Optimal ß-galactosidases for producing high-titer 3,6-anhydro-L-galactose from red-algal agarobiose.

3,6-Anhydro-L-galactose (L-AHG) is a monomeric sugar in agarose derived from red macroalgae. Owing to its various physiological activities such as anti-inflammation, moisturizing, skin whitening, anti-colon cancer, and anti-cariogenicity, L-AHG is a potential functional ingredient. In our previous study, a simple and efficient two-step L-AHG production process was designed for high-titer L-AHG production, where a single enzyme was used after the liquefaction of agarose by acid prehydrolysis. However, the enzyme used did not completely hydrolyze agarobiose (AB). Therefore, in this study, for the efficient hydrolysis of AB and the high-titer production of L-AHG, various ß-galactosidases belonging to glycoside hydrolase families 1, 2, 35, and 42 were compared by testing their substrate specificities and kinetic parameters. Among the five ß-galactosidases, Bga42A, originating from Bifidobacterium longum ssp. infantis ATCC 15,697, showed the highest substrate specificity. Consequently, the two-step process utilizing Bga42A as a single enzyme resulted in a high-titer production of L-AHG at 85.9 g/L, demonstrating the feasibility of producing L-AHG from agarose. KEY POINTS: • L-AHG derived from red macroalgae has various physiological activities. • Various ß-galactosidases were evaluated to efficiently hydrolyze agarobiose. • Bga42A showed the highest substrate specificity against agarobiose. • The highest amount of L-AHG with 85.9 g/L was simply produced.

Carrier-Free Immobilization of α-Galactosidase as Nano-Biocatalysts for Synthesizing Prebiotic α-Galacto-Oligosaccharides.

α-Galacto-oligosaccharides (α-GOSs) have great functions as prebiotics and therapeutics. This work established the method of batch synthesis of α-GOSs by immobilized α-galactosidase for the first time, laying a foundation for industrial applications in the future. The α-galactosidase from Aspergillus niger L63 was immobilized as cross-linked enzyme aggregates (CLEAs) nano-biocatalyst through enzyme precipitating and cross-linking steps without using carriers. Among the tested agents, the ammonium sulfate showed high precipitation efficacy and induced regular structures of α-galactosidase CLEAs (Aga-CLEAs) that had been analyzed by scanning electron microscopy and Fourier-transform infrared spectroscopy. Through optimization by response surface methodology, the ammonium sulfate-induced Aga-CLEAs achieved a high activity recovery of around 90% at 0.55 U/mL of enzymes and 36.43 mM glutaraldehyde with cross-linking for 1.71 h. Aga-CLEAs showed increased thermal stability and organic solvent tolerance. The storage ability was also improved since it maintained 74.5% activity after storing at 4 °C for three months, significantly higher than that of the free enzyme (21.6%). Moreover, Aga-CLEAs exhibited excellent reusability in the α-GOSs synthesis from galactose, retaining above 66% of enzyme activity after 10 batch reactions, with product yields all above 30%.

Exploring the edible gum (galactomannan) biosynthesis and its regulation during pod developmental stages in clusterbean using comparative transcriptomic approach.

Galactomannan is a polymer of high economic importance and is extracted from the seed endosperm of clusterbean (C. tetragonoloba). In the present study, we worked to reveal the stage-specific galactomannan biosynthesis and its regulation in clusterbean. Combined electron microscopy and biochemical analysis revealed high protein and gum content in RGC-936, while high oil bodies and low gum content in M-83. A comparative transcriptome study was performed between RGC-936 (high gum) and M-83 (low gum) varieties at three developmental stages viz. 25, 39, and 50 days after flowering (DAF). Total 209,525, 375,595 and 255,401 unigenes were found at 25, 39 and 50 DAF respectively. Differentially expressed genes (DEGs) analysis indicated a total of 5147 shared unigenes between the two genotypes. Overall expression levels of transcripts at 39DAF were higher than 50DAF and 25DAF. Besides, 691 (RGC-936) and 188 (M-83) candidate unigenes that encode for enzymes involved in the biosynthesis of galactomannan were identified and analyzed, and 15 key enzyme genes were experimentally validated by quantitative Real-Time PCR. Transcription factor (TF) WRKY was observed to be co-expressed with key genes of galactomannan biosynthesis at 39DAF. We conclude that WRKY might be a potential biotechnological target (subject to functional validation) for developing high gum content varieties.

Galactofuranose-Related Enzymes: Challenges and Hopes.

