towards galactose accumulation in dairy foods fermented by conventional starter cultures: challenges...

Trends in Food Science & Technology xx (2014) 1e13

Review

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Please cite this article in press as: Wu, Q., et al., Towards galactose accu

strategies, Trends in Food Science & Technology (2014), http://dx.doi

Towards

galactose

accumulation in

dairy foods

fermented by

conventional starter

cultures: Challenges

and strategies

Qinglong Wu, Christine K.W.
Cheung and Nagendra P. Shah*
Food and Nutritional Science, School of Biological

Sciences, The University of Hong Kong, Pokfulam

Road, Hong Kong (6N-08, Kadoorie Biological

Sciences Building, The University of Hong Kong,

Pokfulam Road, Hong Kong. Tel.: D(852) 2299 0836;

fax: D(852) 2559 9114; e-mail: [email protected])

Galactose normally accumulates in dairy foods after milk

fermentation with conventional starter cultures and gives rise

to serious effects on functionalities of fermented dairy foods.

Tagatose-6P pathway (lac operon) and Leloir pathway ( gal

operon) are responsible for lactose/galactose metabolism in

lactic acid bacteria (LAB). Leloir pathway is a common

pathway in LAB whereas Tagatose-6P pathway is mainly found

in Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus

lactis subsp. cremoris, Enterococcus faecium, and Entero-

coccus faecalis. This review provides new insights into using

specific above LAB as starter culture for galactose reduction

in fermented dairy foods.

mulation

.org/10.1

IntroductionConventional starter cultures, especially Streptococcusthermophilus, Lactobacillus delbrueckii subsp. bulgaricus,and Lactococcus lactis including Lc. lactis subsp. lactisand Lc. lactis subsp. cremoris, are classified into lacticacid bacteria (LAB) that have been extensively used formilk fermentation due to their generally regarded as safe(GRAS) status, capability to metabolize milk lactose, ex-hibiting rapid acidification of milk, and giving new func-tionalities to fermented dairy foods (Savijoki, Ingmer, &Varmanen, 2006). These conventional starters have beenconsidered as essential for the manufacture of yogurt,cheese and other fermented milk products, and are gov-erned by regulations in regards to their usage as conven-tional starters for manufacturing fermented dairy foods(Wessels et al., 2004). In general, S. thermophilus and Lb.delbrueckii subsp. bulgaricus are used for the manufactureof yogurt whereas Lc. lactis is mainly used in cheese-making. In some products, they may be combined formilk fermentation (Leroy & De Vuyst, 2004). Other speciesof LAB such as Lb. casei, Lb. helveticus, Lb. acidophilus,Lb. plantarum, Lb. rhamnosus, and Bifidobacterium sp.have been used as probiotic starters, however, these speciesare usually used as adjunct starters with conventional start-ers for the development of fermented dairy foods withnovel functions (Hati, Mandal, & Prajapati, 2013; Leroy& De Vuyst, 2004; Shah, 2007).

LAB are able to metabolize milk lactose and its moietygalactose, however, a large amount of galactose normallygets accumulated after milk fermentation (Johnson &Olson, 1985; Michel & Martley, 2001; Turner & Martley,1983). This is mainly due to the strain-specific capabilityof metabolizing lactose/galactose via Tagatose-6P pathwayor Leloir pathway (Neves et al., 2010; Thompson, 1987).However, high galactose level leads to several serious prob-lems in regards to functionalities of fermented dairy foodssuch as cheese (Daly, McSweeney, & Sheehan, 2010;Dattatreya, Lee, & Rankin, 2010; Johnson & Olson,1985; Matzdorf, Cuppett, Keeler, & Hutkins, 1994;Rudan & Barbano, 1998; Zoon & Allersma, 1996). Thus,any efforts to reduce galactose would be of great impor-tance for improving functionalities of fermented dairyfoods.

In this review, side effects of residual galactose in fer-mented dairy foods, especially in cheese, are summarized,and general pathways for metabolism of lactose and

in dairy foods fermented by conventional starter cultures: Challenges and

016/j.tifs.2014.08.010

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http://dx.doi.org/10.1016/j.tifs.2014.08.010



2 Q. Wu et al. / Trends in Food Science & Technology xx (2014) 1e13

galactose are highlighted. This review also surveys theirdistribution in completely sequenced and released LAB,and discusses solutions for galactose reduction in dairyfoods.

Effects of galactose accumulation on textural defectsof cheese

An optimum production of gas (mainly CO2) duringcheese ripening is responsible for the formation of eyes,which is an important quality parameter for most semi-hard cheeses such as Swiss-type and Tilsiter-type cheeses(Guggisberg et al., 2013; Martley & Crow, 1996; Schuetzet al., 2013). However, eye formation is not expected forfresh cheese such as Mozzarella cheese, and hard cheesesuch as Cheddar cheese. Production of carbon dioxide(CO2) during cheese ripening may come from: 1) decarbox-ylation of glutamic acid into g-aminobutyric acid (GABA)by dairy starters such as GABA-producing LAB (Martley& Crow, 1996; Nomura, Kimoto, Someya, Furukawa, &Suzuki, 1998); 2) heterofermentation of carbohydrates(lactose, galactose, glucose and citrate) by heterofermenta-tive LAB (El Attar, Monnet, & Corrieu, 2000); 3) over pro-duction of CO2 via metabolizing lactic acid byPropionibacterium used for manufacturing Swiss-typecheese during ripening process.

