eviews REVIEWS a Fateneadt aety Galacto-Oligosaccharides: Production, Properties, Applications, and Significance as Prebiotics Duarte PM. Torres, Maria do Pilar F. Goncalves, José A. Teixeira, and Ligia R. Rodrigues Abstract: Galacto-oligosaccharides (GOS) have now been defintely established as prebiosic ingredients after in vitro and animal and human in vivo stadies, Currently, GOS are produced by lycoside hydrolases (GH) using lactose as substrate ‘Converting lactose into GOS by GH results in mixtures containing GOS of different degrees of polymerization (DP) unreacted lactose, and monomeric sugars (glucose and galactose). Recent and future developments in the production of GOS aim at delivering purer and more efficient mixtures. To produce high-GOS-content mixtures, GH should not cory have good ability to catalyze the transgalactorylation reaction relative to hydrolysis, but also have low affinity for the GOS formed relative to the affinity for lactose. In this article, several microbial GH, proposed for the synthesis of GOS, are hierarchized according to the referred performance indicators, In addition, strategies for process improvement are discussed, Besides the differences in purity of GOS mixtures, differences in the position of the glycosidic linkages ‘occur, because different enzymes have different regiochemical selectivity. Depending on oligosaccharide composition, GOS products will vary in terms of prebiotic activity, as well a other physiological effects. This review focuses on GOS production ftom synthesis to purification processes. Physicochemical characteristies, physiological effects, and applications “of these prebiotic ingredients are summarized, Regulatory aspects of GOS-containing food products are also highlighted swith emphasis on the cusrent process of health cliims evaluation in Europe, Introduction duced worldwide w Lactose (f-D-galactopyranosyl-{I-+4)-a-D-plucopytinote) is rate {CAGR) between 3% and 5% until 2010 (Affe a diacchavide preset in the milk of all mammals, with only few 2007). Lactose has many uses i foods (infant formulas, choco- r exceptions, in the rine of concentrations of 2.0% to 10% ate and confectionery product, baked item, and other procerted {ov/e). The average lactose content of bovine ml ir about 4.8%, foods including men product) snd pharinaceutical (a expe ‘ranging between 4.4% and 5.2% (Ganale and others 2008), During of blew and, in finely granulated form, a8 2c cheese production, almost all the lactose in mulk is transferred. in éry powder inhalation preparations) (Sl into whey. Unlike in the past, whey is not considered a waste tn humans, lctose maldigestion increases with age, reaching anymore. Nowadays, economic and environmental considerations reported adult levels of approximately 70% of the world’ adult dictate chat whey should be wed eficiently. Whey can be dried population (Paige 2005). Theve individaal tend to avoud milk to produce vasious whey powders or fractionated by membrine consumption becatse of the risks of serious abdominal discom= technology to produce whey protein products and a pertieate fart. ‘The observation of this physiological event was the main sezeam rich in tose (Fox 2009; Lifan and others 2009) dhiving force forthe development of com The most common method for large-scale production of lac- g-pulactsidase activity that permitted the sore directly fom whey oF from whey permeste i erytaiation tose products (Gekas and Loper-Lewa 1985, Pivarik and othets ofa supersaturated solution (Yang and Silva 1995; Paterson 2009). 1995, Nakayama and Amachi 1999; Rehman 2009). Since lac~ In 2006, about 70000 metric tons crystilline lactose Were pIO- tose has low solubility and low sweetnes, its hydrolysis be performed to decrease unwanted etre erytalization events che range of lactose applicstions when sweetnes is desired (Gekas snd others 1995; Nakayama and xp expected compound annual grovth ‘sholt-Allen [MS 20100121 Submited 2/3/2010, Aspe 4/11/2010, thon inate Shae Trl ue OFT? Guna Pom ay 284 Lopez-Leia 1985; Piva {Be Tie, ond Rednus a wih IBB Into Botehslgy and Buenp> tachi 1999; Rehman 2009) necing Cento Bgl Bngncng, Uric te Mik, 4710087 Bg, B-Galictosduse isa hydrolase that ‘Mor Trew yo Nato od Fd Stony, Uo Reser us, 420063 Pr Pra uo Congas ah REQUIAGTE es the o-glacosyl group of lactose, The general mechanism of enzymatic lactose hydrolysis : oa has a transgalactosylc nature, involving + multitude of sequen Dept de Engehania Culm, Pune de Egehana du Un do Pr, Rus Dt [Relers Fran 1200-405 Por, Pa. Dit nguns tutor Toes (Emad tal zeactions with disaccharides (other than lactose) and higher storeistenp com) saccharides, collectively named galacto-oligosaccharides (GOS), 4 intermediate products (Wallenfels and Malhotra 1950), tn 438 comprehensiveReviewsinFoodScience and FeodSafety + Vol.9,20Galacto-oligosaccharides Gowramoecular transgalactoslation) Gal-B-(Ia)-X + (©) Gal- Gaerne Gal-B-(lb)-x + (©) Oya 1,0 X+@-Gal = ®+Gat ti Gevamokevbr renga) Y I~ iveminiss progress ofthe reaction, generated products ate Potential substrates forthe enzyme Y ean have ofthe flowing structures: lucese, Cal, {al al, or[all,cle (with 1 =" <6}. © + Gal-p-(e-¥ processes for which lactose hydrolysis tequited, such at for low Ihetose or lactose-free products, GOS appear as undesirable by- products at they can alto potentially produce secondary effect However, the abilty of those oligosaccharides, when added co infant milk formulas, to replicate the bifdogenic effect of hu- ‘man milk, not only in bacterial numbers, but also with respect £0 the metabolic activity af the colonic microbiota (Knol and others 2008), has significantly increased interest in thei production and, pplication in various food and pharmaceutical processes, GOS were recently defined a5 “a mixuue of those substances produced fiom lactose, comprising between 2 and 8 saccharide ‘unite, with one ofthese unite being a terminal ghicose andthe re- ‘maining saccharide units being galactose and disaccharides com- prising 2 units of galactose” (Tzortais and Vulevic 2009). The global market size of GOS was recently estimated to be about 20.000 tons with a CAGR of 10% to 20% (Affertsholt-Allen 2007). ‘This review on GOS focuses on production processes fom synthesis to purification. Physicochemical characteristics, physio- logical effects, and applications of these prebiotic ingredients are sommarized Synthesis Strategies for Production of GOS Te is well known that oligosaccharides can be formed from monosaccharides by the action of mineral aids (chemical syathe- 5). This process, known a “reversion,” exphins the production of oligosaccharides during acidic hydrolysis of lactose, frst observed inthe 1950s (Aronson 1952). The conditions suitable for ligosae~ charide production during acidic hydrolysis of lactose and the re= tuling oligosaccharide stactares formed have bees well sudied (Buh and others 1990, 1991), Ie was reporced that there is for ration of a complex mixture of disaccharides and tisacchardes, with a variety of linkages wth a~ and B-anomeric configurations, and anhydeo-sogars, a resule of tis chemical process (Huh and ‘others 1991). Probably due to the lick of product specificity and ‘extreme contltions applied during aidic hydrolysis of lactose, chis GOS production process not used on a age sale “The prefered mode for GOS synthesis is by enzymatic eatalk ypis from lactose using glvcosyeansferases (EC 2.4) or glycoside hhydeolises (EC 32.1) (De Roode and