Due to Fermentation by Selected Lactic Acid

Transcription

1 Fd Sci. Technl.. Int., 2 (4), , 199 hanges in arrt Juice mpnents Bacteria Due t Fermentatin by Selected Lactic Acid Hideki SAKAMOT0,1'2 Masaru KOGUHI,2 Yuki IsHIGUR02 a Tkichi MIYAKAWA1 1 Department f Fermentatin Technlgy, 2Research Institute, Kagme., Ltd., 17, Faculty f Engineering, Hirshima Nishitmiyama, Nishinasun(mach University, Higashihirshima 739, Japan Nasu-gun, Tchigi , Japan Received May 1 7, 199 Lactic acid bacteria were evaluated fr suitability in prducing a fermented carrt juice. All nine strains f lactic acid bacteria studied grew well in the juice. Sensry evaluatin iicated that L. delbrueckii subsp. bulgaricus IAM-1120 a L. helveticus JM-1120 prduced the mst favrable juice. These strains prduced less diacetyl, suggesting that the lack f diacetyl prductin is an imprtant factr fr acceptable fermented juice. Mrever, decreased aldehyde a increased ketne cmpnents f fermented carrt juice by these selected strains was bserved by gas chrmatgraphy/mass spectrmetry, a this was suggested t cntribute t taste imprvement. Althugh the clr f the fermented juice changed in fresh range clr, there was n significant difference in cartenid cmpsitin a their cntents. Keywrds: carrt juice, Iactic acid fermentatin, diacetyl, clr, cartenid Lactic acid bacteria have a lng histry in fd prductin, because lactic acid fermentatin cntributes t imprved strage qualities, physical prperties a flavr. Recently, the physilgical functins f lactic acid bacteria, such as relieving intestinal disrders a having an immunstimulatin effect, have attracted public attentin (Benn, 1994). Typical examples f fds prduced using lactic acid bacteria include prcessed dairy prducts (e.g., cheese, ygurt), traditinal feamented cereal prducts (e.g., mis, sy sauce, sake) a prcessed meat prducts (e.g., sausage, etc.). It is interesting t nte that lactic acid bacteria selected fr these fds vary depeing n the fd materials (Nakan, 197). Fr prductin f lactic acid fermented vegetable prducts such as sauerkraut a pickles (Daeschel & Fleming, 1984), haltlerant lactic acid bacteria f the grups Pedicccus r Lactbacillus are used a give the prducts a specific fiavr. Hwever, there were few reprts abut the lactic acid bacteria suitable fr vegetable juice applicatins. In this study, Iactic acid fermentatin was examined using carrts as a substrate. Amng vegetables, carrts are the richest in p-cartene cmpus, which are attributed t prviding certain health effects (Hart & Sctt, 1995). Because carrts cntain nly a small amunt f rganic acids, carrt juice (ph 5.-.0) is a lw acid fd a requires retrt sterilizatin after canning fr strage. Hwever, this sterilizatin methd causes quality deteriratin f carrt juice due t severe heat treatment a is an inefficient prcess fr cmmercial prductin. In cntrast, Iactic acid ferrnentatin changes the fiavr a results in a mre acidic juice which may nt require as much heat prcessing. T date, mallactic fermentatin f carrt juice by the additin f malic acid (Takanami et al, 199 l) a traditinal Iian lactic acid fermented beverage (kanji) frm black carrt (Berry et al, 1 989) have been reprted. Hwever, rganleptically-preferable lactic acid fermentatin a the change in the cmpnents f carrt juice due t fermentatin have nt been adequately studied. In this study, Iactic acid bacteria suitable fr the prductin f carrt juice were selected, a subsequent changes in the carrt juice cmpnents due t fermentatin were evaluated. Mre detailed chemical analysis was dne n the juice prducts f specific strains which sensry panelists preferred. Materials a Methds Preparatin