Currently, most of commercial cheeses are manufacturedby adding S. thermophilus or Lc. lactis or mixed starters de-pending on the types of cheese (Giraffa & Rossetti, 2004;Leroy & De Vuyst, 2004). These starters are responsiblefor rapid acidification of milk due to the efficient productionof acids (mainly lactic acid) and their proteolytic activity inorder to shorten the duration of fermentation duringmanufacturing processes (Savijoki et al., 2006). However,short-term fermentation leads to accumulation of galactosein cheese due to limitation of galactose utilization by conven-tional starters, and its content remains high even after drain-ing of whey and ripening process (Fox, Lucey, & Cogan,1990; Johnson & Olson, 1985; Michel & Martley, 2001;Turner & Martley, 1983). This in turn contributes to largeamount of CO2 production during ripening leading to thedevelopment of undesirable slits and fractures in cheese(Daly et al., 2010; Lucey, Johnson, & Horne, 2003;Martley & Crow, 1996; Zoon & Allersma, 1996). Hence,galactose reduction helps improving the texture of cheese.

Effects of galactose accumulation in Mozzarellacheese on browning of pizza

Pizza is widely consumed around the world and is madewith Mozzarella cheese (pizza cheese) as an essentialcomponent (Jana & Mandal, 2011). Dairy starters areable to possess extracellular proteolytic activity resultingin considerable concentration of free amino acids and smallpeptides in products after fermentation (Savijoki et al.,2006). The formation of browning in pizza cheese is mainlydue to the non-enzymatic reactions such as caramelizationand Maillard reaction during baking process (Dattatreya

Please cite this article in press as: Wu, Q., et al., Towards galactose accumulation

strategies, Trends in Food Science & Technology (2014), http://dx.doi.org/10.1

et al., 2010; Johnson & Olson, 1985; Matzdorf et al.,1994; Rudan & Barbano, 1998). Hence, high content ofgalactose in cheese has an adverse effect related to the for-mation of dark brown color, which gives an undesirableappearance for pizza.

Mozzarella cheese is a fresh type of semi-soft cheese andis generally white (Jana &Mandal, 2011). Low galactose (orno galactose) in Mozzarella cheese is desirable for pizza-making as per consumers’ preference for cheese on pizzato have low or moderate brown color (Matzdorf et al.,1994; Rudan & Barbano, 1998). Development of modernlow-galactose Mozzarella cheese involves three importantsteps: firstly, the usage of galactose-utilizing dairy startersfor galactose reduction during milk fermentation (Baskaran& Sivakumar, 2003; Hutkins, Halambeck, & Morris, 1986;Ma, James, Balaban, Zhang, & Emanuelsson-Patterson,2013; Mukherjee & Hutkins, 1994); secondly, the drainingof whey after coagulation of milk proteins by addition ofrennet and production of acids from dairy starters, whichcould remove much of galactose dissolved in the whey(Jana & Mandal, 2011); thirdly, ripening, which is a post-proteolysis stage for Mozzarella cheese resulting in anincreased content of peptides and amino acids (Feeney,Fox, & Guinee, 2001). Meanwhile, there is limited meta-bolism of residual galactose by live LAB cells includingdairy starters in Mozzarella cheese during storage(Coppola, Blaiotta, Ercolini, & Moschetti, 2001; Ercolini,Mauriello, Blaiotta, Moschetti, & Coppola, 2004).

Effects of galactose on individuals with galactosemiaGalactosemia, a genetically determined disease, is rare

condition with an incidence of approximately1:40,000e60,000 among general population (Leslie,2003). Some individuals have near total absence of activ-ities of galactokinase (GalK) or galactose 1-phosphate uri-dyltransferase (GalT) or UDP-galactose-4-epimerase(GalE), an essential enzymes for galactose metabolism inthe Leloir pathway, leading to accumulation of galactoseor galactose 1-phosphate in body tissues (Leslie, 2003;Reichardt & Woo, 1991). This leads to an acute deteriora-tion in liver function, mental retardation and septicemia, ifthe condition of patients is not well recognized and treated(Leslie, 2003).

Lactose is the main carbohydrate in mammalian milks,which have been largely consumed around the world.Lactose is hydrolyzed into glucose and galactose upon up-take by human with the action of ß-D-galactosidase. Hence,individuals suffering from galactosemia are not recommen-ded to have any non-fermented or fermented dairy prod-ucts, and should strictly avoid any galactose-containingfoods, especially general fermented dairy foods containinghigh galactose (Portnoi & MacDonald, 2009; Varga,Palvolgyi, Juhasz-Roman, & Toth-Markus, 2006). Howev-er, milks or their manufactured products are considered tobe the only foodstuff that provides approximately all nutri-ents for humans, especially infants. Moreover, evidence


016/j.tifs.2014.08.010

3Q. Wu et al. / Trends in Food Science & Technology xx (2014) 1e13

shows that milk products could maintain good bone anddental health, and prevent major cardiovascular diseasessuch as hypertension, type-2 diabetes and metabolic syn-dromes (Davoodi, Esmaeili, & Mortazavian, 2013). Hence,some lactose- or galactose-free milk products, such as ValioZero Lactose� and specific dairy foods fermented bygalactose-utilizing dairy starter cultures (Hutkins et al.,1986; Varga et al., 2006) have been manufactured for indi-viduals with galactosemia. These kinds of dairy foodswould benefit the health of the patients havinggalactosemia.

Metabolism of lactose and galactose in LABDue to problems associated with galactose accumulation

in fermented dairy foods, galactose metabolism has beenextensively documented for some important dairy starterssuch as S. thermophilus and Lc. lactis. General pathwaysfor lactose/galactose metabolism in LAB are shown inFig. 1.