others 2003), Gyeosstrnse ferses and glycoside hydrolases are enzymes that ate responsible forthe tanser of elycosyl moieties om a donor sug to an accep tor (Ly and Withers 1999). Glycosytransferases use sugar donors containing a nucleoside phosphate ora hpid phosphate remaining ‘roup (Coutinho and others 208; Larson and others 2008). Al. ‘though highly regio-elective, stereoselective, and efficient, these ‘envymes ate not used for industrial GOS production due to theit ‘unavalabilty, prohibitive prices of commercial enzyme prepari- tions and the need of specific sugar nucleotides as ubsmates De Roode and others 2003) Gurrently, GOS are indussally produced sing the eatae scsity of pyconde hydrolaes These enzynes ate more teade diy salable than lycoylansfeases but ae generally ls stereo= selective (Troris and Valevc 2009), Enzymatic hydrolyisof the tGycondie bond ss performed by 2 extytc residue ofthe one Syme acting st gee acd and 2 nocleophile/bur,eopecaely. Depending on the spit poston of there etc reais, y= drolys can occur wach 1 of2 posible stereochemical outcomer: inversion of momerie configuration, if the average distance be- tween the 2 exaysi reside i approsimately 10 Ai of seenton of anomeric configuation, ifthe merage dance between the 2 cata seues is about 53 A. This subject has been exon ssvelyevewed by other reseschers (Koshland 195%; Sinnor 1990 Dives and FHenriat 1995, Zechel and Withers 2001) Some reining glycoside hydrolases are able to catalyze GOS sywhens. Mechaniriily, thee enaymes ae thought t0 ope ste by a 2atep rection mechanion. The Ii ieverable ep involves formation of 4 configrsionaly inverted galactonl- fnryme intermediate, followed by the exit of the “larng group” ‘The covalent intermediate i subsequently hydrolyzed, un with inversion, completing the reaction with tention of configuration na gene acd-byse calyte mechanism (Koshland 1983, Sine hot 1990; Divies and Liennsat 1995, Zechel and Withers 2001), Inavwllknovn variant ofthe 2nd ep the glctonated enzyme nay be intercepted by nucleophiles ether than water, potentially dy supa in slituon to fem tansgalactosylaton products (GOS) (Figure 1) (Wallenfels 1951; Pazur 1953). “Therefore, converting lactove into GOS by fogalactoniates it 4 hinescally contolled reaction, by means of the competion lewween hydra and tinzpaaconltion. Specifically daring ths comveron, the thermodynamical favored hydlys of lex tose, which generates D-paactose and D-glucose, compete with the wane activity that generates a complex mixture of varie ous glsctoecbued ie and oigoaccharides of deren race {fvortas and Valevi 2099). Hence, knowledge of the reaction time courte (oF lactove conversion) ie required to determine the point of maxim yield ofthe deed prodct Tranigaactonyaon involves both intrmoleedar and in teamoleclr factions. Inamolecular or drectglactosy transfer te D-gacose yields reio-somers of lctove. Intermolecular oF indirect tanalactonytion i the route by which dicchaider, ttincchandes and tetacchardes, nd evensually longer GOS, ste produced fom liste (Huber and others 1978) (ig 1) ‘Alternatively to teansglictorytion from disaccharide de- scibed previously, GOS can be obuined by eevee hydiolyss ‘ Vol 92010 + Comprehensive Reviews in Foed Science and Food Safety 439Galacto-oligosaccharides, Table 1-Archaeal sources of ye hydrolases used fo the production of CO. RGAE aire pa rm" Y Process Ref Senarchacata Sufllobussovataricus .GalacesidaseltacSj cored GHT_—=«7Ot080—«S5t96S —Tte80 8B 3 SGalacosdase (as) cores = GH 75 63 50 3s 5 BGalacesdase lac cores = GH 70 35 Sse? 23 GAR Euyarchaeate Pyrococcus ross B.Glucosdase (Cela). ened GH] 7Ot095 $0055 Trego aa 4 Clues aase(Cels) nursed GHI_—=«StG85 SSO Natasa . E.chucosdase (Cela) cones GH] 70 55 45tel7 25 OMAR : Siannosidase (Srna) cones G4i__—$0t095__SOt055 1070 ad 4 hi pity. aa 80 ci fi ann cyte (caquiibriumn synthens) fiom monosaccharides (i hammer and others 1991; Monsan and Paul 1995; Pivanike and athers 1995; Crout and Vie 1998). Equilibrium yields are mach lower, as mainly disaccharides (ound 10% to 25%, w/w) and. few percentages of trisaccharides (or higher) are formed. How ‘ever, rom this reaction no hydrolytic side-produecs are obtained, and when combined with an effective separation process, a hypo- ‘thecial 100% yield is posible (Brains and others 2003). GOS Production The catalysts: glycoside hydrolases “The rpid increse since the late 1980 in che numberof asilsble mine acid sequences of plycoside hydrolises (GEL) and the ava ablly of new sequence comparison methods permit the clasfi- ‘ation ofthese enzymes bated om amino acid sequence similarities (GHensissat 1991; Henrisiat and Bairoch 1996; Cantazel and others 2008). Currently, these enzymes constitute 115 protein families (CAZy 2009). Enzymes with B-palactosidase activity (classified as EC 32.1.23/108, according to substrate specificity) are grouped ‘within the GHI, GH2, GH35, and GH42 fanslies, Lactose i ‘the natural subetste only to some enzymes belonging to GH and GI, while fimilies GH85 and G12 include enzymes that act on different galactose-containing glycosides (Adam and others 2004; BESA 2010b). Some B-glucosidass fiom the GH Emily ‘which are involved in the hydrolysis of terminal, nonreducing B— Deghucosyl residues (EC 3.2.1.21), and fl-mannosidases from the GH and GH2 families, which ate involved in the hydrolysis of terminal, nonteducing B-D-mannosyl residues (EC 5.2.1.25), also attack the o-glucosyl group of lactose, what make them suitable for GOS synthesis (20 Table 1 to 3) Glycoside hydrolases with B-galactosidase activity occur in a variety of microorganisms from the superkingéoms Archaea, Bac- teria, and Eukaryota, Some of theve enzymes have been expresied in host organisms, and/or purified by a combination of several, ‘conventional techniques, such at salting-ovt fractionation, ion ex- change, gel filration, hydroxyapatite, and hydrophobic interse— tion chromatographies (Nakayama and Amachi 1999), A variety ‘of commercial glycoside bydrolates with A-galactosidare activity sre commercially avslable for we in food processing (Richmond and other: 1981; Gekas and Loper-Leiva 1983; Pivarnik and oth= ‘ets 1995; Panesar and others 2006), and some of them are ateady ‘used in the industrial production of GOS as disewsted in a section, previously: Nevertheless, there is continous interest in finding. Inieroorganians with adequate properties for industrial uses and shle to produce specific GOS mixtures with better yields ‘The product spectrum obtained daring lctore conversion, the linkage between the galactose units, and the efficiency of trans sgalactorylation all depend on the enzyme source and the physic= 440. comprehensive Reviews in Food Science and Food Safety + Vo, 9,20 sao 9,7 2s Gum ea td anssh, chemical conditions in the catalytic environment. Interactions betwen the galactosyl acceptor and the active site of the enzyme: are thought to phy a major role n the formation of intermolect= lar galactosyl transfer products (Perzelbauer and others 20003) In ‘other words, the enzyme ability ro accommodate the galactosyl acceptor in the neighborhood of the active site during the eat= lytic moment, and the spatial orientation ofthe galactosyl accep tor, are probable key factors in the eanegalactosyition efciency and in the position of new glycosidic linkages (xe Figure 2 and “Table 4), Glucore/galactote rato at the maximum GOS yield war de- termined to quantify the ability of different enzymes