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GalactoseStudy - DAIRY FOODS Specillclty Inhibitory Studios...

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Unformatted text preview: DAIRY FOODS Specillclty. Inhibitory Studios, and Oligosaecharide Formatlon by B-Galactosldase from Psychrotrophlc Bacillus subtiiis KLaa KHALID A. A. RAH!!! and mono fl. LEE1 mmarmsmmmwmcmm A BSTRACT fl—Galactosidase from psychmtrophic Bacillus rubrih‘s KL88 was specific to the B-D-glycosidic linkage normally nesenthtlactose.’['heenzymewascom— pletely inhibited by transition metal ions $051+. Fe“. Fen Zn“) and partially tnhrbited by high concentrations of glu- cose and galactose as well as Ca”. It was activated by most of the alkaline earth metal ions 011“. K“. Li"). Ohgosaccharides were formed at the dif- ferent level; of lactose concentraliims rubbing mme than 20% for high lactose Doncentration (20%). Three types of Ohgosoocharides were formed in signifi- cant concentrations detected by HPLC analysis. (Kay words: oligosaccharide formation. B—galactosidase, psychrotrophic Bacillus rubtih‘s KL88) giglweviation key: ONP = a-nitrophenol. _ PG == o-nitrophenyl—B-D—galactopyrano- “(16. OS = oligosaccharides. INTRODUCTION Th“ enzymatic hydrolysis of lactose using 8.de is a complex process involving of thifrm 0f sequential reactions. The action “it!!!“ is believed to be of a dual “me because it has nanaferase activities, as 83;) by the fonmtion of oligosaccharides 'v‘flfills types and amounts of OS are its on during the lactose hydrolysis depend- u“ Mme source, lactose concentra- -——_._ We! My 21. 1990. in“ Serum: 21. 1990. new "“11 Ao'lnltm Cmdl Food Research :3 (Corn _ Cm.5LHyaohtthe.PQ.CamdllZS I‘M Numb: 213). 199] . 1 but so 143773—177: WWO'MGGIIUHWW Sm.AnmchoBevue.F0.0Ml-BX1OO tion, temperature. pH. and the degree of lac- tose conversim (20). The formation ofOS was reported early in the 19505 (1, 13). The mechanism ofthe m tion was first proposed by Wallenfels and Mill- ltotra (18), who used B—galactosidase from Es- cherichia coli. The formation of OS required a prolonged hydrolysis time. The transgalacto— sidic nature of ladose enzymatic hydrolysis seemstobethemajorfactorbehindtheos fonnatioo (6. 14). The detection and characterinfion of these 08 requiretheuseofcomplicatedandtedious techniques. Several investigators have reported thefomtation ofOS inmilkandtnilkproducts treeted with fl-galactosidase from most of the sources (19). Considerable interest has developed in the use of OS as growth factors for Bifidabacteriwn spp. in foods because of their roles in controlling pH of the large intes- tines by generating lactic acid and acetic acid, which restricts the growth of pathogens and putrcfaotive bacteria (12). Theaimsofthis workwete 1)todemon— strate the specificity of lactase from psycho- trophic. food grade Bacillus subtilis [(1.88 (15), 2) to investigate the effect of the inhibi- tors ontheenzymeaaion,and3)tofollow OS formation at different lactose levels to control their production in a specific dairy product. MATERIALS AND METHODS Mlcroorganlsm, Gromh. and Enzyme Productlon Bacillur rub (16) was grown in 3 containing 5% yeast extract for 7 d at 10‘C. Cells were harvested at the end of the logarith- mic gmwth phase by centrifugatim at 10.000 x g for 15 min. the pellet obtained was washed twice