f carrt juice arrt juice was prepared by the fllwing prcess in the Kagme Research Institute pilt plant, Tchigi. arrts (variety: Kurda-5-sun) btained frm a lcal market were washed, blanched in a ht water at 90' fr 30 min a lye-peeled by a rller-type peeling machine. They were crushed int small pieces (average size 2-3 mm) using a micrgrader Mdel MR-130 (5 mm screen size, Seiken., Ltd., Tky) heated t 85' a centrifuged by decanter Mdel HS-204LS (IHI., Ltd., Tky) at 3,000 rpm fr 10min. After pasteurizatin at l05' fr 30 s by a plate-type heat exchanger Mdel MRS-2 (Hisaka, Wrks Ltd., Osaka), the juice was cncentrated t 3% Brix by evapratr Mdel T-1B (Alfa Laval., Ltd., Lu, Sweden) at 0' 55 cmhg pressure. The mass btained was packed in a plyethylenefilm bags a stred at -20' in a freezer. Befre each experiment, cncentrated carrt juice was thawed in a cld rm (5') a diluted with distilled water t.5% Brix, which crresped t raw carrt juice. This carrt juice was pured int Erlenmeyer flasks, pasteurized in ht water (95', 5 min) a cled rapidly uer running tap water. Micrrganisms a media Lactic acid bacteria used in this study belng t types f genera Lactbacillus,

2 Lactic Acid Ferrnentatin f arrt Juice Entercccus faecalis I FO , Leucnstc mesen terides IFO , a Streptcccus thermphilus IF The six Lactbacillus strains used were L. acidphilus IFO , L. delbrueckii subsp. bulgaricus (L. bulgaricus) IAM- I 1 20, L. casei IAM-1045, L. delbrueckii subsp. delbrueckii (L. delbrueckii) IFO- I 085, L. helveticus JM a L. plantarum IF MRS brth (Man et al, 190) was used fr lactic acid bacteria grwth. BL agar medium (Eiken hemical., Ltd., Tky) was used fr the determinatin f the viable cell ppulatin as clny faming units (cfu)/ml after cultivatin fr 48 h at 30' uer anaerbic citins (BBL Gas Pak, H2+02 anaerbic system, Japan Bectn Dickinsn., Ltd., Tky). Fermentatin f carrt juice Lactic acid bacteria were first pre-incubated in MRS medium. The bacteria were inculated int 100ml f medium in a 200 ml fiask a statinarily Incubated at 37' fr 20 h in a water bath. The bacteria shwing slwer grwth were activated by repeating the abve prcedure. Precultures were prepared by inculatin f MRS culture int 100 ml f recnstituted carrt juice in a 200 ml Erlenmeyer fiask at an inculum rati f 3% (v/ v) a by incubatin uer the same citins as mentined abve. Fr the fermentatin test, these precultures were used by inculatin similarly int 200 ml f recnstituted carrt juice in a 500 ml Erlenmeyer fiask at an inculum rati f 3% (v/ v) a statinary incubatin at 37' fr 20 h in the incubatr. After fermentatin, all juices, including nn-fermented cntrl samples were filled int 200 ml glass bttles a pasteurized at 95' fr 5 min. They were stred in a 5' cld rm until each analysis. hemica/ analysis Ttal acidity was determined by titratin with O. I N NaOH a expressed as percent lactic acid. The ph measurements were made with a ph meter F- 12 (Hriba Ltd., Kyt) equipped with a glass electrde. Iividual rganic acids (citric, acetic, Iactic a malic) were determined by HPL using a Shdex A0-30 system (Shwa Denk K.K., Tky) a an 8cX300 mm Lnpack K-81 1 separating clumn at 80'. Samples were diluted I :3 a a 20 pl aliqut was injected after filtratin using a membrane filter (0.45 pm). The mbile phase was I mm HI04 at I.O ml/min; the eluted acids were passed thrugh a pst clumn reactin system where they were mixed with ST3-P reagent (Shwa Denk K.K.) a detected at 430 nm by a Shdex VD- I detectr. arbhydrates (sucrse, glucse a fructse) were determined by HPL n an L-3A system (Shimadzu rp., Kyt). Samples were diluted I :3 a 20 pl was injected after filtratin using