Firstly, uptake of lactose and galactose is an importantfactor influencing the capacity of galactose utilization inLAB. Transportation of sugars relies on specific permeaseor phosphoenolpyruvate-dependent phosphotransferase sys-tem (PEP-PTS) in cell membrane (Devos & Vaughan,

Fig. 1. General pathways of lactose/galactose metabolism in lactic acid baMeyerhof-Parnas (EMP) pathway (dash dot), and Leloir pathway (long dash dfor lactose and galactose metabolisms. Leloir pathway for galactose metaboliimportant for providing substrate for glycolysis (also known as EMP pathwaytosidase; 2, Galactose 6-phosphate isomerase; 3, Tagatose 6-phosphate kinasglucose isomerase; 7, Phosphoglucomutase; 8, b-Galactosidase; 9, Galactok

pyrophosphorylase; 12, UDP-galactose 4 epimerase



1994; Thompson, 1987). So far, two widely recognizedpathways including Tagatose-6P pathway (lactose/galac-tose uptake via PTS) and Leloir pathway (galactose uptakevia galactose permease) contribute to the metabolism oflactose and galactose by LAB depending on strains(Devos & Vaughan, 1994; Neves et al., 2010; Thompson,1987). However, some conflicting results regarding galac-tose uptake via a galactose-specific PTS (PTSGal) orlactose-specific PTS (PTSLac) in LAB have been reported(Crow, Davey, Pearce, & Thomas, 1983; Devos &Vaughan, 1994; Park & Mckay, 1982; Thompson, 1980),but a gene encoding PTSGal was identified in very few spe-cific LAB strains such as Streptococcus lactis, Lb. casei andLc. lactis (Bettenbrock, Siebers, Ehrenreich, & Alpert,1999; Chassy & Thompson, 1983; Neves et al., 2010;Park & Mckay, 1982). Although it has been demonstratedthat permease and PTS have different affinities to lactoseor galactose, both pathways play different role in metabo-lizing lactose and galactose (Neves et al., 2010). Sincemany enzymes are involved in both pathways and theirenzymatic activities are strain-dependent, it is generallyhard to define which pathway metabolizes more lactose/galactose than the other pathway in LAB (Degeest & DeVuyst, 2000; Van Laere, Abee, Schols, Beldman, &

cteria. Pathways include Tagatose-6P pathway (square dot), Embden-ot dot). Enzymes 1, 8 and 9 have been regarded as priming indicatorssm is involved in the EPS biosynthesis, whereas tagatose-6P pathway is). The numbers refer to the enzymes involved: 1, 6-phospho-b-galac-e; 4, Tagatose 1,6-di-phospahate aldolase; 5, Glucokinase; 6, Phospho-inase; 10, Galactose 1-phosphate-uridylyltransferase; 11, UDP-glucose; 13, galactose mutarotase; 14, phosphatase.


016/j.tifs.2014.08.010


Voragen, 2000; de Vin, Radstrom, Herman, & De Vuyst,2005). Apart from the type of lactose/galactose uptake,both Tagatose-6P pathway and Leloir pathway are impor-tant for lactose/galactose metabolism in LAB.

Sugar metabolism and exopolysaccharidesbiosynthesis in LAB

Exopolysaccharide (EPS) produced by LAB is verypromising for dairy industry because of its texture-modifying property and beneficial effects (Ruas-Madiedo,Hugenholtz, & Zoon, 2002; Ruas-Madiedo, Salazar, & delos Reyes-Gavil�an, 2009). Exopolysaccharides (EPSs)have been generally classified into homo-EPS and hetero-EPS based on their chemical composition. Homo-EPSssuch as glucans and fructans contain only one type ofmonosaccharides which are mainly either glucose or fruc-tose, while hetero-EPSs consist of repeating units of at leasttwo types of monosaccharides or other molecules whichmay include glucose, galactose, rhamnose and fucose(Boels, van Kranenburg, Hugenholtz, Kleerebezem, &de Vos, 2001). Depending on the position of polysaccha-rides in LAB, EPS could be cell wall-associated polysac-charides termed as capsular polysaccharides (CPS), ormay be directly secreted into the medium as ropy EPS(Ruas-Madiedo & de los Reyes-Gavilan, 2005). Moreover,the producers of homo-EPS could be Lb. reuteri, Lb. fer-mentum and Lb. sanfranciscensis etc., whereas mesophilicand thermophilic LAB normally produce hetero-EPS(Badel, Bernardi, & Michaud, 2011). Thus certain typesof EPS from LAB may have different biofunctionalitiesin regards to their chemical composition, molecular sizeand structure (Ruas-Madiedo et al., 2009; Welman &Maddox, 2003).

Sugars such as galactose, glucose, fructose, sucrose, andlactose could be substrates for the biosynthesis of EPS(Boels et al., 2001). From Fig. 1, two pathways includingLeloir pathway and Embden-Meyerhof-Parnas (EMP)pathway are able to provide priming substrates such asfructose-6P, glucose-6P and galactose-1P for synthesis ofnucleotide sugars. It appears that Tagatose-6P pathwaydoes not contribute to the production of EPS from LAB,while EMP pathway and Leloir pathway are able to providenucleotide sugars such as UDP-glucose and UDP-galactosefor assembly of homo- or hetero-EPS. For the EPS produc-tion in milk, EPS production is closely associated withlactose and galactose metabolism in mesophilic and ther-mophilic LAB, which are normally conventional startersincluding Lc. lactis, S. thermophilus and Lb. delbrueckiissp. bulgaricus. Thus galactose could be more consumedfor EPS production if the enzymatic kinetics of Leloirpathway were improved via metabolic engineering as sug-gested by Boels et al. (2001).

Leloir pathwayA gal operon for Leloir pathway in LAB is usually

located in chromosome (Table 1). Firstly, b-D-galactose,



which comes from extracellular medium after hydrolysisof lactose by a cytoplasmic b-D-galactosidase in LAB, istransported by a highly galactose-specific permease orlactose-galactose antiporter (also known as lactose/galac-tose permease; bifunctional), and then is converted intoa-D-galactose by galactose mutarotase for following enzy-matic reactions in Leloir pathway (Fig. 1). Interestingly,it was found that nucleotide sugars including UDP-glucose and UDP-galactose as basic substrates for the as-sembly of repeating units of hetero-EPS are extensivelysynthesized in Leloir pathway (Boels et al., 2001;De Vuyst, De Vin, Vaningelgem, & Degeest, 2001;Degeest & De Vuyst, 2000; Welman & Maddox, 2003).Although partial galactose is converted into glucose-6P asstarting substrate for the entry into glycolysis reactions(known as EMP pathway), less galactose is metabolizedthrough glycolysis due to the activities of enzymes suchas phosphoglucomutase and phosphoglucose isomerase(Levander & Radstrom, 2001; Levander, Svensson, &Radstrom, 2002). To best of our knowledge, UDP-glucoseand UDP-galactose are the main substrates for synthesizingmost types of hetero-EPS in dairy starters during milkfermentation. And the production of above two nucleotidesugars from milk lactose is possibly via Leloir pathwayin LAB. Thus, Leloir pathway contributes most to EPS pro-duction from LAB during milk fermentation.