Galactofuranose is a rare form of the well-known galactose sugar, and its occurrence in numerous pathogenic micro-organisms makes the enzymes responsible for its biosynthesis interesting targets. Herein, we review the role of these carbohydrate-related proteins with a special emphasis on the galactofuranosidases we recently characterized as an efficient recombinant biocatalyst.

An unusual GH1 ß-glucosidase from marine sediment with ß-galactosidase and transglycosidation activities for superior galacto-oligosaccharide synthesis.

A novel ß-glucosidase, BglD1 with high ß-galactosidase and transglycosidation activities, was screened and cloned from the deep-sea bacterium Bacillus sp. D1. BglD1 exhibited the maximal ß-glucosidase and ß-galactosidase activities at 55-60 °C and pH 5.5-6.0. The enzyme maintained approximately 50% of its original activity at 35 °C and pH 6.0 after 120-h incubation. When applied to synthesize galacto-oligosaccharides (GOS), BglD1 generated 118.3 g/L GOS (33.8% (w/w)) from 350 g/L lactose, with trisaccharide Gal-ß(1 → 3)-Lac and disaccharide Gal-ß(1 → 4)-Gal as the main components. Furthermore, BglD1 could hydrolyze lactose in milk and produce GOS simultaneously. Using milk as the substrate, BglD1 hydrolyzed 88.5% lactose and produced 3.3 g/L GOS after incubation at 30 °C for 1 h. To improve the transglycosidation activity, a mutant BglD1:E224T was generated based on the semi-rational design. The GOS yield of BglD1:E224T was 11.5% higher than that of BglD1 when using lactose solution as the substrate. Thus, BglD1 and the mutant could be used as beneficial alternatives of the existing ß-galactosidases for the production of GOS.

Defects in Galactose Metabolism and Glycoconjugate Biosynthesis in a UDP-Glucose Pyrophosphorylase-Deficient Cell Line Are Reversed by Adding Galactose to the Growth Medium.

UDP-glucose (UDP-Glc) is synthesized by UGP2-encoded UDP-Glc pyrophosphorylase (UGP) and is required for glycoconjugate biosynthesis and galactose metabolism because it is a uridyl donor for galactose-1-P (Gal1P) uridyltransferase. Chinese hamster lung fibroblasts harboring a hypomrphic UGP(G116D) variant display reduced UDP-Glc levels and cannot grow if galactose is the sole carbon source. Here, these cells were cultivated with glucose in either the absence or presence of galactose in order to investigate glycoconjugate biosynthesis and galactose metabolism. The UGP-deficient cells display < 5% control levels of UDP-Glc/UDP-Gal and > 100-fold reduction of [6-3H]galactose incorporation into UDP-[6-3H]galactose, as well as multiple deficits in glycoconjugate biosynthesis. Cultivation of these cells in the presence of galactose leads to partial restoration of UDP-Glc levels, galactose metabolism and glycoconjugate biosynthesis. The Vmax for recombinant human UGP(G116D) with Glc1P is 2000-fold less than that of the wild-type protein, and UGP(G116D) displayed a mildly elevated Km for Glc1P, but no activity of the mutant enzyme towards Gal1P was detectable. To conclude, although the mechanism behind UDP-Glc/Gal production in the UGP-deficient cells remains to be determined, the capacity of this cell line to change its glycosylation status as a function of extracellular galactose makes it a useful, reversible model with which to study different aspects of galactose metabolism and glycoconjugate biosynthesis.

Production of galactooligosaccharides using various combinations of the commercial ß-galactosidases.

Galactooligosaccharides (GOS) are currently attracting considerable interest as prebiotic substances and can be prepared by transgalactosylation reactions from lactose using ß-galactosidase. We applied various combinations of the commercial ß-galactosidases, such as Nola Fit 5500, Saphera 2600 L, Maxilact LGI 5000 and Maxilact A4 MG to achieve the highest yield of GOS and reduced lactose content. The combination of the Maxilact LGI 5000 and Nola Fit 5500 resulted in amount of GOS 105 g L-1 with lactose content lower than 5 g L-1, whilst the combination of the Maxilact A4 MG and Maxilact LGI 5000 enzymes led to an increase in GOS to 141,1 g L-1 and decrease of the lactose content to 46,9 g L-1. The combination of enzymes produced a higher yield of GOS, reduced the concentration of lactose, eventually, increases the efficiency of galactooligosaccharides purification that could be potentially used in the further investigations.

Co-culture of Lactobacillus delbrueckii and engineered Lactococcus lactis enhances stoichiometric yield of D-lactic acid from whey permeate.