a membrane filter (0.45 pm). Injected samples were separated n a 4.c x250 mm Shdex NH2P-50 clumn a detected by a refractive Iex detectr ER The mbile phase was 75% acetnitrile slutin a the fiw rate was I ml/min. The amin acid cmpsitin was determined using amin acid analyzer mdel L-8500 (Hitachi, Ltd., Tky). Samples were diluted I : 5 with 3% (v/v) sulfsalicylic acid slutin, a after filtratin thrugh a membrane filter (0.45 /tm), aliquts (20 pl) were injected int the analyzer. Vlatile analysis Vlatile cmpnents were analyzed by G-MS using an HP 5972A (Hewlett-Packard., Pal Alt, A, USA) mass spectrmeter fitted with an HP 5890 series 11 G a an HP hemstatin data system. arrt juices were centrifuged at 8,000 rpm fr 5 min, a the supernatants were diluted 1:10 with distilled water. The mixture f 10 ml diluted sample a 5 pl -dichlrbenzene/ benzyl alchl (0.012%, v/v) slutin as an internal staard were put int a 25 ml glass tube. The tube was cnnected t a Purge & Trap ncentratr LS-2000 (Tekmar., incinati, OH, USA) a held at 0'. On purging with helium gas (40 ml/min) fr 0 min, the gases generated were trapped nt a Tenax-TA (GL Science Inc., Tky) tube. After 15 min f helium gas aeratin fr dehydratin, the tube was heated at 200' fr min t desrb the vlatiles. Then the vlatiles were cryfcused at - 100' a directly intrduced int the analyzer with a capillary clumn J & W DB-WAX (30 mx0.25 mm i.d., df=0.5 pm) at 200' fr 2 min. The temperature prgram was isthermal fr I min at 40' a was then raised t 190' at 3'/min. The detectr temperature was 250'. The inizatin vltage was 70 ev (EI mde), a the mass range was m/z Determinatin f the diacetyl cncentratin was dne based n staard curves btained by the same methd, using O. l, 0.5, I.O a 2.0 ppm diacetyl slutins. Sensry evaluatin Each fermented carrt juice was evaluated by the paired-preference test (Yamaguchi, 1973), using nn- fermented carrt juice as a cntrl with 1 5 trained sensry panelists. Samples were kept in a glass bttle at 20' a were tested uer a red lamp t distinguish the difference in clr. Panelists were als asked t describe their reasns fr selectin. Statistical analyses were judged by the tw-tailed test. lr a cartenid analysis The clr f treated carrt juice was measured by a Hunter clr difference meter ND-80 (Nippn Denshku I.., Ltd., Tky). The absrptin spectra f the juice were analyzed using a U-32 lo spectrphtmeter (Hitachi, Ltd.). The analysis f a-, a -cartene cntent a the cmpsitin f cartenids was dne by the reversed phase HPL technique, after extracting the cartenids frrn the juice samples. Fr extractin, 2 g f each sample were accurately weighed ut a mixed with 10 ml pyrgalll slutin (3%, w/v), I ml KOH slutin (0%, v/v) a I.8 g KOH. Subsequently, the mixture was incubated at 70' fr 20 min. After additin f 5 ml HI slutin (%, v/v) a 15 ml ethyl acetate/n-hexane (1 : 9, v/v), the mixture was shaken fr lomin. After centrifugatin, the supernatant was dried with a stream f nitrgen gas. T the residue, n-hexane was added t reach an accurate vlume f loml; this slutin was used fr HPL analysis. HPL analysis was carried ut using a PR-18-5 clurnn (Merck &., Inc., NJ, USA) a slvent (methanl : acetnitrile : dichlrmethane : water=7:7:2:0.1, v/v). Each cartenid was quantified at 450 nm wavelength. T determine cy-, a -cartene cncentratin, calibratin curves based n peak area were established using the staard samples (SIGMA hemical., St. Luis. MO.. USA), which were prepared as 0.5, 1.0, 2.0, 5.0 mg% slutin in n-hexane. 247