Tagatose-6P pathwayIn LAB, a lac operon for Tagatose-6P pathway may be

plasmid-encoded or of chromosomal origin. Normally,Tagatose-6P pathway in Lc. lactis is plasmid-encoded,whereas in S. thermophilus and Lb. delbrueckii subsp. bul-garicus it is located in their chromosomes (Table 1).Lactose uptake is achieved via PTSLac and then lactose-6P is hydrolyzed into galactose-6P and glucose throughthe action of phospho-b-galactosidase. Then entry intoTagatose-6P pathway is achieved by galactose-6P isom-erase-catalyzed isomerization of galactose-6P intotagatose-6P (Fig. 1). For lactose metabolism viaTagatose-6P pathway, less enzymatic reactions areinvolved; however, there are complex reactions for galac-tose metabolism via Leloir pathway, and normally, theenzymatic kinetics are limited by the activity of enzymes.Thus less glucose-1P will be provided for glycolysis via Le-loir pathway. As mentioned above, Leloir pathway contrib-utes most to EPS production. Hence, it appears thatTagatose-6P pathway may provide more substrate, e.g.glyceraldehyde 3-P, for glycolysis (EMP pathway; seeFig. 1) than Leloir pathway.

Distribution of Tagatose-6P pathway and Leloirpathway in LAB

To date, thousands of LAB strains including the speciesfrom Lactobacillus sp., Lactococcus sp., Bifidobacteriumsp. Leuconostoc sp. and S. thermophilus have been fullysequenced and their genomic data has also been released


016/j.tifs.2014.08.010

Table 1. Distribution of Tagatose-6P and Leloir pathway in complete sequenced and released lactic acid bacteria.

Strain Tagatose-6Ppathway

Leloirpathway


Leloirpathway

Lc. lactis subsp. lactis IL1403 e þ(B) Lb. johnsonii N6.2 e þ(B)Lc. lactis subsp. lactis KF147 e þ(B) Lb. acidophilus NCFM e þ(B)Lc. lactis subsp. lactis CV56 e þ(B) Lb. acidophilus 30SC e þ(B)Lc. lactis subsp. lactis IO-1 e þ(B) Lb. acidophilus La-14 e þ(B)Lc. lactis subsp. lactis KLDS 4.0325 e þ(B) Lb. sakei subsp. sakei 23K e þ(B)Lc. lactis subsp. cremoris SK11 þ(A) þ(B) Lb. salivarius UCC118 e þ(B)Lc. lactis subsp. cremoris MG1363 e þ(B) Lb. salivarius CECT 5713 e þ(B)Lc. lactis subsp. cremoris A76 þ(A) þ(B) Lb. delbrueckii subsp.

bulgaricus ATCC 11842e incomplete (B)

Lc. lactis subsp. cremoris NZ9000 e þ(B) Lb. delbrueckii subsp.bulgaricus ATCC BAA-365

e incomplete (B)

Lc. lactis subsp. cremoris UC509.9 þ(A) þ(B) Lb. delbrueckii subsp.bulgaricus ND02

þ(B) þ(B)

Lc. lactis subsp. cremoris KW2 e þ(B) Lb. delbrueckii subsp.bulgaricus 2038

e incomplete (B)

Lc. garvieae ATCC 49156 e þ(B) Lb. brevis ATCC 367 e þ(B)Lc. garvieae Lg2 e þ(B) Lb. brevis KB290 e þ(B)S. thermophilus CNRZ1066 e þ(B) Lb. casei ATCC 334 þ(B) þ(B)S. thermophilus LMG18311 e þ(B) Lb. casei BL23 þ(B) þ(B)S. thermophilus LMD-9 e þ(B) Lb. casei Zhang þ(B) þ(B)S. thermophilus ND03 e þ(B) Lb. casei BD-II þ(B) þ(B)S. thermophilus JIM 8232 e þ(B) Lb. casei LC2W þ(B) þ(B)S. thermophilus MN-ZLW-002 e þ(B) Lb. casei W56 þ(B) þ(B)Lb. plantarum WCFS1 e þ(B) Lb. casei LOCK919 þ(B) þ(B)Lb. plantarum JDM1 e þ(B) Lb. paracasei N1115 þ(B) þ(B)Lb. plantarum ZJ316 e þ(B) Lb. paracasei subsp.

paracasei 8700:2þ(B) þ(B)

Lb. plantarum 16 e þ(B) Lb. gasseri ATCC 33323 þ(B) þ(B)Lb. plantarum subsp.plantarum ST-III

e þ(B) Lb. reuteri DSM 20016 e þ(B)

Lb. plantarum subsp.plantarum P-8

e þ(B) Lb. reuteri JCM 1112 e þ(B)

Lb. johnsonii NCC 533 e þ(B) Lb. reuteri SD2112 e þ(B)Lb. johnsonii FI9785 þ(B) þ(B) Lb. reuteri I5007 e þ(B)Lb. johnsonii DPC 6026 e þ(B) Lb. reuteri TD1 e þ(B)Lb. helveticus H9 e þ(B) B. longum NCC2705 e þ(B)Lb. helveticus H10 e þ(B) B. longum DJO10A e þ(B)Lb. helveticus R0052 e þ(B) B. longum subsp.

infantis ATCC 15697e þ(B)

Lb. helveticus CNRZ32 e þ(B) B. longum subsp.infantis 157F

e þ(B)