with .05 M sodium phosphate bufl’et (pH 7.0), suspended in 10 m1 of the same bufi'er, and dimrpted by three passages I773 1774 throughaFrenchPressCellathOMPa (American Instrument Co.. IL). The disrupted cells were centrifuged at 15,000 x g for 35 min. The supernatant was then subjected to togaphy. (Phannacia. Piscataway. NI) with Mono Q HR SIS column for ion-exchange and Snperose—lZHR 10130 for gel filtration chro- matographyasdcectibedbyleeandleefl). Enzyme Assay and street of inhibitor: The mzyme assay mixture was composed of 1.9 ml sodium phosphate buffer (50 mM. pH 7.0) containing 3 mM o—nt'trophenyl-fl-D— galactopyranoaide (ONPG). and .1 ml of the partially purified (ion-exchange chromatogra- phy) enzyme solution containing .01 mg pro- tein [Michaelia constant values for this enzyme are 2.21 and 28.08 mill for ONPG and lactose, respectively (15)]. After the mixture was in- cubatedat 10'C for30min,thereactinnwas stoppedbylheadditionoflMsodiumearbon- ate'lhebasisfortheassayconditionsiaom- lined elsewhere (15). Absorbance readings were ' determined at 410 nm. The values obtained were converted to molar concentrations using o-nitrophenol (GNP) standard me. This assay was used to determinethe effectofthe ionic mvironment mmadivhyoffl-galactoaidaseaswellnsthe 617061 Ofglttcose andgalactose.Forthe speci- ficuy studies, the same procedure was fol- lowed anew the substrates. Unless other. Wise main , all trials m m ' triplicate. One writ of B—galactoailfifse activit‘; nocfimduflleamotmofthecuzymro- ‘ tomleasel [haul of ONlein lmdenhe specific activity of each substrate compared wrth cmtrol without adding the en— zyme. 53mm Preparation Different lactom solutions (5. 10, 15. and 20% wtlvol) wereptepared hydissolv‘ lac- tose 1n sodium phosphate buffer (50 mmtg, pH 1.0).1heteaohrfionswerefleatedwithlactaae producedbyB.aubriIi:atadoseof501mitr GNP/ml. Samples wan incubated for 20 h at WdWySdmecV¢14_No.6,1991 RAHIMANDLEE 10'C with continuous stirring in I Ihakerinm— bator to facilitate hydrolysis and avoid [note precipitation. Samples for analyses were with- drawn at 2-h intervals and trnmedtately' ' heated in water-bad! (so'c. 10 min) toward-u zymeamon‘ .The sampleswetethmdilutedto a concentration as predetctmtnod' by the stan- dard auves of lactose. glucose. andgalamu- The samples were diluted trains 4-5 m" H2504 (the mobile phase), filtered. and u jeeted for carbohydrates analysis using Hm: (Bio-Rad. Richmond. CA). ngh Performance Liquld Chromatography meHPLCsystemconsisu ofarcfiaflth index monitor model 170 A. an “00;: sampler-com sedofaninjection5Y5?“l _ Radmodel7€lOLandeccntmlmoduiem asanintcrfneebetweentheIlflmlllfli':mumr and the compments of the . system. The system is 01111in m a ”fi- startHPLC pumpModel 1350 miwmv“ the cohunn is With ion-300 (Dower).1heflowratewasadju5‘°d_'°‘4mv minwithliizsousmemoblblm’ atamaximumpnssureoflePI-Weh' fiontimewasprogrammodtobemfflng systemwas connectedtoa processor and a control unit. RESULTS AND DISCUSSION Specificity of B-Gnhetosldul The hydrolysis of selected Mug galaclosidnae from psycluomphlc EM was studied to tbtennim them!!!“ 3"” towardsthcfi-D-anomericw W tore in the glyconefotm WM 7.0 were the same for all sum and will 10'C, .1 ml of the utzyme mlww ONPG was replaced with the ”firm mm. This M enzyme 9!” a,” bin preference to tlus specific WW in'l‘able 1.1nespecincity farm“ ”Ir moietywaanotdetecuflc.whwgul;md stimtiononthephenolic ringdu we. had no efi’ect on the Wag-W ever. replacemmt of the $153"de fl, 3 group with a methyl one W421.)