to catalyze the tranegalactosylation reaction (0 letose or galactose acceptors) teltive to complete hydrolyse (Figuse 2). This ratio should be higher than 1 because some of the galactose produced by lic= tose cleavage was transferred to oligosaccharides, whereas all the glucose was released, It should be noted that ths ratio does not take into account the transfer ofthe galactosy] moiety to fice glt= cote via intermolecular reaction or vis inttamolecular reaction (see Figure I for dew) Process improvement Generally, the yield of GOS synthesis from Lactose wing ely= coside hydrolases can be increased by: using highly concentrated starting lactore solution; decreasing water thermodynamic acav= ity (fr example, using a micro-aqueous environment); removing, the final product and/or ishibitoss fom the reaction medium; and modifying the enzyme (Monsan and Paul 1995; Czermak and others 2008) Using highly concentrated starting lactose solution. Data gleaned fiom the literstute show that maximus GOS yield i largely infiaenced by initial lactore concentration (La) mainly, n= til the concentration range i 30% to 40% (w/v), For La > 30%: the iniluence of Ly in GOS yield markedly decreases (Figare 3). Data fiom A. oryzae, A, avulatus and B, longum (aot represented) fare more seatered but show a similar behavior for Lo > 40% Since hetose solubility is relatively low at room tempersture but manigey increases with increasing temperature (R00: 2009), high temperstuzes are generlly desiced, Some studies have been, focused on sourcing thermostable glycoside hydrobses, Glycoside hydsolises from S,solfitancs (Petzelbauer and others 2000; ians= ton and Adlererewty 2001; Splecitna and others 2001; Bask and. thers 2008), P furore (Petzelbaver and others 20008; Hanston and others 2001), Thermus sp. Akayama and others 2001), T cal= saphilus (Choi and others 2003), C, serhariytios (Stevenson and others 1996), T manta (Kizn and others 2008; Ji and others 2005) ate examples of enzymes from hyperthermophil microorganisms ‘used a temperatures around 80 °C and higher ° © 2010 isu of Food Technoloiss®Galacto-oligosaccharides ‘Table 2-Bacterial sources of glycoside hydolases used forthe production of GOS, BACTERIA cn Teo ry Y 1 Process Ret S-Actinebacteria Saccharopeyspoa ectvingula @-Calactosiase purified on 7 1080 “4 a Hindaboctetum adolescent Crude enayne'aeton 373 ao ae 5 Bifdobacterum angulatum Crude enayre fraction 37S St030 4 ‘ Bifdabactenum bifaum” —.Calatsizse(B\"3).mutared = CH2_ «37D Oto aa ‘ ‘Cude eye feet.on 37S 30 cn * els resting ele 3850 2 i Gels fluenestested resting cee 40 dma Asms0 Aa 9 (els toluene tested resting eels 40 S105 Whey, 25t050 38 f Bihdobacterium longum subsp. S-alacosiease(Sal)cones# = CHD 30S 6 1 ‘tants (Galactosidase (.Call,cloned CH? 301060 75 2010308 i alacsidase,(5-all. coned Cha? 377320 To k ‘ude ene traction 37S 30 8 t Gelsrincature S73 Mikasa = Bifdobacterium ongum Crude enaye fraction 4 68 51050 6 2 [iidabactenum pseudolongum Cruse enzye ation 373 a0 2 ° Deincoceus Thermus Thermus -Clucosidase, cloned ch 70 79 7Ste30 40 p Thermus enidophits [Clucstdase (BQN) ores CH For80 60 © S0wS0 77 4 >thermotogae Thermetoga maritima (pCalacosdase Lac} cloned cH 80 60 20t050 19 ' Fiemicutes Bociluscrealans ‘sCalocasidase (), purifed 4 604g 50 : 4 60 a3 40 sc t (2alactsiase (purified 4 6045 a a 4 69 48020 dk sc pak 4 60 4 33 PR PAR Crude enzyme faction fowss 69 © 63m38 x Bocius sp. ude enyme faction Sos 50 0 3m3e a y rr ne 41 chitosan r Ceobocilussterathermophius 9-Calactasiase (8938) ce 37637 24 & BGalactsidase (BgaB),mutaed CHA? 37 BST So % Caldcevesaptor ‘Clucsidase (8g), cloned CH §5:080 63 107283 = ‘occharojyicas"* octobocusccidophius ——@-Calactosidase LacLsLaM.coned CH2 30S 21 39 x “aetobocius eter Balacosidase(faccslacl cored CK2 23 GOO 26 a (BAalactosdase LaciciacM}puried CH2 30037 601065 451021 38 # Gear? go? ah Fe oman 5 Septoccuthempins —Calacaituselapuited GH? Mikes 95 a Sproteabacers Enterobacter agglomerons"** 9-Galactsiase (Le), cloned cr 5013 38 s Enterabatercloncae (3.alactsidase,(8ga/Lacz) cloned Ch? 2 8 a els resting 2 55 % Escherichia colt Galactosidase (Laz) purited CH dar 56 I a pu bm sect gh ‘te tear ott he nthe if em st [2004 ete ODS ee ase Stina tae sg ets aE vse nd oad an ance Ree eee Re Sea ey opt tonsa ee High temperature alto seems to favor tranigalactosylation rel- ative to hydiolyss. An increase in reaction temperature sgaifi- cantly increases GOS yield when glycoside hydeolses from A. scales: (Cazdelle-Cobas and others 2008), 5. solfataricus, or P fu ross (Hansson and Adlercreutz 2001) were used. However, other researchers found only a slight or no correlation between tem- perature and GOS yield sing P fares: (Boon and others 1998; Perzelbauer and others 2002), S. sofas Perzelbauer and others 2002), B.cvalons, A. oryzae, K. las, and K. marcianas (Boon and ‘others 20003), “The use of microwave irradiation as a nonconventional energy source during GOS synthesis was also auccenfuly attempted using. an immobilized crude enzyme preparation from K. lactis at 40°C (Maugued and others 2003), Maximum GOS yield inereated from 224% to 28% when inital ietose concenteation was 168% (w/¥} 2010 stitute of Food Technologists? vol wees {20059 Cals yews 2007ah Cols a eee ae Arties 082 Meni and sine 0 Mes yeaa ths Near he es fae, gaa pa apache acetal Loses ale feet ceroppones nes eee Decreasing water thermodynamic activity. Ax example of using, 2 micro-agqueots environment to decrease water thermodynamic setivity during GOS synthesis is the use of reverse micelles. A ssmalar wansgalactosylation capability was obtained at low Lactose concentrition when reverse micelles were wed, a compated co ‘that reported 3¢ high lactore concentration in an aqueaut system, (Chen and others 2001, 2003), “Aqueous 2-phase systems appear to inctease the transpalactoay~ lasion yield in comparison with uswal aqueous solution probably clue to the partitioning of the desired product, inbibitor, and the enzyme between the 2 phases ofthe systers. The plucose/alactose ratio at maximum GOS yield wing 2 crude enzyme fiaction fiom, A. soulatus increased ftom 2.2 co 127 (Figure 2} when an aqueous 2ephase system is used, rellecting an advantageous environment for ‘galactoryl transfer reactions (Del-Val and Otero 2003) 2010 + Comprehensive Reviews in Food Science and Food Safety 441Galacto-oligosaccharides, DEAROTA ae GY) Pree Apeaiisacien ——Caudeeayme tain 451060 4st26S lows? 28 : uso oso Nee ans 3 se so Sune : Asp iger tule name frcion 13 ate 3 pu ofene ade rage acon owe tSu70 ees : Bee 73°" Notes aw ft eo 8 » moe Sowss asa Trm30 tan 3$t038 430080 wheyaiozs Te ; BP" Aeks Tea HE cts i a 4S" ssa? ay en Hl ® i FF i cos 43 Slop Be Gon Pk 43 Ste5e Be nrozhva : 43 $88 3e rosa : 43 imyeso20 He Metin ae Penctumetpanan ——_Cudeenyretacion ans $2 SOBA? IS Aas 5 SosSochoompctareive, GS 8 Stns ; enim hits catenin ws $0 2 ‘ Fetal phim — Ssicentae hes $5 Hote do i Tolromeerdermophis — cascansn putes & be x ‘ $3 Sbm20 a ‘ ® de a a ed Tietedema harzanim cae 8 is 3 x Mpetompet aye racion Mose $9073 Sbico 38 ; we? sE78 toe 8 aun "7 abe Rae ts & 8 Te An ow 88 down He Eatin Mate Bios 19 Whey ttto22 42 Fa Bee a nyze aur Kuperomyesmanionas* —Caateenyne action S104 G2u70 Sowse as i Bee TBE Gay ttto2a Be 3 > baonye Srcbaidiomagnun ——_alacosiae, pues & 50 2 a8 * Est cure 88 S : Ele hunetesegresingeete 3D S y Seignatonyesehice lesan ped &@ § 3 a eat were %& ge se i Ele whunetenedsesiogeale = 3 se in Atodtnsaminta ‘ein