly optimal speed the can screw contents, conditions anthocyanin extrusion total most reduce Although B-type. exclusively were of procyanidins lower-level the un- the All over control. seed grape extruded in increase 80% and pomace in- grape 120% in with crease contents monomer in increase highest the in resulted xrdrada h aefe osuecnet Kaa and (Khanal contents moisture feed same same the 2009). the using others blueberry at conditions when and similar previously under extruder loss extruded ACY was total pomace 42% We to 2007). others 33% and observed Camire 2003; others others and and (Camire Chaovanalikit 2002; extrusion 90%) during and concentrates 64% (between blueberry to observed with (18% previously content than ACY re- lower total is was in 53%) that reduction temperature The loss. high ACY to for exposure sponsible bar- the extruder minimizing the thus inside material rel, such the of rpm of time 200 residence to extent the 100 reduced from the speed lin- screw minimized the content Increasing speed linearly. ACY reduction screw total the the increasing decreased early, temperature extrusion ing ( otn fgaepomace grape of content anthocyanin total on extrusion of Effect nesnR,Bodus L oasyM,ShitW,Ka ,Faaa VP, Flanagan A, Khan WF, Schmidt MM, Polansky CL, Broadhurst RA, Anderson Ham- BG, Kremers PH, Jacobs G, Cao RL, Prior AE, Mitchell SA, Lazarus GE, Adamson by grant. funded Inst. Biosciences was Arkansas study an This pomace. grape providing for U.S.A.) (Fayetteville, Ark., program Enology Arkansas provid- of Univ. for and seed U.S.A., grape Wash., ing Prosser, FruitSmart, and sorghum decorticated providing white for U.S.A., Tex., Station, Texas the College Univ., at Laboratory A&M Quality Cereal the of Rooney L. Dr. thank We E otnsi rp edadgaepmc shsbe observed been 170 has at as Extrusion pomace pomace. blueberry grape in and previously seed grape in contents aSla20) ohtmeaueadsrwsed u o their ( not contents but ACY speed, total screw affected and interaction, temperature Ricardo- and Both Fuleki 2003). 1996; are others Da-Silva de- amounts and (Auw are appreciable juice ACY and the into grape steps expressed as mashing grapes expected juice whole was during for which graded earlier 2006), reported others val- DW values and the the mg/kg (Wu Both than 530 speed. lower screw to were lowest reduced ues and was temperature unextruded highest it of DW, the content at mg/kg ACY 1134 total was the pomace Whereas 5). (Figure ditions P con W rvsD.20.Ioainadcaatrzto fplpeo type-A polyphenol of characterization and Isolation 2004. DJ. Graves NW, Schoene total to correlation 47:4184–8. and Chem quan- Food samples the Agric chocolate J for capacity. and method antioxidant cocoa HPLC in 1999. procyanidins HH. Schmitz of KA, tification Ritter RB, Rucker JF, merstone xrso fgaepmc eue h oa C content ACY total the reduced pomace grape of Extrusion ≤ tuinapast eeulyefciefrehnigthe oligomer enhancing lower-level for and effective monomers equally important be biologically to appears xtrusion .5 n h euto curdars l xrso con- extrusion all across occurred reduction the and 0.05) o.7,N.6 2009 6, Nr. 74, Vol. Acknowledgments Conclusions References — ORA FFO SCIENCE FOOD OF JOURNAL P ≤ .5.Wieincreas- While 0.05). ◦ nvivo in n 0 rpm 200 and C .Theex- H 181

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Y, Sorghum extrusion Waters increases EJ, bioavail- Cheynier V, Herderich MJ, VidalHolt S. RR, 2003. Lazarus Characterization SA, Sullards MC, Zhu QY, Schramm DD, Hammerstone JF, Fraga Baba S, Osakabe N, Natsume M, Terao J. 2002. Baba absorption and urinary excretion H Grape procyanidins and extrusion . . . H: Health, Nutrition, & Food