Lb. helveticus DPC 4571 e þ(B) B. longum subsp.longum JDM301

e þ(B)

Lb. fermentum IFO 3956 e þ(B) B. longum subsp.longum BBMN68

e þ(B)

Lb. fermentum CECT 5716 e þ(B) B. longum subsp.longum JCM 1217

e þ(B)

Lb. fermentum F-6 e þ(B) B. longum subsp.longum KACC 91563

e þ(B)

Lb. rhamnosus GG þ(B) þ(B) B. longum subsp.longum F8

e þ(B)

Lb. rhamnosus Lc 705 þ(B) þ(B) B. adolescentis ATCC 15703 e þ(B)Lb. rhamnosus ATCC 8530 þ(B) þ(B) B. animalis subsp.

lactis AD011e þ(B)

Lb. rhamnosus LOCK900 þ(B) þ(B) B. animalis subsp.lactis Bl-04

e þ(B)

Lb. rhamnosus LOCK908 þ(B) þ(B) B. animalis subsp.lactis DSM 10140

e þ(B)

Lb. crispatus ST1 e þ(B) B. animalis subsp.lactis BB-12

e þ(B)

(continued on next page)


Please cite this article in press as: Wu, Q., et al., Towards galactose accumulation in dairy foods fermented by conventional starter cultures: Challenges and

strategies, Trends in Food Science & Technology (2014), http://dx.doi.org/10.1016/j.tifs.2014.08.010

Table 1 (continued )


Leloirpathway


Leloirpathway

Lb. amylovorus GRL 1112 e þ(B) B. animalis subsp.lactis BLC1

e þ(B)

Lb. amylovorus GRL1118 e þ(B) B. animalis subsp.lactis CNCM I-2494

e þ(B)

Lb. buchneri NRRL B-30929 e þ(B) B. animalis subsp.lactis V9

e þ(B)

Lb. buchneri CD034 e þ(B) B. animalis subsp.lactis B420

e þ(B)

Lb. kefiranofaciens ZW3 e þ(B) B. animalis subsp.lactis Bi-07

e þ(B)

Lb. ruminis ATCC 27782 e þ(B) B. animalis subsp.lactis Bl12

e þ(B)

Lb. sanfranciscensis TMW1.1304 e incomplete (B) B. animalis subsp.lactis ATCC 27673

e þ(B)

Leuconostoc mesenteroides subsp.mesenteroides ATCC 8293

e þ(B) B. animalis subsp.animalis ATCC 25527

e þ(B)

Leuconostoc mesenteroides subsp.mesenteroides J18

e þ(B) B. dentium Bd1 e þ(B)

Leuconostoc mesenteroides KFRI-MG e þ(B) B. bifidum S17 e þ(B)Leuconostoc citreum KM20 e þ(B) B. bifidum PRL2010 e þ(B)Leuconostoc kimchi IMSNU 11154 e þ(B) B. bifidum BGN4 e þ(B)Leuconostoc gasicomitatumLMG 18811

e þ(B) B. breve ACS-071-V-Sch8b e þ(B)

Leuconostoc sp. C2 e þ(B) B. breve UCC2003 e þ(B)Leuconostoc carnosum JB16 e þ(B) Pediococcus pentosaceus

ATCC 25745e þ(B)

Leuconostoc gelidum JB7 e þ(B) Pediococcus clausseniiATCC BAA-344

e þ(B)

Pediococcus pentosaceus SL4 e þ(B) Enterococcus faecalis V583 incomplete (B) þ(B)Oenococcus oeni PSU-1 e þ(B) Enterococcus faecalis 62 þ(B) þ(B)Weissella koreensis KACC 15510 e þ(B) Enterococcus faecalis OG1RF incomplete (B) þ(B)Enterococcus faecium Aus0004 incomplete (B) þ(B) Enterococcus faecalis D32 þ(B) þ(B)Enterococcus faecium Aus0085 þ(A) þ(B) Enterococcus faecalis

Symbioflor 1þ(B) þ(B)

Enterococcus faecium DO incomplete (B) þ(B) Enterococcus faecalis DENG1 þ(B) þ(B)Enterococcus faecium NRRL B-2354 þ(A þ B) þ(B) Enterococcus casseliflavus EC20 incomplete (B) þ(B)

Note: e, pathway is absent in the organism; þ, pathway is available in the organism; A, pathway is plasmid-encoded; B, pathway is located inchromosome. Only the genome of lactic acid bacterium that has been fully sequenced and released in NCBI database was included in this table.


in public databases such as GenBank maintained by Na-tional Center for Biotechnology Information (NCBI) andEuropean Nucleotide Archive (ENA) from European Bioin-formatics Institute at European Molecular Biology Labora-tory. Kyoto Encyclopedia of Genes and Genomes (KEGG)is a database resource for understanding high-level func-tions in biological systems based on molecular datasetsgenerated by genome sequencing and other high-throughput sequencing technologies (Kanehisa & Goto,2000). KEGG pathway (http://www.genome.jp/kegg/pathway.html) is able to predicate and map all of the meta-bolic pathways based on genomic information in a microor-ganism. Hence, we compiled information regarding thedistribution of Tagatose-6P pathway and Leloir pathwayin sequenced LAB strains by searching galactose meta-bolism in KEGG pathway. The result of pathway distribu-tion is shown in Table 1.



Remarkably, most LAB strains possess Leloir pathwayfor metabolizing galactose, whereas the distribution ofTagatose-6P pathway in LAB is species-dependent. It hasbeen very well documented that UDP-glucose and UDP-galactose is the main substrate for EPS biosynthesis asglucose and galactose are normally found in EPS composi-tion (Jolly, Vincent, Duboc, & Nesser, 2002; Laws, Gu, &Marshall, 2001; Welman & Maddox, 2003). Since mainnucleotide sugars such as UDP-glucose and UDP-galactose are synthesized in Leloir pathway, wide distribu-tion of Leloir pathway in LAB suggests that most LABstrains are able to produce EPS, but their capabilities ofproducing EPS is highly strain-dependent (Boels et al.,2001; Welman & Maddox, 2003).