3 (//1](./"=;1i' i.:;1/(':;:/;; Table 1. mparisn f acidity, ph, viable cell number a sensry evaluatin f carrt julce btained by fermentatln with nine different lactic acid bacteria. Acidity Micrrganism (%) L. acidphilus L. bulgaricus L. casei L. delbrueckii L. helveticus L, plantarum E. faecalis Leu. mesenterides S. thermph ilus l O O ell number Sensry ph (cfulml) test") 4.5 l 4,09 4,5 I I 2. I X 108 l.3x I X I I X I X I 09 4.X I I X I X I X I 08 a) Number f panelists wh preferred fermented t nn-fermented carrt juice. * Significant difference (p<0.01 ), ** significant difference (p<0.05). Results a Discussin Selectin f lactic acid bacteria suitable fr fermented carrt juice Strains suitable fr the femaentatin f carrt juice were selected frm 9 Iactic acid bacteria based n the values f acidity, ph, vlable cell number a sensry evaluatin after 20 h f femaentatin (Table l). All fthese stralns grew well In the juice. Acid prductin level was very high in L. delbrueckii IF0-l085 a L. plantarum IF (ver 0.5%, v/v). Feamented carrt juice by L. bulgaricus IAM- I 1 20 a L. helveticus JM-il20 were preferred by all f the panelists, a als the juices prepared using L. acidphilus 12** 15* O* 1* 15* 7 O* 12** IF a Streptcccus thermphilus IF were selected as shwing significant difference. Based n the majrity f descriptins, these fermented juices had a gd sur a citrus fruit-like taste. The ther strains, especially L. casei IAM-l045, L. delbrueckii I FO a Entercccus faecalis IFO- 1294, prduced strng ff-flavr taste a were cncluded t be unsuitable fr carrt juice. T date, mallactic ferrnentatin f carrt julce with L. plantarum has been reprted. In this case, the fermentatin was dne by additin f malic acid a required a lng perid (4 days) (Takanami et al, 1991). Similarly, Iactic acid fermentatin f black carrt beverage (mixed with salt water a seasning) fr 10 days was reprted (Berry et al, 1989). In the present study, the fllwing tw fiings were cnsidered t be interesting. The preferred species (L. bulgaricus, L. he/veticus, L. acidphilus a S. thermphilus) fr carrt juice fermentatin have generally been used fr prductin f ygurt a sur milk; whereas, the unsuitable species (L. delbrueckii, L. casei, L. plantarum a Leu. mesenterides) had been detected in ther fermented vegetable prducts a cereal prducts. Secly, in this study an rganleptically suitable lactic acid-fermented beverage withut any additive culd be btained within a shrt perid f time (20 h). Speafuatin f fuvr cmpnents affecting rganleptic dlfferences f fermented carrt juice It was assumed that amng flavr cmpnents f fermented carrt juice, dlacetyl wuld be mst infiuential in the rganleptic differences. Diacetyl Is regarded as a spilage iicatr in tmat juice (Juven & Weisslwicz, 1981) a range juice (Sadler et a[, 1992); whereas, it is well-knwn as a psitive E.* a) = - I.O ' > /'(/S/' r, +-//^ rtl i l* t//' OO i io Micrrganism H. SAKAMOTO et al Fig. 1. mparisn f diacetyl fermented by nlne lactic acid bacteria a nn-fermented carrt julce. ( I ) L. acidphilus, (2) L, bulgaricus, (3) L. casei, (4) L, delbrueckii, (5) L, helveticus, () L. plantarum, (7) E. faecalis, (8) Leu. mesenterides, (9) S. thermphilus, ( lo) nn-fermentatin. (/':../ i= p / / '/'.i// / "' *,,; "41' "'/ /, """',,/.,=::,.; :' ;.; S flavr cmpnent in milk prducts such as ygurt a cheese (Kanek et al, 1990). Therefre, the amunts f diacetyl in carrt julce fermented by varius lactic acid bacteria were cmpared by the G-MS methd. Figure I shws the frmatin f diacetyl after fermentatin with the 9 strains examined a nn-fermented carrt juice. As these figures clearly shw, the strains preferred by sensry evaluatin (L. helveticus JM a L. bulgaricus IAM-1 120) frmed nly a small amunt f diacetyl. On the ther ha, rganleptically unsuitable