D-Lactic acid (D-LA) is an enantiomer of lactic acid, which has a niche application in synthesis of poly-lactic acid based (PLA) polymer owing to its contribution to the thermo-stability of stereo-complex PLA polymer. Utilization of renewable substrates such as whey permeate is pivotal to economically viable production of D-LA. In present work, we have demonstrated D-LA production from whey permeate by Lactobacillus delbrueckii and engineered Lactococcus lactis. We observed that lactose fermentation by a monoculture of L. delbrueckii yields D-LA and galactose as major products. The highest yield of D-LA obtained was 0.48 g g-1 when initial lactose concentration was 30 g L-1. Initial lactose concentration beyond 20 g L-1 resulted in accumulation of glucose and galactose, and hence, reduced the stoichiometric yield of D-LA. L. lactis naturally produces L-lactic acid (L-LA), so a mutant strain of L. lactis (L. lactis Δldh ΔldhB ΔldhX) was used to prevent L-LA production and engineer it for D-LA production. Heterologous over-expression of D-lactate dehydrogenase (ldhA) in the recombinant strain L. lactis TSG1 resulted in 0.67 g g-1 and 0.44 g g-1 of D-LA yield from lactose and galactose, respectively. Co-expression of galactose permease (galP) and α-phosphoglucomutase (pgmA) with ldhA in the recombinant strain L. lactis TSG3 achieved a D-LA yield of 0.92 g g-1 from galactose. A co-culture batch process of L. delbrueckii and L. lactis TSG3 achieved an enhanced stoichiometric yield of 0.90 g g-1 and ~45 g L-1D-LA from whey permeate (lactose). This is the highest reported yield of D-LA from lactose substrate, and the titres can be improved further by a suitably designed fed-batch co-culture process.

Genome-wide association analysis identifies a natural variation in basic helix-loop-helix transcription factor regulating ascorbate biosynthesis via D-mannose/L-galactose pathway in tomato.

Tomato (Solanum lycopersicum) is one of the highest-value vegetable crops worldwide. Understanding the genetic regulation of primary metabolite levels can inform efforts aimed toward improving the nutrition of commercial tomato cultivars, while maintaining key traits such as yield and stress tolerance. We identified 388 suggestive association loci (including 126 significant loci) for 92 metabolic traits including nutrition and flavor-related loci by genome-wide association study from 302 accessions in two different environments. Among them, an ascorbate quantitative trait locus TFA9 (TOMATO FRUIT ASCORBATEON CHROMOSOME 9) co-localized with SlbHLH59, which promotes high ascorbate accumulation by directly binding to the promoter of structural genes involved in the D-mannose/L-galactose pathway. The causal mutation of TFA9 is an 8-bp InDel, named InDel_8, located in the promoter region of SlbHLH59 and spanned a 5'UTR Py-rich stretch motif affecting its expression. Phylogenetic analysis revealed that differentially expressed SlbHLH59 alleles were selected during tomato domestication. Our results provide a dramatic illustration of how ascorbate biosynthesis can be regulated and was selected during the domestication of tomato. Furthermore, the findings provide novel genetic insights into natural variation of metabolites in tomato fruit, and will promote efficient utilization of metabolite traits in tomato improvement.

Evaluation of ß-galactosidase from Lactobacillus acidophilus as biocatalyst for galacto-oligosaccharides synthesis: Product structural characterization and enzyme immobilization.

ß-Galactosidase is an important industrial enzyme that catalyzes reaction of lactose hydrolysis and recently more interesting reaction of transgalactosylation, yielding a highly valuable group of prebiotic compounds named galacto-oligosaccharides (GOS). In this paper, parameters for achieving high yields of tailor-made GOS using crude ß-galactosidase obtained from Lactobacillus acidophilus ATCC 4356, probiotic bacteria regarded as safe for human consumption, were optimized. At the same time, detailed structural elucidation of obtained GOS was conducted, and it was concluded that ß-galactosidase from L. acidophilus shows a particular specificity towards the formation of ß-(1→6) glycosidic bonds. In order to develop more stable and economically cost-effective preparation, crude enzyme was successfully immobilized on a methacrylic polymer carrier Lifetech ECR8409, leading to its simultaneous 2-fold purification. This immobilized preparation showed unchanged specificity towards the transgalactosylation reaction, thus yielding 86 g/l GOS under the previously optimized conditions (lactose concentration 400 g/l in 0.1 M sodium phosphate buffer, pH 6.8 and temperature 50°C).

Heterologous Expression of a Thermostable ß-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides.

A thermostable ß-1,3-galactosidase from Marinomonas sp. BSi20414 was successfully heterologously expressed in Escherichia coli BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD600 of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme ß-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg-1 at 37 °C using ONPG (o-nitrophenyl-ß-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3'-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with ß-1,4 linkage and ß-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with ß-1,3 linkage.