Additionally, Tagatose-6P pathway is found consistentlyin five species of LAB including Lc. lactis subsp. cremoris,Lb. casei, Lb. rhamnosus, Enterococcus faecium, and


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http://www.genome.jp/kegg/pathway.html

http://www.genome.jp/kegg/pathway.html


Enterococcus faecalis (Table 1). This suggests that thesefive species may exhibit better capability of utilizinglactose and galactose than other LAB species. In Lc. lactissubsp. cremoris, Tagatose-6P pathway is plasmid-encodedwhereas the gal operon for Leloir pathway occurs in chro-mosome (Table 1). The reason why both Lc. lactis subsp.cremoris MG1363 and NZ9000 do not possess Tagatose-6P pathway is possible due to the absence of plasmids inthem. For Lb. casei and Lb. rhamnosus, the lac operonfor Tagatose-6P pathway occurs in their chromosomes(Table 1). Interestingly, Lc. lactis subsp. cremoris, togetherwith S. thermophilus, is commonly used for the manufac-ture of cheeses while Lb. casei and Lb. rhamnosus aregenerally used as probiotics for manufacture of probioticsupplement and functional fermented dairy foods (Leroy& De Vuyst, 2004). Also, Bifidobacterium sp., which iswidely recognized as probiotic bacteria in human gut(Fanning et al., 2012; Martinez, Balciunas, Converti,Cotter, & Oliveira, 2013; Picard et al., 2005), does nothave the ability to metabolize lactose via Tagatose-6Ppathway but possesses Leloir pathway for galactose meta-bolism after intracellular hydrolysis of lactose. This isalso confirmed by previous studies of genomic analysis ofBifidobacterium sp. (Parche et al., 2007; Pokusaeva,Fitzgerald, & van Sinderen, 2011). For Ent. faecium andEnt. faecalis, both are the most prevalent species in thegut of humans, and have been generally classified asLAB. It was found that some strains of both species haveincomplete Tagatose-6P pathway in their genome (seeTable 1). Although both species show the potential toreduce the content of galactose in milk, further efforts areneeded to confirm their GRAS status before commerciali-zation, because they have been regarded as opportunisticpathogens as well.

Regarding the definition of galactose-positive and -nega-tive LAB in previous reports based on their examination ofgalactose content in the medium after lactic acid fermenta-tion (Hutkins et al., 1986; Mukherjee & Hutkins, 1994;Thomas & Crow, 1984; de Vin et al., 2005), attentionsare paid to define the capability of utilizing galactose inLAB at genomic level. From Table 1, it is clear that mostsequenced LAB strains excluding the strains with incom-plete Leloir pathway such as the listed strains, Lb. del-brueckii subsp. bulgaricus and Lb. sanfranciscensis, weregenetically identified as galactose-positive strains becauseof the presence of Leloir pathway in their genome. Howev-er, it is hard to define how much of galactose they could uti-lize. As stated above, Leloir pathway is responsible forproviding nucleotide sugars for EPS biosynthesis (Boelset al., 2001; Welman & Maddox, 2003). It appears that theirability of utilizing galactose is closely associated with theirEPS production (Degeest & De Vuyst, 2000).

Strategies for enhancing galactose utilization in LABDue to serious problems associated with high galactose

content in fermented dairy foods, endeavors have been



made to investigate how to enhance galactose utilizationby LAB strains, such as metabolic engineering of theirpathways or selection of specific starters that could metab-olize lactose and galactose completely.

Metabolic engineering of LABRemarkably, EPS produced by LAB has multi-functions

such as immune modulation (Fanning et al., 2012; Liuet al., 2011; Makino et al., 2006; Wu et al., 2010), anti-oxidant activity (Liu et al., 2011; Pan & Mei, 2010;Sengul, Isik, Aslim, Ucar, & Demirbag, 2011; Xu, Shang,& Li, 2011; Xu, Shen, Ding, Gao, & Li, 2011), anti-microbial activities (Fanning et al., 2012; Wu et al.,2010), and has important applications for improving func-tionalities of yogurt and cheese (Amatayakul, Halmos,Sherkat, & Shah, 2006; Amatayakul, Sherkat, & Shah,2006; Bhaskaracharya & Shah, 2000; De Vuyst et al.,2003; Purwandari, Shah, & Vasiljevic, 2007;Ramchandran & Shah, 2009; Zisu & Shah, 2005, 2007).Hence, most of current genetic modifications for enhancedgalactose utilization by LAB are closely associated withEPS production. As stated above, Leloir pathway for galac-tose metabolism is mainly for the production of nucleotidesugars for EPS biosynthesis in LAB. Thus, this pathway inconventional dairy starter cultures, especially S. thermophi-lus and Lc. lactis subsp. cremoris, has been widely modifiedto improve the EPS production which in turn enhancesgalactose utilization by mutants of dairy starters(Bouazzaoui & LaPointe, 2006; Levander et al., 2002;Neves et al., 2010). In general, Lb. delbrueckii subsp. bul-garicus does not possess both Leloir pathway andTagatose-6P pathway (Table 1), whereas it could simplyhydrolyze lactose into glucose and galactose; this is diffi-cult to perform genetic manipulations and fewer studieshave been conducted for this species.

Specific LAB as adjunct dairy starters for galactosereduction

In general, Leloir pathway normally occurs in thegenome of LAB while Tagatose-6P pathway is species-specific in Lc. lactis subsp. cremoris, Lb. casei and Lb.rhamnosus (Table 1). We found that these three speciescould possibly utilize more lactose and galactose than otherspecies during milk fermentation. Although some specificLAB strains possess high capacity of utilizing galactosevia Leloir pathway (Thomas & Crow, 1984; Vaillancourtet al., 2004; de Vin et al., 2005), it is difficult to obtainthose high galactose-fermenting LAB strains via screening,and is not a common solution to address issues related togalactose accumulation in dairy industry. For instance, thepotential solution for the undesirable defects of cheesecaused by carbon dioxide from heterofermentation ofgalactose is to reduce its content. Here, we propose a spe-cific approach of using above corresponding species asadjunct starters to reduce galactose content in fermenteddairy foods. For example, Lb. rhamnosus was added during


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the manufacture of Cheddar cheese which was made usingdairy starter S. thermophilus and utilized all the galactose(24 mmol per 1 kg of cheese before ripening) during the3-month ripening (Michel & Martley, 2001).