- decrease in the overall WWW Very little is known about this _—______———-— _______‘____—————— m Hydmlyfll specific-em" (5) Rim .1 mil 1.0 an "x” s]; x 5D X SD W 53 2 cud? u .2 u .3 W 32 1 NI‘ 129 J 131 l W 26 1 K’ "5 1 17-9 '3 WW 0 u’ [81 .l [6.9 A W 34 l as“ '13 3 c1 2 W 0 Ma” 52 .1 4.9 1 W an 2 m2“ ‘5 J 53 1 W 0 O ”I“ 0 0 W 0 “52*- R” 0 0 -—‘_‘——_——_—_1 012" o o Kmnhu arm “mam m“ o o 0.1+ o o 2-Wl 162 1 1:4 I 202 :2 214 1 ‘ Mummers ‘ Thelkfti‘l'itycafB—galmt:|'.uxi¢:lnsethumps)!— I Wammmmmagmmme A mm W fiomthe ’MfiademmmmcmmMyfiisWh I 5mm: let-Ivy (Hails/mu mum) GhreeseeVOcloetM-lx) Piglnez.11reflPt.Crepruartarrvecllunatoyantlor the auger: running Iran the enzymatic hydrolysis of lactoaetl=nnlnnwn;Z-rolvmt:3.4.and5=ohgoaae- eh-iadeq6=laetore7=glncose.nd8=gataetose). atthisstage. Peak lontheehromatogramisan lath-town; peak 2 is the solvent; peaks 3. 4. and Srepresent OS; sndpeaks6.7, and 8 are lactose, glucose, and galactose, respectively. The hydrolysis of 5% lactose was followed for a maximum of 20 h. As shown in Figure 33. the OS concentration reached its maximum after 4 h. The glucose concentration was higher than that of gslactose. which indicates thumeOSaremorelikelytobedominatedby galactose moieties. In similar expel-imam for 10% lactose over 20 h, high levels of OS were formed (Figure 3b). However. the OS levels dmppedtolessthanS‘XaafierabomZOhof inwhation in favor of glucose and galactose production. At 15% lactose. higher levels of 08 were formed; lactose probably becomes an receptor for the galactose released by the en- zyme actim (3). High lactose concentration along with its low solubility at low tempera- tures (lO'C) could be attributed to the decreased hydrolysis by substrate inhibition and the by-product inhibition of the enzyme, presumably by competitive inhibition at the active site of the enzyme (Figure 3c). The same trend was observed at 20% lactose. and high rates of hydrolysis were not achieved (Figure 3d). The increase in lactose concentration obvi- ously is a major factor for OS formation due to the We hydrolysis of lactose. Roberts and Pettinati (17) reported that more than 44% OS wa'eformedatalsctoseccneentratinn of 35%, whereas Greenberg and Mulroney (5) showed that levels up to 22% OS could be Journal of Daily Saturn: Vol. 74. No. 6. 199! Wmh Figure 3.011ng tmdonbvflm dasefrmnflocrflurrnbriblrudifl'erelflmdl C: WulO‘CtAzflzlmosefli 195%., tsst lactose. and o; 20% lactose). O=W silicone-splintereA-wnli framed with 5% lactose using Strepm thermophilus B-galactosrdase' at 37 C_~ .5 we observedinthlsworkwemnctashlsh, eJEPectcd, even at high lactose _ (Figure 4). This may be due one 10:30: lion temperature ofithe reaction (1 - low solubility of lactose at this M “d high concentrations of subsoil“. inhi- could have led to a subsequent submite bitten. According to Burval et al. (4). l1“ ”3: Produced by B—galaclosidase in?!” W‘ Wmmycer lactis caused gastrointestinal OLIGOSACCHARIDE FORMATION BY fl-GALACI‘OSDASE cold-storage refirigeratiou and 2) bifidogenic '- al-‘Uwuyvtur..., Irma It figure 4. The overall otigouochatide formation try 5- M” maxilla; tubtflisat IO'C u a function glean): coneentntim: 5% (O). 