strains (L. casei IAM-l045, L. delbrueckii IFO a Entercccus faecalis IFO- 1294) prduced a large amunt f diacetyl. This research shwed that an imprtant factr fr the selectin f an acceptable strain suitable fr lactic acid fermentatin f carrt juice was the lack f diacetyl prductin. The changes in ther fiavr cmpnents were bserved by the cmparisn f G-MS spectra. The gas chrmatgrams fcarrt juice fr nn-fermented, fermented with L. helveticus JM a with L. casei IAM-l045 are shwn in Fig. 2, where the peaks shwing significant changes are summarized in Table 2. Many f the aldehyde cmpus such as peak 3, 3-methyl butanal; peak 5, pentanal; peak, hexanal; peak 8, heptanal; peak 10, ctanal; peak 14, nnanal; peak 1, decanal, etc., which were cmmnly knwn as a fiavr cmpnents (Maarse & Visscher, 1989), remarkably decreased, whereas ketne cmpus such as peak 9, 2-heptanne, a peak 1 3, 2-nnanne, were prduced by fermentatin f L. helveticus JM Similar changes were als bserved with the juice f L. bulgaricus IAM- I These ketne cmpus exhibited a fruity a sweet arma n smelling the staard sample. On the ther ha, many f these cmpus prduced by L. casei IAM-1045 were nt much different frm thse in nn-fermented juice except fr a large increase in diacetyl. In the present study, it has been shwn that a decrease in aldehydes a an increase in ketnes in carrt juice by selected lactic acid bacteria were characteristic a might

4 l f I l Lactic Acid Ferrnentatin f arrt Juice 249 bu8nce ntrl 2 3 L. lo ime--> l 12 Li ULL, -N 20. OO 5. OO lo. OO 15. OO 25. OO LLk L , O 50. OO , OO 55, buance L.helveticus 18 9 ime-> l lo ] U r L_ AJ 15. OO 30, OO 35, OO 40. O 45. O 50. O 55. O 5. OO lo. OO 20. OO 25. OO buance 4 L.casei lo ime--> L -- l l L' va. L"t" J u JI' ' ^ ll 15 I ' ul\l 28 [ ' r ', 5. OO 10. OO 15. OO 20. OO 25. OO 30, OO 35. OO 40. OO 45, OO 50, O 55. OO Fig. 2. Gas chrmatgram f vlatile cmpus f nn-fermented carrt juice, carrt juice fermented by L. helveticus a carrt juice fermented by L. casei. cntribute t the imprvement f juice quallties. The factrs respnsible fr these changes remain t be slved. Quantitative characteristics f carrt juice during lactic acid fermentatin Figure 3 shws the time curse changes in the viable cell number, the titratable cncentratin f lactic acid a ph, as well as glucse, fructse a sucrse cntents during the lactic acid fermentatin f carrt juice using the selected strain, L. helveticus JM This strain grew well in the carrt juice, a the viable cell number increased frm 10 t 109 cells/ml during 20 h fermentatin. In additin, the increase in lactic acid cntent a ph Iwering caused by the feamentatin were significant. This lwering effect f ph (ca. 4.0) suggests that carrt juice prductin withut retrt sterilizatin after canning is feasible. The sugar cntent during fermentatin decreased with time, a the rati f glucse, fructse a sucrse did nt change significantly.

5 250 Table 2. Odr carrt juices. Peak number l lo cnstituents f nn-fermented carrt juice a fermented mpnent 2-Methyl prpanal 2-Methyl butanal 3-Methyl butanal D i acety l Pentanal Hexanal n-butanal Heptanal 2-Heptanne Octanal 5 Z -Octa- I, 5-dien E-Heptenal 2-Nnanne Nnanal 2 E -Octenal Decanal Benzaldehyde trans-arylephyne 2-Franmethanl 2-Uecanl Unknwn Unknwn (Y-Humulene 2E,4E-Decadienal Hexanic acid, I O-Dimethyl-5 E,9 E- uecadien-2-0ne Unknwn l -Ddecanl Relative peak area (%)* a) b) c) l, ,29 2.lO 3, , l.84 l,35.35 l l I. l, O.97 l.19 0,70 2. l l.2 l,07 3,3 l 0.90 l,97 l l.53 l0.02 3,74 l * The relative peak area t that fthe internal staard (1.S. peak area= 100). a) Nn-ferrnented carrt juice, b) fermented by L. helveticus IAM-1 