ped @ Se FH e Exist % 2 e se * Elle uneiestdreiogeele 98D 0 FA = Srostobmyessngiaic ‘Sluatevelgnygutee ot) S50 S360 TBs 8 3 Shonda patee a as Ege Ip 55 chiens oak ch G8 hey20 3 x te ring cals SS we ie = ec resing el S50 ao 40_algaste au See ele suta yucatan Sea eet acl ig hate rece seen ate soc ees eae Bare St ico atria eet te 18h a ines beet 0, bese ib phen Tobe Sr an sa 990, my eb sthrs 0) a acer ces ON Eretmyc em one Scho Sp sna el Modifying the enzyme, Optimization of the enzyme saucture can contribute to increasing the maximum GOS yield Gom la~ tose. A protein engineering approach was aplid to Boplicosidae fiom P furious. An ineewse in GOS yield was observed by change ing phenylalanine 426 residue to tyrosine (Table 1). By changing sn additonal residue, methionine 424 to aysne, beter transgalee tosyliton properties at low lactose concentrations were obtined (Hinson and others 2001). More cently, ini approach was spplied to the Besilactondave fom G. steesthermaphiu. When 442 comprehensive Reviews in Food Sconce and a Safety + Vol.9,20 ised a otc slne enh ea Tate Baonit a eh as bah sree ace era hie (an Sesto et 29, ah Pecuaneus ena 2} a eben as ae ale ener the arginine 109 residue om the active site was changed to lysine, valine, or teyptophan, trisaccharide yield increased from 2% to 12%, 21% and 23%, eespectively (Phcier and others 2008). Deleon mutagenesis of the C-terminal part tamed the B-puactosidase fom B. bifidum into a high-effcient transgalac— tomylating enzyme (Table 2) characterstieally noniniluenced by lhctose concentration Jorgensen and others 2001). At maxianam, GOS yield the glucose/galictose fatio in the reaction media is ‘much higher using this truncated galactosidase from B, bm © 2010 isu of Food Technoloiss®Galacto-oligosaccharides than that obtained with other Pogaactosidaes from the genus Bifidobacterium (Figure 2) ‘Transgalactosylition activity of the A-galctosidase-1 fom B. ciralas was increased by modification of the enzyme with glu- taaldehyde (Table 2}, probably due to conformational changes near the active site of the enzyme (Mozaffir and others 1987). ‘This is an example of a chemicalnduced structural change, pat= siculaly intereming since glutaraldehyde i commonly wed at a ‘roztinking agent for covalent enzyme immobilization, Removing the final product and/or inhibitors from the reaction medium, Membrane-assted, continuously stirred tank reactors have been used for GOS syntheds. An advantage of this type of scheme is that the product is continuously removed (long. with water, some substrate, and simple sugir by-products) Gor the sired tnk using ctostflow membrane wluaéltration, while the enzyme is retained. This configuration ako allows variation of the residence time aiming at the optimization of the yield and. composition of the oligosaccharides fraction (Czerinak and othets 2004). The yield of GOS produced by glucosidase from S. sal- eters 4¢ 30% and 70% lactose conversion wat 3- and 1 3-fold higher, respectively, inthe continuous reactor as compated to the results obtained inthe batch eactor Petzelbauer and others 2002) ‘When compazed with the batch process, higher yields of GOS se ing membrane-asisted continuously sized tank reactors were also ‘observed during lactose conversion (in solution of in whey) with ‘enzymes from K, latis (Table 3). Increases in GOS yield can be exphined by a mote efficient uansls of galactosyl residues to glu= cose and lactose, and a smuller extent of secondary degradation ‘of GOS in the steady sate, a compared to batch reactor (Ret= aalbauer and others 2002; Czermnak snd others 2004), However, this advantage was not observed wing glycoside hydrolates fou P fiisus (Petzelbauer and others 2002), L.reuten (Table 2), and A. ‘nyzue (Table 3), There differences result from the ditinet kinetic properties ofthe enzyines involved Contrary tothe batch operation mode, where the composition ofthe different saccharides is changing constantly over the reaction tume/lactose conversion, isthe steady state of contisuous system the composition of the mixture stays constant over time. The ‘composition of GOS mixtares can be finely toned by controlling the residence time ofa continuous systems (Petzelbauer and others 2002; Czermak and others 2004; Chockchaisawardee and others 12005; Splechtna and others 2007). GOS yield and lactose conversion ‘As already discussed, the efficiency of the tanspalactorylation reaction is kinetically controlled, a5 GOS are potential substrates af the glyconide hydrolase enzyme. The formation of eligosac- charides reaches a maximum time-courte during a batch reaction. 3 . 21 enn g 5 = fy s za 2 i 2? é o om a a Bone LA gM ey re Enayme source (se legend) Figure 2-Clucose/ galactose ratios at maximum GOS yield of glycoside hydrolases from diferent microbial sources, ARCHAEA: I 5 solfataricus {(Petzelbauer and others 20008); 2. furosus (Petzelbauer and others 2000b}; BACTERIA: 3 5.rectivirgua (Nakao and others 1994); 4,5. bitum (B13, mutated) Uorgensen and others 2001) 5B. infants {(B-Gall (Hung and Lee 2002); 6,6. infonti(2-Calll) (Hung and Lee ‘Thermus 5p. (Akiyama a fs 2006¢); 11, © stearothermophius witd-type (Placier land ethers 2009}; 11°, steorothermophius (mutated) (Placie and ‘others 2008); 12,1. acidophilus (Nguyen and athers 2007); 13, reteri, {Splechtna and others 2006); 14,5. thermophius(Creenberg a Mahoney 1983); 15, ¢ saccharolyticus (Stevenson and others 1896); 16, T. maritima (i and others 2005); FUNGI 17, A. aculeats (Cardelle land ethers 2008); 17°", A, aculeatus (aquesus 2-phase systems) (Det-Val hemophis Nabhan lei 2007) 20, 7 pena and ‘thers 2008); 21. funiculosum (Shin and Yang 1986) {impicissimam (Crsz and others 1999) 23, K mars Pettnati 1957}; 24, K lactis (Chockchaisawasdee and others 2005); 25, ‘S-magnum (Onishi and Tanaka 1997) 26,5. singuaris(shikawa and ‘others 2005); 27, R. miata (Onishi and Tanaka 1996); 28,5. elvae {Onishi and Tanai 1995) (Dats extracted fromthe iterature) After the maximum point, GOS concentration decreases until ap- proximately zero when lactore conversion is around 100%, The Incteate in the maximum amount of oligosaccharides can be due cither to the enzyme ability to catalyze the manspalactorylation reaction relative to hydrolysis (as discussed previously) and/or to a lower sate of breakdown of the oligosaccharide products relative to Inctose, probably because ofthe nyuch higher km (afinity) values for GOS than for lactose. The enzyme ability could explain the differences observed in Figure 3 regarding the GOS yield (y axis), while the lower tate of GOS breakdown may explain the diferent lactose conversion rates obtained (x axis) ‘As reported inthe literature, maximum yields of GOS between 159% and 77% are achieved when lactose conversion is between 45% and 95% depending on the enzyme source and the production, process Figure 3). If, with 2 given enzyme, a maximum yield of GOS is obtained ora high lactose conversion (examples: glycoside Ihydsolstes from caldophius, S. thermophilus, or K. mersianus), that is probably because it has higher km valtes for GOS than for lactose Enzymes, cells, or resting cells Processes for GOS production from lactose can be roughly ela sified into those employing enaymes extracted fom microbial cell, with variable purity; those where a specific microorganism, is cultored in a laetose-containing medium, and finally those that ‘use resting cells of a lctosenutiliving microorganism acting as a so-called enzyme bag (Dombou and other 1992) ‘The former process requires the extraction of enzymes, which ‘means additional costs and time consumption, Moreover, ashy drolyss of lactose and GOS is, to some extent, ineviuble, ac- ‘cumulation of glucose and galactose as by-products occurs. The presence of these monosaccharides is generally reported as unde= sinible ance the objective i ta produce tue and Larger oligorac- charides, Galactose and glucose were reported at inhibitors of flycoside hydrolases fom K; lactis (Chockchaisawasdee and oth- 14 2005) and from S. ebiae (Onisht and others 1995), respec- tively regarding GOS formation, However, an unexpected postive ‘ Vol 92010 + Comprehensive Reviews in Foed Science and Food Safety 443Galacto-oligosaccharides, a z z ie 07 61 3 mn a Fi ay p> pue Hi igo hq poses suena kg asr3e © 2010 Isitute of Food Techn Safety + V 444 comprehensive Reviews in Food Sconce andGalacto-oligosaccharides aa Maximum yeld/maximum yield when 20% _ a 1 . 