Generally, S. thermophilus and Lb. delbrueckii subsp.bulgaricus are used for Mozzarella cheese making. Howev-er, since both organisms do not have Tagtose-6P pathway(Table 1), they normally metabolize the glucose moietyof lactose while galactose is largely accumulated in milk af-ter excretion. Apart from the removal of lactose and galac-tose via draining of whey during cheese-making, there arevery limited studies in regards to the removal of accumu-lated galactose. Moreover, the duration for fermentation un-til the draining of whey is about 3e4 h for Mozzarellacheese, and the ripening or storage is about 28 days at4 �C. Thus the industry faces a challenge to reduce thegalactose content, and it appears that modification ofcheese-making process especially the fermentation timemay be necessary. The potential strategy is to use dairyLb. casei and Lb. rhamnosus as adjunct starters at adequateconcentration for fermentation.

Lc. lactis subsp. cremorisThis species is a sub-species of Lc. lactis, which along

with Lc. lactis subsp. lactis, has been widely used for themanufacture of various types of cheeses. In Table 1, thisspecies is a potential candidate that possesses bothTagatose-6P and Leloir pathways. However, Tagatose-6Ppathway is not fully distributed and was found to beplasmid-associated in this species. This suggests that Lc.lactis subsp. cremoris may obtain Tagatose-6P pathwayvia lateral gene transfer (LGT) during co-culturing withother LAB or bacteria (Bolotin et al., 2001). Thus, strainswith Tagatose-6P pathway as dairy starters are very prom-ising for removal of galactose during cheese-making.

Lb. caseiThis species is normally found in milk environment and

is involved in the manufacture of dairy foods such askoumiss (Wang, Chen, Liu, Yang, & Zhang, 2008). It isnot a conventional dairy starter culture, but is usually re-garded as probiotic starter for functional dairy foods.Currently, a widely studied strain, Lb. casei Zhang, isolatedfrom koumiss was used as probiotic. Numerous functionalstudies were carried out for this strain and it showed anti-oxidant effects and immunoregulatory activities for thehost (Ya et al., 2008; Y. Zhang, Du, Wang, & Zhang,2010; W. Y. Zhang et al., 2010). Remarkably, Tagatose-6P pathway consistently occurs in the chromosome of allcompletely sequenced L. casei (Table 1). This impliesthat this species may be a good starter for manufacturingprobiotic yogurt and reducing galactose in milk.

Lb. rhamnosusThis species was originally considered to be a sub-

species of L. casei by physiological and biochemical



characterizations, and was renamed as L. rhamnosus aftergenetic identification (Collins, Phillips, & Zanoni, 1989;Ward & Timmins, 1999). L. rhamnosus GG (LGG) is themost studied strain and is a widely-recognized probiotic or-ganism and has been marketed in the form of probiotic sup-plement and also used in dairy foods (De Keersmaeckeret al., 2006; Gruber et al., 2007; Kankainen et al., 2009;Kumpu et al., 2012; Lebeer, Claes, Verhoeven,Vanderleyden, & De Keersmaecker, 2011; Nase et al.,2001; Pessi, Sutas, Hurme, & Isolauri, 2000). Interestingly,all sequenced strains of this species possessed Tagatose-6Ppathway as indicated in their chromosomes (Table 1). Also,previous studies reported that the addition of L. rhamnosusor when combined with conventional starters promotedreduction of galactose during manufacture of cheeses(Michel & Martley, 2001; Oliveira, Perego, de Oliveira,& Converti, 2012).

Ent. faecium & Ent. faecalisSome certain strains of Ent. faecium and Ent. faecalis

possess probiotic traits which may confer human health,and these non-clinical and GRAS-grade Enterococcusstrains haven been involved in the production of fermentedfoods (Bourdichon et al., 2012; Franz, Huch, Abriouel,Holzapfel, & Galvez, 2011; Franz, Stiles, Schleifer, &Holzapfel, 2003). However, both species are normallyrecognized as opportunistic pathogens, and are the maincause for nosocomial infections and transfer of virulencedeterminants and antibiotic resistance genes (Rathnayake,Hargreaves, & Huygens, 2012; Valenzuela et al., 2013; X.L. Zhang et al., 2012). For instance, Ent. faecalis accountedfor 90e95% of clinical enterococcal isolates, followed byEnt. faecium which is more frequently resistant to vanco-mycin and ampicillin than Ent. faecalis (Arias & Murray,2012).

Nowadays, there has been an increasing interest in usingGRAS-grade bacteria without antibiotic resistance genesfor producing fermented foods. This is mainly due to theattention paid to the spread of antibiotic resistance genesvia the food chain. Thus additional safety evaluation forEnt. faecium and Ent. faecalis is necessary to confirm theirGRAS status before they are used as starters for milkfermentation.

Proteolytic system in Lc. lactis subsp. cremoris, Lb.casei and Lb. rhamnosus

Due to the conflicting status of Ent. faecium and Ent.faecalis as food starters, using Lc. lactis subsp. cremoris,Lb. casei and Lb. rhamnosus is highly recommended formilk fermentation because of their natural GRAS status.

Extracellular proteinases (Prt) including cell envelope-associated proteinase, subtilisin-like serine protease andtrypsin-like serine protease from both starter and non-starter LAB are very important for cells to adapt to milkenvironment (Goh, Goin, O’Flaherty, Altermann, &Hutkins, 2011; Savijoki et al., 2006). These proteases could


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Table 2. Overview of potential extracellular proteinases from sequenced Lactococcus lactis subsp. cremoris, Lactobacillus casei and Lactoba-cillus rhamnosus.