120, c) fermented by L. casei IAM-l , , l 2, l.2 1.lO , , ,2 l.02 5,20 2, ):( O,Q ' :,D O O,!5 O1 :5 U) = E 5 c, *O G E :: :: ; '- -e- -HI- - -lt Sucrse lucse Fructse ph Acidity Fermentatin time Fig. 3. Grwth f L, helveticus a changes cntents in carrt juice during the fermentatin, (hrs) in ph, H. SAKAMOTO et al :.' > < acidity a sugar The cmparisn f qualitative analyses f carrt juices withut fermentatin a fermented by L. helveticus JMl 120 are shwn in Tables 3 a 4. ncerning the changes in rganic acids, the carrts cntained a small amunt (0.2%) f malic acid a lst it during the fermentatin prcess. It is knwn that lactic acid bacteria, such as L. plantarum, Leu. ens a Leu. mesenterides, have mallactic fermentatin activity, cnverting malic acid t lactic acid by decarbxylase reactin (Radler & Brauhl, 1984). Thugh the activity f mallactic fermentatin in L. helveticus has nt been reprted, the present study suggests its reactin. Hwever, judging frm the amunt f lactic acid prduced (ca. 0.58%), it was cnsidered that the acid was mainly prduced by sugar cnsumptin (ca. 0.54%). In terms f the amin acid cntents f the juice, nly a decrease in aspartic acid cntent was bserved. hanges in clr a structure f cartenids due t lactic acid fermentatin Similarly, changes in clr a cartenids in carrt juice befre a after the lactic acid fermentatin were examined. The lactic acid ferrnentatin caused a large change in clr, Ieading t a light, favrable clr with raised L, a a b values as shwn in Table 3. Hwever, this change is almst the same fr acidificatin by acid (data are nt shwn). In terms f absrptin spectra, the spectral pattern was nt changed by the fermentatin, but the absrbance after the fermentatin was higher than befre Table 3. hemical cmpsitin a clr parameters in nn-fermented a fermented carrt j uice (L. helvettcus ). ph Refractive iex (%) Acidity (%) Ttal amin acid (mg%) L value a value b value a/b value Sucrse (%) Glucse (%) Fructse (%) itric acid (%) Malic acid (%) Lactic acid (%) Acetic acid (%) c!-artene (mg%) fi-artene (mg%) : Nn detected. Nn-fermentatin l lo.7 l l l.54 l. 0.2 l Fermentatin l l.34 l O.8 l 2.07 (data are nt shwn). Mrever, the amunt f a- a p-cartene, which are the main cartenids in carrt juice, was nt changed (Table 3) a, as shwn in Fig. 4, the carrt juice had a very small amunt f xanthphylls, a 5,-epxy-cartenid was hardly detected. Recently, the clr change in carrt juice caused by

6 Lactic Acid Ferrnentatin f arrt Juice 251 Table 4. Amin acid cmpsitin f nn-fermented a fermented carrt juice (L. helveticus). mpu Aspartic acid Threnine Serine Asparagine Glutamic acid Glycine Alanine Valine Methinine Isleucine Leucine Tyrsine Phenylalanine Lysine Histidine Arginine Prline Nn-ferrnentatin (mg%) l l O. l Fermentatin (mg%). l 3.7 l acidificatin in the pressing prcess was reprted (Sims et al, 1 993). Althugh this change was cnsidered as an imprvement caused by extractin a/r retentin f cartenids, the determinatin f cartenids cntents was nt reprted. Fr clr change caused by rganic acids such as citric acid, there were reprts n the clr change in itrus unshiu during strage (Tada, 1987) a in live berry by lactic acid feamentatin (Minguez-Msquera & Gaul-Rjas, 1994). These changes were cnsidered t be caused by the cnversin f 5,-epxy-cartenid in juice t 5,8-franxy-cartenid. In the present study, it was shwn that the clr changes in carrt juice uer acid citins culd nt be attributed t the change in cartenids. It might be changes in prteins, pectin r ther cmpnents in the juice that resulted in the spectral changes. The factrs respnsible fr these changes remain t be slved. nclusins Fr the