60 . = x a 2 so a ze |e oF 0 Oo ee 30 a . 20 6 o ° 2 Py 20 o> 0 mm mm m0 ita cto ocean ae Late comerion Figure 3-The efecto intial lactose concentration on maximum COS Yields, 5 sotfataricu (Petzelbauer and others 2000b;Splechtna and ‘thers 2001; Park and athers 2008); MP friosus (Petzelbauer and ‘thers 2000b; Hanston and others 2001) - 8, ongulatum (Raia and ‘others 2001); a, T.caldophils and Thermus sp. (Akiyama and others 2001; Choi and ethers 2003); , 7 marie Kim and others 2004; Ji and ‘others 2005}; 0, niger (Toba and Adachi 1978; Prenesi and others 1987}: expansum (and others 2008); oP simplissisum (Cruz and ‘thers 1989); 6, K facts (Burvalland others 1978; Boon and others 2000s; Chockchasawasdee and others 2005; Cheng and others 2006s; Martinez Villauenga and others 2008): x, K mardanus Roberts and Pettinat 1957); +, 5. singularis(Shn and ethers 1998; cho ana ethers 2003: shikawa and others 2005), Maximum GOS yield is normalized to reduce the enzyme soure effect. effect of ides on GOS yield wat observed when ‘using B, bifidum reeting cells (Goulae and others 2007: CCalure-based processes can be conducted if glycoside bydro- lases expresied by the microorganism are cell-hound or secreted into the culture medium. One ofthe advantages of caltured-bate processes is that glucose and galactose can be consumed during. growth reducing their content in the final saccharide mixture, ‘thus improving GOS yield. Higher GOS yields weve zeported ss= ing fermentation systems with microorganisms from the species S.elriae, . magnim, or R. minuta growing on lactose compar- cells ot purified enzymes from the igure 4) (Onishi and others 1995, 1996; Onishi and Tanaka 1996, 1997). These researchers concluded that these improved GOS yields rule from the fact the glucose is connimed for cell growth, since ghicose har been found to inhibit GOS eynthesis, More recently, an inno- vative stategy for GOS synthesis by anchoring Begalactosidase from P expansum on the cell surice of S. mobilized enzyme was also stccewflly apphed (Li and others 2008), (On the other hand, GOS mutt be separated fiom accumulate secreted microbial products and ffom other ingredients essential for useful for cell growth incoxporated in the culture median (estrogen sources, vitamins, trace elements, among others) Industrial GOS producers use either cells ot enzyme extract For example, © laurent resting cells entrapped in calium alginate {gels ae uted for indusral-seale production of GOS, Furthermore, GOS aze also produced by means of a sequential reaction of a Figure 4-Maximum reported yield of GOS as a function of reported Conversion. enzyme; *, cells. Data extracted fom the itersture. ARCHAEA: 1,5 sofetarcus (Park and ethers 2008}; 2, .friesus (Petzslbauer and others 20008) BACTERIA: 2,5, ectvirgla (Nakao and thers 1994). 4, bifidum (BFS, mutated) (Jorgensen and others 2001), 5,8. bifidum (Tzortzis and others 2005b);6,8. infantis (B-Call (Hung and 1 2002}; 7,8 longum (Hsu and others 2007);8 7. coldophils (Choi and thers 2003);9, Thermus sp. (Cheng and others 2006¢); 10,8. crculans {jecali (Mozafar and others 1984) 11,8. cculas (8 11999}; 12, Bacup, (Cheng and others 2006) 13,6. -earothermophilu (Pacier and others 2009) 14, c saccharolyticum {Stevenson and others 1996); 15,L acidophilus (Nguyen and others 2007); 16,1 reste (Splechtna and athers 2006); 17,5 thermophilus (Greenberg and Mahoney 1983) 18, . cll (Huber and others 1976); 19, maria (and others 2005); FUNGI: 20, A, aculeatus (CardlleCobas and others 2008) 21, niger (Toba and Adachi 1978); 22, oryzae (iwasaki and others 1996) 23, .expansum (Land others 2008); 24, ? ‘expansim (f-galactaidaze gene from P expansum expressed onthe cell Surface of cerevisiae) (Ui and ethers 2009), 25, .funiculesum (shin and Yang 1896); 26, smplicisimum (Cruzand others 1998) 27,7. thermophilus (Nakkharat and Halteich 2007); 28 Tactis (Cheng and ‘thers 20060); 29, K marxanus (Raberts and Petinat 1957} 30,5. ‘magnum (Onishi and Tanaka 1997); 31, 5. magnum (Onishi ana others 11986}; 32, 5 elvige (Onishi and Tanaka'1995) 33,5 lize (Onishi and thers 1995); 34, minute (Onishi and Tanaka 1996): 35, R- minuto (Onishi and Yokozeki1996};36, 5 singulrs (Shin and others 1998); 37, Ssingularis (Sak and others 2008), Lhetore solsion with A. oryzae Begaactosidase snd S.themmaphilur B-palactosidase, sespectively (Nakayama and Amachi 1999), Nevertheless, itis amportant to notice that the production of GOS mixtures using microbial cultures. sa consequence of the multiple glycoside hydzolases expressed by some micro ‘ania, ‘Thus, this combined activity ofveveral enzymes nts the ide hydzolses from B. crus (Morar ad others 1984), 3 from A. niger (Widener and Leuba 1979), 3 fiom B. bifidum DSM20215 (Moller and others 2001), and B. bifidum NCIMBS3171 (Gouls and others 20076) have been reported High-content GOS production GOS mixtures produced by transgalactosyation always co considerable amounts of nonreacted lactose and monosaccharides. The efficient removal of these non-GOS impurities allows the commercialization of added-vilue GOS products (Crittenden and Phyne 2002) 2010 + Comprehensive Reviews in Foed Science and Food Safety 445Galacto-oligosaccharides, Table 5-Chemical composition of commercial GOS (w/w dry mat in Glucose Galactose Lactose DP2__DPS___DPs DP Total Enayme source fet Grote 751030 ~ ~ - = 70 Grypioeccus owen * Sligemate 53 181038" 1ote22 151017 18926 10116 254 —SOt0G0 Aeyeveanes themophis Tost" ot Otero 55. 331035 2014 9910100 AonzseancS themophils —& VivialGos 192022 aBto13 10t23. 91027 22:023 11 6ot7s 571559 Badluscreuans 4 Binune 1D 22°"? 