Evolution of bidirectional costly mutualism from byproduct consumption.

Mutualisms are essential for life, yet it is unclear how they arise. A two-stage process has been proposed for the evolution of mutualisms that involve exchanges of two costly resources. First, costly provisioning by one species may be selected for if that species gains a benefit from costless byproducts generated by a second species, and cooperators get disproportionate access to byproducts. Selection could then drive the second species to provide costly resources in return. Previously, a synthetic consortium evolved the first stage of this scenario: Salmonella enterica evolved costly production of methionine in exchange for costless carbon byproducts generated by an auxotrophic Escherichia coli Growth on agar plates localized the benefits of cooperation around methionine-secreting S. enterica Here, we report that further evolution of these partners on plates led to hypercooperative E. coli that secrete the sugar galactose. Sugar secretion arose repeatedly across replicate communities and is costly to E. coli producers, but enhances the growth of S. enterica The tradeoff between individual costs and group benefits led to maintenance of both cooperative and efficient E. coli genotypes in this spatially structured environment. This study provides an experimental example of de novo, bidirectional costly mutualism evolving from byproduct consumption. The results validate the plausibility of costly cooperation emerging from initially costless exchange, a scenario widely used to explain the origin of the mutualistic species interactions that are central to life on Earth.

Coimmobilization of ß-Agarase and α-Neoagarobiose Hydrolase for Enhancing the Production of 3,6-Anhydro-l-galactose.

Here we report a simple and efficient method to produce 3,6-anhydro-l-galactose (l-AHG) and agarotriose (AO3) in one step by a multienzyme system with the coimmobilized ß-agarase AgWH50B and α-neoagarobiose hydrolase K134D. K134D was obtained by AgaWH117 mutagenesis and showed improved thermal stability when immobilized via covalent bonds on functionalized magnetic nanoparticles. The obtained multienzyme biocatalyst was characterized by Fourier transform infrared spectroscopy (FTIR). Compared with free agarases, the coimmobilized agarases exhibited a relatively higher agarose-to-l-AHG conversion efficiency. The yield of l-AHG obtained with the coimmobilized agarases was 40.6%, which was 6.5% higher than that obtained with free agarases. After eight cycles, the multienzyme biocatalyst still preserved 46.4% of the initial activity. To the best of our knowledge, this is the first report where two different agarases were coimmobilized. These results demonstrated the feasibility of the new method to fabricate a new multienzyme system onto magnetic nanoparticles via covalent bonds to produce l-AHG.

Synthesis of galacto-oligosaccharides derived from lactulose by wild-type and mutant ß-galactosidase enzymes from Bacillus circulans ATCC 31382.

Oligosaccharides derived from lactulose (ß-D-Galp-(1 → 4)-D-Fru) are drawing more and more attention nowadays because of their strong resistance to gut digestion, and the interest to discover novel prebiotics. Compared to galactooligosaccharides, currently known structures of lactulose oligosaccharides are very limited. In this study, the wild-type ß-galactosidase BgaD-D of Bacillus circulans ATCC 31382, as well as the derived mutant R484H, were used to synthesize oligosaccharides from lactulose. In total, 9 oligosaccharide structures were identified by MALDI-TOF-MS and NMR spectroscopy analysis. Trisaccharide ß-D-Galp-(1 → 4)-ß-D-Galp-(1 → 4)-D-Fru was the major structure produced by the wild-type enzyme, while the R484H mutant showed a preference for synthesis of ß-D-Galp-(1 → 3)-ß-D-Galp-(1 → 4)-D-Fru. Our study greatly enriched the structural information about oligosaccharides derived from lactulose.

The allelochemical trans-cinnamic acid stimulates salicylic acid production and galactose pathway in maize leaves: A potential mechanism of stress tolerance.

In this study, the effects (5 days) of the secondary metabolite trans-cinnamic acid on maize leaves (Zea mays L.), through a physiological and an untargeted metabolomic approach, were evaluated. A reduction in leaf growth and development accompanied by a decrease in protein content was observed in treated seedlings. Besides, trans-cinnamic acid stimulated the photosynthetic machinery with a significant increment in pigment content (chlorophyll a, b and carotenoids), a stimulation of the light adapted PSII efficiency (ɸII) as well as the chlorophyll a fluorescence (YNO), the apparent electron transport rate, and the regulated dissipation of the energy (YNPQ). By contrast, the dark adapted PSII parameter (Fv/Fm) was not affected suggesting that no physical damages to the antenna complex were caused by trans-cinnamic acid. These results suggested that maize seedlings were experiencing a stress but, at the same time, were able to cope with it. This hypothesis was confirmed by both the increment in benzoic and salicylic acids, important molecules involved in stress response, and the metabolomic results, which pointed out that the seedlings are directing their metabolism towards galactose production modulating its pathway, which is pivotal for the production of the antioxidant compound ascorbic acid (ASA). Indeed, in treated plants, a significant increment in total ASA content (28%) was observed. The results suggested that the main strategy adopted by plants to cope with trans-cinnamic-induced stress consisted in the modulation of their metabolism in order to increase the total ASA and carotenoids concentration, radical scavenging species.