develpment f an acceptable lactic acid-fermented carrt juice, the prducts using the strains L. helveticus JM-ll20 a L. delbrueckii subsp. bulgaricus IAM-1120 were preferred by sensry panelists. These strains hardly prduced diacetyl; therefre, it was cnsidered that the lack f diacetyl prductin activity was the mst imprtant factr in the sensry selectin. Mrever, a decrease in aldehydes a the ccurrence f ketne cmpus in fermented carrt juice prduced by these selected strains was bserved, a it was suggested t cntribute t the taste imprvement. Althugh the clr f the fermented juice changed t a fresh range clr as a result f lactic acid prductin, there was n significant difference in the cartenid cmpsitin a their c ntents. Acknwledgments We gratefully acknwledge Dr. Michinri Nakarnura, Emeritus Prfessr, Tky Univ., Dr. Hiryasu Fukuba, Emeritus Prfessr, Ochanmizu Univ., a Dr. Michi Ksaki, Emeritus Prfessr, Tky Univ. f Agriculture, fr valuable advlce, Shuji Ubukata f the Kagme Research Institute fr technical l0,9 2, l 5. l 2,9 2,8 l.8 l,o 0.3 7,8 4.9 Q) cs :2 :: < Q; : q; O U e q; = a) S,Q V U) q, = q; P e V a Retentin 1 O time (min.) 30 Fig. 4. Elutin patterns f cartenids frm carrt juice (L. helvettcus). assistance in G/MS, a Drs. Karen Agriculture a Agri-Fd anada terminlgy. Lapsley a Jhn Mullin f fr assistance with English References Benn, Y. ( 1994). Effect f lactic acid bacteria a ygurt n human health. Up-t-date Fd Prcess., 29, 5-7. Berry, S.K., Jshi, G.J., Saxena, A.K. a Kalra,.L. (1989). Preparatin a evaluatin f ready-t-serve black carrt beverage (Kanji). J. Fd Sci. Technl., 2, Daeschel, M.A. a Fleming, H.P. (1984). Selectin f lactic acid bacteria fr use in vegetable fermentatin. Fd Micrbil., 1, Hart, D.J. a Sctt, K.J. ( 1995). Develpment a evaluatin f an HPL methd fr the analysis f cartenids in fds, a the measurement f cartenid cntent f vegetables a fruits cmmnly cnsumed in the UK. Fd hem., 54, l. Juven, B.J. a Weisslwicz, H. ( 1981). hemical changes in tmat juices caused by lactic acid bacteria. J. Fd Sci, 4, Kanek, T., Watanabe, Y. a Suzuki, H. ( 1990). Enhancement f diacetyl prductin by a diacetyl-resistant mutant f citrate-psitive Lactcccus lactis ssp. Iactis 30 a aerbic citins fgrwth. J. Dairy Sci., 73, Maarse, H. a Visscher,.A. (Eds.) (1989). "Vlatile mpus in Fd " TNO IVO Fd Analysrs Instrtute Zeist, pp Man, J.. de, Rgsa, M. a Sharpe, M.E. (190). A medium fr the cultivatin f Lactbacilli. J. Appl. Bact.,, Minguez-Msquera, M.1. a Gaul-Rjas, B. (1994). Mechanism a kinetics f cartenid degradatin during the prcessing f green table lives. J. Agric. Fd hem., 42, Nakan, M. (197). "Fermented Fd," Krin Shin, Tky, pp. l 12- I 15. Radler, F. a Brauhl, K. ( 1984). The metablism f several carbxylic acids by lactic acid bacteria. Z. Lebensm Unters Frsch., 179, 8-1. Sadler, G.D., Parish, M.E. a Wicker, L. (1992). Micrbial, enzymatic, a chemical changes during strage ffresh a prcessed range juice. J. Fd Sct, 57, I Sims,.A., Balaban, M, a Matthews, R.F. (1993). Optirnizatin f

7 252 carrt juice clr a clud stability. J. Fd Sci., 58, I 129- I 131. Tada, M. ( 1987). nversin f epxy-cartenids a clr changes f itrus unshiu juice. Bul/. Faculty Agric. Okayama Univ., 9, Takanami, S., Kuribayashi, T., Osawa, K. a Yshida, T. ( 1991). A new carrt beverage cntaining fermented lactate; Studies n sur H. SAKAMOTO et a/. fruit beverage prduced by mal-lactic fermentatin (Part IV). The Kanzume Zih (anners J.), 70, (in Japanese). Yamaguchi, S. (1973). Direct cmparisns amng samples. In "Sensry Evaluatin Habk" ed. by Research mmittee f Sensry Evaluatin. Unin f Japanese Scientists a Engineers. JUSE Press. Tky, pp