954029 I2%14 G7t—077 38toA4 484953. Bifobocterumbifum ——€ Purimune fo 1.0 D805 7Ote10 16102) 3BtaST — 2Se—79" ADEN Bacllurcrcuions : Promouts cos 9" : . . : - - s " Seperate erent ner eaeln ect ear eae rete ye dat eet Beier ee LA sistema ses 38) Bayete ie PUIG eh sabi ie ages arty Large-scale sepustion of monoaccharides is ually con ducted by 2 chromatographic process with ion-exchange resins dr activated charcoal (Hemander and others 2009; Nobre and ‘other: 2009). Regarding ion-exchange chromatography, the cation-exchange reins are the most used ar they have the highest afioity for monosaccharides, and therefore oligosaccharides are the first to elute fiom the coluusn, Activated charcoal has been reported o possess a higher afinity for oligaraccharides compared, to mono- and diiccharides, which makes their operation at the industrial level more advantageous, since regeneration can Uke plice off-line without large substrate loses. Recently, a comparison of ftactionation techniques to obtain high-content GOS mixtures, a preparative sale, concluded that size-exclusion chiomatography was the most appropriate method to obtain fiacions with high purity, enabling the purification of GOS with different degrees of polymerization (DP) (Hernander and others 2009), Although not in a mature stage, membrane techniques, particularly asnofitestion, alto show potential for large-scale fractionation of oligosaccharides from complex mix- tures (Goulas and others 2002, 2003; Feng and others 2008), Su- peteritical uid extraction technology also has shown satisfactory performances in the isolation of monosaccharides, disaccharides, and higher saccharides fom complex mixtures (Montanes and ‘thers 2009). Furthermore, other approaches based on the selective fermen- tation characteristics for a given microorganism have been pro- poted, During these fermentation process, depending on the ‘microorganism, ethanol can be produced and, ifn toxic concen- trations, its activity can be compromised, S-coviie wae wed to improve the purity of a commercial mixture of GOS obtained swith f-galactosdase ftom B. ciulons by completely removing the monosaccharides (Hernandez and others 2009). The same ap- proach was applied co 2 GOS mixture produced by B. bid biomass (Goulas and others 20073). K. marsiamus was used to it~ prove the purity of 1 GOS mixture produced by B-galactoidate from B, crlans from 38% to 97% by selective fermentation of amono- and disaccharides (including Lactose) (Cheng and others 2006). A combination of S crenisae and K. luis was used to im- prove the purity of 1 GOS mixture produced by f-galactosidave from P expansum fom 29% to 98% by selective fermentation of ‘monosaccharides and lactose (Li and others 2008) ‘The sepurition of ctore fom 3 diaccharie fection has proven to be dificult by all the reported processes, and usually zeslts in lage loses of GOS product, mainly nonlactose disaccharides, ‘To overcome this difficulty, 2 process where lactose is efficiently separated from other sugars by anion-exchange chromatography, afer its selective oxidation into lactobiouie acid using a fangal 446. comprehensive Reviews in Food Science and Food Safety + Vo, 9,20 est 2004 20) lena nen oD pdt 30 ean eae a Beaks a abl Ears a cellobiose dehydiogenate, was proposed by Splechtna and others (2001). However, with this proces, total lower of GOS around 17% occus, since the fangal cellobiose dehydrogenases used are not specific for lactote (Splechtna and others 2001; Maischberger and others 2008) Commercially available GOS GOS have been manufievured and commercialized by Yakolt Hossha and Nissin Suge ManuGitaring Gom Japan, by Corn Products Int. fiom the United State, and by Borculo Doma la- sgredients and Clisido from Europe. Also, some companies have been reported to produce GOS for incorporating in their own products rather chan fer commercial purposes Playne and Crit= tenden 2009). Recently, new players have come into the market, Dut available information on their products is scarce (Table 5). Food-grade GOS are usually transparent syrups or white powders, In both cases, they are mixtures containing oligosaccharides of different DP, the nonreacted lactose and monomer sugars (ghicore and galactose) (Phyne and Crittenden 2009; Tzortis and Vulevie 12009). Purified products with moze than 90% (ov/) GOS are svuilable ftom some manugacrrers Besides the differences in the purity amongst the commercially offered products, chere ae diferences alo an the linkages of the oligosaccharide chain due to the diferent enzyme used in their production (Table 3). The Oligomate range products offer mainly GOS with B1-+6 linkages; the Bamuno product contains mainly 1-3 linkages, whiltt Cup-Olgo, Vivinal GOS, and Purimone offer mainly 61-4 linkages (fee Table 4 for more details about alycosidic linkage according to enzyme source). GOS Properties Physicochemical properties of GOS Since commercially avaiable food geade-GOS are mixtures (able 5), itis expected that their physicochemical properties and physiological actions will, to some extent, depend on the ‘mixture composition, which in tara will determine its proper ap~ pheation. Sweetness, solubility, osmolality, crystal formation 2b diy, and rescvity (Mallrd reactions) decreae athe moleculit size increases, contrary to viscosity (Phyne and Crittenden 2009), “Tle 6 presents summary ofthe commonly eported GOS prop- Physiological properties of GOS Indigestibilty. Several m vino and in vivo experiments have demonstrated the indigestibilty and stability to hydrolysis by digesuve enzymes of GOS. In a consensus report, wis cone ‘cluded that more than 90% of GOS pases into the colon (van Loo snd others 1999) ° © 2010 isu of Food Technoloiss®Galacto-oligosaccharides 1 from Macfarlane and others (2008), Playne and Crittenden (2009), and Tzortzis and 30 “Cfo: 10min at p 7, stable to 100°C fr 10min at pH2; stable to 37 *Cat 2 for several months Table 6-6 schemicl properties of COS. Ad Vulevie (2000) Sola soluble, about 80% (w/in) ‘Appearance centealarle eos To that of igh fuctose com sytup Hest stably Stableto Freezing point Reduces te reezig pint of anes Humane properties High motture retaining capacty preventing ecassve dying Sweetness "Typically 0.3 tg. times tat of ease ‘This and teua- GOS were not hydtelyzed in vino by human salivary a-amylase, artificial gastric juice, @-amylse of hog pan- ‘reas, and rat intestinal acetone powder, Contraily, disaccharides ‘were partially digested by the intestinal enzymes (Chonan and, ‘others 2004). In another study, only a very small amount of 4'- sgalactosylactose was digested by the homogenate of the rat small, Intestinal mucosa (Ohtsuka and others 19906) ‘Most ofthe in vivo human dats regarding the nondigestibility of GOS were obtained by hydrogen breath tess. Breath hydro- ‘gen excretion i likely to be a dose-dependent effec, Using this noniavasive technique, several studies eparted an nctessed breath hydrogen excretion when the ingested GOS amount ws berween, 15 to 35 g/d, indicating that GOS escaped digestion and were fer- amend by the colonic microbiots (Tanaka and others 1983; Alle and others 1999; Chonan and others 2004). However, Boubailk sind others (1997) showed reduced breath hydrogen after admin- Intation of a 10-g dally dose of GOS but, at the same ume, bili dobacterial numbers were increased, indicating thatthe GOS were fermented by colonic microbiota (Boubnik and others 1997). It thas ato been shown that dietary GOS can be detected in feces of infants fed with formula containing GOS (Moto and others 2005). ‘European regulation on food labeling obliges the mancfictur ‘ert of GOS-contsining food products to clearly identify these Ingredients as dieu fiber. According to the recent legal def- inition, fiber means “carbohydrate polymers with 3 or more ‘monomeric units, which are neither digested nor absorbed in the Inuman small intestine obtained from food raw material by phys- ical, enzymatic, or chemical means and which have a beneficial, physiological efect demonstrated by generally accepted scientific ‘evidence’ (European Directive 2008). Usually, the amount of die ‘cary fiber that appears on food labels ¢ determined acconding, to the methodology outlined in the Official Method of Analy- tis fom the Anociation of Official Analytical Chemists (AOAC 1995; DeVries 2004). However, his methodology is not suitable for