10% (A). 1591: (I), and den and flatulence. Some studies indicated ‘hlllqsareaftutnothydmlyzedbyhmmn diam“? enzymes but are utilized by some ““9131! intestinal bacteria such as Br'fidobac- "NW" we (10). The beneficial roles of Bi- dobactm'wn spp. for the host organism are to W vitamins to assist in digestion and mm. to thwart the growth of putn'fab “W bacteria. and to stimulate the immune WP system (11). Consequently, ftmhel' Investigations are necessary into the OS mined due to lactose hydrolysis because of the: roles in stimulating the gromh of Bi- fidtibtfcterfum spp. and into the possibilities of g Incorporation in cultured dairy products. .HH-‘C Systems pennitted the general de- med of as with high smsitivity and good m0": thus. more attention can be paid to “if-If emu-01 in all the applications of enzy- m lactose hydrolysis. The l-IPLC also pro— . a means for establishing a sotmd analyt- Mmflflflwfidflhe roles orOSasgmwth m“ for Blfidobacrzrr'um .5131:! can be studied. CONCLUSION lwfljqaiactosidm from psychrouophic B. ”'“ '3 a Possible alternative in hydrolyzing News: the 05 formed with 5% lac- We level in milk at 10'C were at low levels mm WM the other sources. The ramifica- gmmfiflhi' work are that fl-galaetosidase from of :"bul" K138 can be used in the production )m‘m‘rredttced mutt tow in os during 17'” factors with high levels of OS. This can be achieved by varying lactose concentration so as to favor either the low or high levels of OS as dictated by the required product. REFERENCES 1 mafia. M. 1952. mug-1mm during lee- wse hydrolysis. Arch. Biocheln. Binphyl. 39:310. 213110me ‘1'.. M. C. Iohneon. and B. Ray. 1979. Factors influencing symhefi! and activity of 31-ho- toailhle in Iaflobrwfflru neidapflhs. J. Ind. knell)- biol. 2:1. sum-1. A" N. G. Asp. one A. Drhiqvist m9. commode: rot-mum Gm hydrolyl'fl of tre- mewith Sacchmmccslacfltrlaeulemm: part 1: qtmtitalive aspects. Food Chem 4:243. 413ml]. A. N. G. Aqr. IM A. mlqvirt. 1930. Oligosaecharldea formation dining hydrolyak of lac- thee with Sacrament)“: lacu‘: not»: W0): patt3: digesdhilityhyhumanurzymu invilro.Pood um. 5:189. Ginnifer, 11. F... G. Rum. and K. Wallenfell. 1976. A quantitarlun of the fasten Well affect the hydtoh-w and mgrlaetosylne activities of fi-gnlactmidase (E. colt) on lactose. Biochemiefl'y 15(9):1994. ‘Iuee.S.Y..uud1i.H.Lee. 19119. mi chanctaizltion of mime: from acrobat-Etna: met up. pseudoplanm L112. Binlanhmt Appl. Biochem- 11:552. 8Un4.R.R.M.DatfleLD.A.Cowm.IndW.Mor-pn. I989. Mid-um astr-ainofflremuhic thermotthile, Themaobucler. Enzyme Mkaob. “radium. 11:180. 9mm.n. B..de.A4nnclnlck- 1980.311th ltmr.ittrmun.mmruds.om 1987. Lactulese as sugar vndr pin-stomped signifi- eanee. Bull. Int. Dairy Fed. 212269. 12 Media. H. M. R. C. MeKeller. and M. Yuguehi. 1990.31fidohcteria and bifidogenifl tact-m. Catt. Inst. Food Sci. Technol. 1. 23(1):29. The unmade conversion of lac- tose into placiosyl oligasaochn‘ides. sch-nee 117: 1778 manna: Tmmm,lm75b29.m.m IQWWLLMF.V.KMIM.M 17Rohl'fl.fl.k..lnd1.P.Peflimfl. Inflammation miwmdmp-m» efl’mhflumymficmuflonofhmuw flmmm.1.naincL56:l4m. 0W. I. Agric. Food Chm 5:130. 20M 8.. all M. H. Lopes-Lain. 1mm iSWMKWMO.P.MAMn1960.G1-mfi- MMMMWWW m.Mv.Cubdkthh-m.l6:239. finamkwoffimml.hodm53am WMWmvumumqml ...
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