Strain Protein name Aminosequence length

Locus tag Location

Lactococcus lactis subsp. cremorisStrain SK11 Lactocepin I (PrtP) 1962 LACR_C42 plasmid

Subtilisin-like serine protease 1017 LACR_1726 chromosomeTrypsin-like serine protease 407 LACR_2439 chromosome

Strain MG1363 e e e eStrain A76 HtrA-like 407 llh_12405 chromosomeStrain NZ9000 e e e e

Strain UC509.9 Lactocepin I (PrtP) 1974 uc509_p6025 plasmidTrypsin-like serine protease (htrA) 407 uc509_2116 chromosome

Strain KW2 Serine protease HtrA 407 kw2_2196 chromosomeLactobacillus caseiStrain ATCC 334 Subtilisin-like serine protease 1902 LSEI_2270 chromosome

Trypsin-like serine protease 367 LSEI_0933 chromosomeSubtilisin-like serine protease 1637 LSEI_0468 chromosomeSubtilisin-like serine protease 762 LSEI_2660 chromosome

Strain BL23 PII-type proteinase (lactocepin) 1902 LCABL_24520 chromosomeCell envelope-associated proteinase 1809 LCABL_05330 chromosome

Strain Zhang Cell wall-associated proteinase PrtP 1902 LCAZH_2241 chromosomeSubtilisin-like serine protease 1333 LCAZH_0497 chromosomeSubtilisin-like serine protease 1808 LCAZH_0498 chromosome

strain BD-II Cell wall-associated proteinase PrtP 1902 LCBD_2450 chromosomeCell-envelope associated proteinase 1809 LCBD_0531 chromosome

Strain LC2W Cell wall-associated proteinase PrtP 1902 LC2W_2433 chromosomeCell-envelope associated proteinase 1809 LC2W_0532 chromosome

strain W56 Cell-envelope associated proteinase 1836 BN194_05400 chromosomePII-type proteinase (PrtP) 1903 BN194_24060 chromosome

Strain LOCK919 Cell-envelope associated proteinase 1808 LOCK919_0558 chromosomeSerine protease, DegP/HtrA 442 LOCK919_3065 chromosomeSubtilisin-like serine protease 762 LOCK919_2910 chromosome

Lactobacillus rhamnosusStrain GG Cell envelope-associated proteinase 1494 LGG_02734 chromosome

Trypsin-like serine protease 365 LGG_00903 chromosomeStrain Lc 705 Cell envelope-associated proteinase 1494 LC705_02738 chromosome

Trypsin-like serine protease 365 LC705_00953 chromosomeSubtilisin-like serine protease 756 LC705_02680 chromosome

Strain ATCC 8530 PII-type proteinase (PrtP) 1973 LRHK_2273 chromosomeSerine protease do-like htrA 444 LRHK_2915 chromosome

Strain LOCK900 Subtilisin-like serine protease 744 LOCK900_2679 chromosomeSerine protease, DegP/HtrA 444 LOCK900_2795 chromosome

Strain LOCK908 Subtilisin-like serine protease 756 LOCK908_2760 chromosomeSerine protease, DegP/HtrA 444 LOCK908_2888 chromosome


hydrolyze milk proteins into small peptides and free aminoacids, and are important for proteolysis during ripeningstage in cheese (Broadbent et al., 2002; Lane & Fox,1996; Wilkinson, Guinee, Ocallaghan, & Fox, 1994). More-over, co-culture of non-starter LAB along with dairy startercultures would be an efficient way to improve the cellviability of non-starter LAB in milk products particularlycheeses and gives new functionalities to milk products(Martley & Crow, 1993; Phillips, Kailasapathy, & Tran,2006; Settanni & Moschetti, 2010).

Extracellular proteinases in Lc. lactis subsp. cremoris,Lb. casei and Lb. rhamnosus are shown in Table 2. Lc. lac-tis subsp. cremoris strain MG1363 and NZ9000 do not havegenes encoding extracellular proteinases in their genome.In Lb. casei, genes encoding cell envelope-associated



proteinase normally occurred in their genome. This sug-gests that Lb. casei is a good candidate as adjunct dairystarter. In Lb. rhamnosus, all sequenced strains could exertextracellular proteolytic activity. In addition to their capa-bility of metabolizing lactose and galactose, this reviewindicated that most of strains from above three speciescould ferment milk and survive in milk-based environment,which provides clear evidence that they could be regardedas specific adjunct starters for dairy fermentation.

Conclusions and future researchGalactose accumulation in fermented dairy foods when

using conventional starters including S. thermophilus, Lb.delbrueckii subsp. bulgaricus, and Lc. lactis normally oc-curs and could cause serious problems in certain dairy


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foods and to some consumers of fermented dairy foods.Hence, continuous attentions were paid to galactose meta-bolism in these dairy starters, and studies such as metabolicengineering of galactose metabolism in conventional start-ers have been carried out to improve galactose reduction incheese and yogurt. Unfortunately, genetically modifieddairy starters are not easily approved for manufacturing fer-mented milk products. Although some high galactose-utilizing starters have been isolated, we propose a simpleand economical solution of using specific LAB includingLb. casei, Lb. rhamnosus and Lc. lactis subsp. cremoris asadjunct starters for manufacturing fermented dairy foodscontaining low galactose. In addition to their GRAS andnon-GM status, these three species possess both Tagatose-6P pathway and Leloir pathway for metabolizing lactose/galactose. Moreover, most strains of above three speciesexhibit extracellular proteolytic activity for adapting tothe milk environment. However, further studies regardingtheir capability of utilizing galactose during milk fermenta-tion is necessary, and safety evaluation of Ent. faecium andEnt. faecalis as dairy starter is also recommended.

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