measuring nondigestble oligosaccharides requiring the devel- ‘opment of new methods. Specifically, for GOS analysis, a method ‘ised on the enzymatic treatment with f-galactosidsse followed by high-performance anion-exchange chromatography, using pulted amperometric detection (HPAEC-PAD), hat been adopted (De Slegte 2092, AOAC 2005), Because GOS are incigenible, but fermentable (see below), the caloric value of GOS, as well as that of other nondigestible ligosaccharides, ha been eximated to be 1 to 2 keal/g (Rober- froid and athets 1993; Cummings and others 1997), For food Lhbeling purpotes, in Europe, GOS should be given caloric value ‘of 2 keal’g (European Directive 2008). Prebiotic properties. ‘The main physiological eects of GOS are related with their impact on the comporition and activities ‘of the intestinal microbiota, The biwman intestinal tract hatbors a ‘complex community of bacteria, eukaryotic microorganisms, at chaea, viruses, and bacteriophages, collectively seferzeé to as the intestinal microbiota. Bacteria account for the majority of these 2010 institute of Food Tee inieroorganisns: ther total number in dhe buman gut i estimated 210% cellsmaily present in the colon Backed and others 2005; upp and Finy 2005). Colonization ofthe gastrointestinal tract ‘wath intestinal microbiota occurs immediately afer birch and lasts 2 lifeime. In adults, the composition ofthe colonic microbiota is generally estimated to comprise more shan 300 bacterial persis tent and transient species (bated on worldwide observations, not 1 individual person), although iti thoughe that only 80 to 40 of them predominate (MeCartney and Gibson 2008). The majority of the members of the colonic microbiota ae ablgate anaerobic fener, including Baterides, Bifidsbaterm, Closidiom, Entero- cect, Eubacterum, Fussbacerm, Pepococus, Peptstptcos, and Ruminccoeus (MeCarsney and Gibson 2006). Recent advances sn defining the quality, uantiy, and physi- logic activity of the intestinal microbiot have been posible as 4 result of the advent of metagenomics (the analysis of collective {enoues of microbes), an emerging field in which the power of ‘enomic analysis is applied to entse microbial consortia (Gill and thers 2006). Collectively aman microbiota contains atleast 100, times ab many genes 21 the human genome correrponding to 2 degree of meuboie activity that is increasingly being recognized (Gill and others 2006; Wikof ané others 200). This metabolic activity includes: the ability to metabolize, otherwise indigestible, sacchurides of our dit by fermentation to various short-chain fatey acids (SCA): inucncing host genes that regulite energy expenditure and storage; contributing to several biosyatheti path= ‘ways (including vitamins and ioprenoid precursor), production ‘of conjugated linoleic acid (CLA) somes, and bioactive peptides, with recognized health effects; promoting hort homeostasis; and decontaminating the intestine, thereby misimizing exporite co toxic substances that could resuk in malignancies (Sekitov and Finlay 2006; Ross and others 2010), Production of SCFAs is one of the most important physio- logical actions mediated by the microbiota, These are absorbed resuking in energy salvage from indigested food. Moreover, SC= FAs affect colonic epithelial cell transport, energy transdvetion in colonocytes, growth, and cellar diferentiaion. These rophic properties have important physiological implications, in addition {to maintaining the mucosal defense barvier against invading orga~ jams. When absorbed into the bloodsteam, they affect the hepsi contol of hpid and carbohydrate metabolism and provide energy to muscle, kidney, and brain (Cummings 1995) ‘The colonic microbial ecosystem is quite sible but can be influenced by genotype, age, diet, and health states (Lupp and Finlay 2008). Thiee approaches ate used to beneficially modu- late the gusrointestinal microbiots: (1) by dicecly supplementing ‘he intestinal microbiota by consuming live bacteria, “probiotics (2) by consuasing dedicated dietry components selectively wed by resident microorganisms, “prebiotics; or (8) by combining both strategies, “synbiotics” A prebiotc is defined as “a selec~ tively fermented ingredient that allows specific changes, both in the composition and/or activity inthe gastrointestinal microbiota Vol 9.2010 + Comprehensive Reviews in Foed Science and Food Safety 447Galacto-oligosaccharides, that confers benefits upon hot wellbeing and health” (Gibson and Roberffoid 1995; Gibson and others 20044). The selective properties of prebiotics are supposed to relate to the growth of Difdobacteria and letobacil at the expense of other groups of bacteria in the gut (Macfarlane and others 2006). Along with in- ulin, ructo-oligosaccharides (FOS), and lactulose, GOS are also food ingeedients that have been consistently established as pre= Diouc ingredients fiom several studies conducted in vitro and in vivo (imal and human) (Gibson and others 20042; Rastall 2006, Robertroid 2007; Trorteis and Valevie 2009) Te was shown in Table 4 and 5 that the differences in DP and seructures of the oligosaccharides present in GOS mixtures occur ‘mainly due to the different enzymes used for their production, ‘These differences are expected to be imporant when it comes to GOS assimilation by bifidobacteria inthe colonic microbiota (Tzoreis and Vulevie 2009). Ina recent study, the administration af 4 GOS mixture (3.6 g/dl) containing mainly B13, as well x B1->4 and 1->6 linkages, proved co have a better bifidogenic ‘effec than a GOS mixture (6.9 g/d) containing mainly 614, a5 swell B16, afer 1 wk of intake by healthy humans (Depeint and others 2008), Both mixtures had mainly di- and wisaechaties. Both these GOS mixture: had low polymerization degree with DP 24 accounting for les than 12% and 19% of tora sacchardes, respectively, ‘Most of the prophylactic health effects proposed for GOS ase from thei selective conssmption by bifidobacteria and lactobaci, 83 fermentative substrate. These reported health eects have been recently reviewed by other researchers (Gibson and others 2004; ‘Macfarlane and others 2008; Playne and Crittenden 2009; Trortis, and Vulevie 2009) and include protection against enteric infee= tions inctessed mineral absorption; immunomodulation for the prevention of allergies and gut inflammatory conditions; trophic ‘effects of SCFAs on the colonic epitheliim; fecal bulking: and duced toxigenic microbial metabolism that may reduce risk factors for colon cancet GOS in infant milk formulas, The sgnificmce of a Difdobacteria-predominant microbiota in healthy breastfeed in= fats i well accepted (Panato and others 2003), Ar chat time, the ‘microbiota organisms are believed to be particularly important for the correct functioning of the gut and maturation of the immune system. Buidobacteria and, toa leser degree lnczobacll, may ac- ‘count for ar mich af 90% of the total microbiota in breast-fed infants, while formula-fed infant have a more diverse microbiota composition with greater numbers of more potentially harmful ‘organisms sch a clostridia and enterococci (Harmen and others 2000). Oligosaccharides in human milk can reach concentrations as high 3: 8 to 12 g/L, which is 100 caves greater than in cow's ‘milk (Kunz and others 2000), mainly composed of slic acid, N- acetylghucosamine, L-facose, D-glucose, and D-galactose (Boch and others 2005), One characteristic of human mill oligorae- charides i the large amount of galactose. The backbone seruc~ ture is based on lactose (galactose-glacose) plus 2 farther galactose residue forming the 3 different GOS, namely, 3'-palactosyllactos, 4 galactosyL-actose, and 6'-galactosy-lactose Goch and others 12005). The preponderance of bifidobacteria in breast-fed babies is thought to reslt from their abilities to use oligosaccharides in beast milk, including GOS (Newburg 2000; Fanaro and others 2003). ‘The recognized bifidogenic effect of umman milk can be repli- ‘ated using GOS, alone of with FOS, to forufy infant cow's mlk- based formulas (Bakkes-Zierikzee and others 2008; Fanazo and. ‘thers 2005, 2008; Knol and others 20052, 2005b; Bock and. 448 comprehensive Reviews in Food Science and Food Safety + Vo, 9,20 ‘Moro 2008), Additionally the ability of GOS to resemble glyco- conjugate structures on cell sutfice receptors, uted by pathogens for adherence in the got, may also protect the colonization and growth of pathogens ducing this vulnerable period (Kunz and oth= 152000; Newburg 2000; Bochm and Moro 2008; Macfarlane and, others 2008) GOS Applications GOS are mainly used in infant milk: formula, follow-on formula, snd infant foods (Phyne and Crittenden 2009). Supplemented infin formulas usually contain 6:0 t0 7.2 g/L GOS together with 0.6 100.8 g/L FOS (Rastll 2006; Playne and Critenden 2009) Because oftheir stability in addition to infant foods, GOS can ako be incorporated into a wide variety of other foods. Re- cently, they have been wed in beverages (Suit juices and other acid drinks), meal replacers, fermented milks, favored milks, and confectionery products (Affersholt-Allen 2007). Bread, ab most ather baked goods, i a suitable candidate for GOS incorpors- tion because during the fermentation and baking process, GOS, molecules are not cleaved or consumed, Furthermore, due to the high moisture reuining capacity of GOS, excessive product dry- ing is prevented conferring this bread a beter taste and texture Specisived foods for the elderly and hospitalized people ae aio promising fields of application of GOS (Sako and others 1999) ‘As other nondigestble oligosaccharides, GOS have a pleasant ‘arte and can increase the texture and mouthfeel of foods provid- ing bulk properties simular to sucrose. GOS are resistant to salivary degradation and are not used by the oral microbiota and can there fore be wed as low-cariogenic sogar substitutes. Being indigestible they have negligent impact on blood glicose (Prapulla and others 2000), ‘Besides the food sector, other aes, such as the cosmetic and pharmaceutical industries, ean alo exploit the physicochemical and physiological properties of GOS, Indeed, prebsoic oligosac~ charides can selectively simulate “beneficial” bacteria on human, skin and some formulations for that purpose have already been developed (Bockmithl and others 2007; Keutmans 2009) The use of nondigesible oligosaccharides in the vestack feed and pet food industries i alo increasing. GOS are finding in- creased use inthe poultry (Biggs and others 2007; Jung and others 2008; Yang and others 2008), pig (Houdijk and others 1997, 1998; ‘Teortris and others 2008a; Modesto and others 2008), and aqua~ culture @Burr and others 2008; Grsdale-Helland and otters 2008) industeies for improving health and growth; improving gut mi~ crobal ecology, minimizing the use of antibiotics; prevent caly rmorslty; and reduce fecal odor. GOS have alo been applied to suppress methane production by ruminants (Mwwenya and others 20042, 20046; Santoso and others 2004; Sar and others 2004; Iqbal and others 2008) Regulatory and Safety Aspects of GOS GOS have 2 generally ecognized a safe (GRAS) satu in the ‘United States, 2 non-Novel Food satus inthe BU, and are egurded 28 foods for specific health wse (FOSHU) in Jan, (Torts and Valevie 2008) due to the fact chat they are components of human tnilk and teadivional yogurt and can be produced for ingested lhetose by the resident intestinal bacteria, The only adverse effect ‘of GOS known so fir is transient osmotic diaries that occurs ‘when an exces of GOS is consumed, similar to unabsorbed sugar alcohols oF lactose (in symptomatic lactoseantoleant individual) ‘The amount of GOS that docs not induce osmotic diarrhea has ° © 2010 isu of Food Technoloiss®Galacto-oligosaccharides ‘been estimated to be approximately 0.3 0 0.4 g/kg body weight, ‘or about 20 g per human body {Sako and others 1999). Actually, in Europe, health claims on foods, specifically “gen- ‘eral function” claims ate being regulated under Arle 13.1 in Regulation (BC) 1924/2006 of the European Parliament and of the Council of20 December 2006 on nutrition and health claims ‘made on foods (European Regulation 2004), The European Food, Safery Authority (EESA) is evaluating the fandamentsl science beyond the claims provided by all member ster, Although not yet authorized, in the meantime, 3 claims on GOS have already patted EFSA‘s pre-screening, namely. “maintains a healthy normal digestive system,” “prebiotic/bifdogenic,” and “calcium abeorp- tion” (EFSA 20102, 20106). However, the following claims on. GOS: “helps support healthy immune system is a8 ageing pop- ‘ulstion”; “helps to manage the symptoms asociated with ieitable Dowel syndrome"; and “energizes your immunity boosting bacte- 12" or “helps boost your body's seliedefense” did not passthrough the EFSA pre-screening, Conclusions Several microbial glycoside hydtolates have been proposed for the synthesis of GOS from lactote. In this contest, these enzymes fundamensally difer in their ability to catalyze the transgalacto- sybtion reacuon relative to hydrolysis, and in thes afinity for the GOS formed ar compared to the affinity for latose. The fc nal product spectrum obtained during lactose conversion and the glycoridic linkage between the monomers depends on the ex zyme soumce and the physicochemical conditions in the catalytic Current commercially available GOS products ate sgalactote-bated oligoraccharides with varying deguee of polymer- ization and linkage configuration with ghicose, galactose, and Ihetose. GOS mixtures are well-established prebiotc ingredients, However, depending on their oligaraccharide composition, GOS products vary in terms of their bifidogenic and other protective Recent and future developments in the production of GOS aim 2 delivering purer and more efficient mixtures, desirably with narrow and specific target rings. A better understanding of what Constitutes “healthy” intestinal microbiota composition certisly| ‘would contribute to that gol. GOS aze stable wide pH and temperatores ranges and are sit- ble for several applications in the food, feed, and pharmaceutical industries. Infne and geriatric nutrition offer the most promising ‘opporninities for GOS applications GOS are safe and well-tolerated ingredients up to intake levels (of 20 p/d; they have GRAS status an the United States, FOSHU status in Japan, and can be included inthe dietary fiber content of foods nintutes of| Acknowledgments ‘The authors grateflly acknowledge the financial support for this srudy by Project Biolife-PRIME 05/347 of Agencia a Inovacio—Progama IDBIA (Portugal). Duarte P. M. Torzes ac- knowledges Fundagio para a Ciéncia e a Tecnologia (Portugal) for the Ph.D. grant received (reference SERH/BDE/15510/2004). References ‘Ad AC, Rubio-Tecira M, Posi J 2004 Lactose the mal spar ous 2 ‘technologie pespective. Cut Rev Food Set Not 449-8) 3557 2010 stitute of Food Technologists? 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