Purification and characterization of ß-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis.

ß-galactosidase is a commercially important enzyme that was purified from probiotic Pediococcus acidilactici. The enzyme was extracted from cells using sonication and subsequently purified using ammonium sulphate fractionation and successive chromatographies on Sephadex G-100 and Q-Sepharose. The enzyme was purified 3.06-fold up to electrophoretic homogeneity with specific activity of 0.883 U/mg and yield of 28.26%. Molecular mass of ß-galactosidase as estimated by SDS-PAGE and MALDI-TOF was 39.07 kDa. The enzyme is a heterodimer with subunit mass of 15.55 and 19.58 kDa. The purified enzyme was optimally active at pH 6.0 and stable in a pH range of 5.8-7.0 with more than 97% activity. Purified ß-galactosidase was optimally active at 50 °C. Kinetic parameters Km and Vmax for purified enzyme were 400 µM and 1.22 × 10-1 U respectively. Its inactivation by PMSF confirmed the presence of serine at the active site. The metal ions had different effects on enzyme. Ca2+, Mg2+ and Mn2+ slightly activated the enzyme whereas NH4+, Co2+ and Fe3+ slightly decreased the enzyme activity. Thermodynamic parameters were calculated that suggested that ß-galactosidase is less stable at higher temperature (60 °C). Purified enzyme effectively hydrolysed milk lactose with lactose hydrolysing rate of 0.047 min-1 and t1/2 of 14.74 min. This is better than other studied ß-galactosidases. Both sonicated Pediococcus acidilactici cells and purified ß-galactosidase synthesized galactooligosaccharides (GOSs) as studied by TLC at 30% and 50% of lactose concentration at 47.5 °C. These findings indicate the use of ß-galactosidase from probiotic bacteria for producing delactosed milk for lactose intolerant population and prebiotic synthesis. pH and temperature optima and its activation by Ca2+ shows that it is suitable for milk processing.

Optimization of enzymatic production of prebiotic galacto/galacto(arabino)-oligosaccharides and oligomers from potato rhamnogalacturonan I.

A bi-enzymatic system using two multi-enzymatic preparations (Depol 670L and Gamanase 1.5L) was investigated for the production of prebiotic galacto/galacto(arabino)-oligosaccharides and oligomers with well-defined degree of polymerisation (DP) from potato galactan-rich rhamnogalacturonan I. Depending on the reaction condition, yields of low (DP of 2-6) and high-MW oligosaccharides (DP of 7-12) and oligomers (DP of 13-70) varied between 0.1-13.9, 0.0-37.5 and 0.0-75.7%, respectively. Substrate concentration and Depol 670L/Gamanase 1.5L ratio were identified as the most significant linear terms in oligosaccharide and oligomer yield models, respectively. Moreover, interaction between reaction time and substrate concentration had a significant effect on the yield of oligosaccharides, while interaction between reaction time and Depol 670L/Gamanase 1.5L ratio affected significantly the yield of oligomers. Higher yields of both oligosaccharides and oligomers were obtained when equal amount of Depol 670L and Gamanase 1.5L was used in combination. The DP and the monosaccharide composition of the generated galacto/galacto(arabino)-oligosaccharides and oligomers were confirmed.

Improvement of covalent immobilization procedure of ß-galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study.

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by ß-galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane-polyvinyl alcohol (POS-PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL-1 initial lactose concentration and 6 U mL-1 enzyme concentration, obtaining 25.46 ± 0.01 gL-1 yield of trisaccharides. Although below the HPLC-IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL-1 of trisaccharides and 13.79 ± 0.21 gL-1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady-state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568-1578, 2017.

The potential of species-specific tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group for galactose reduction in fermented dairy foods.

Residual lactose and galactose in fermented dairy foods leads to several industrial and health concerns. There is very little information pertaining to manufacture of fermented dairy foods that are low in lactose and galactose. In the present study, comparative genomic survey demonstrated the constant presence of chromosome-encoded tagatose-6-phosphate (T6P) pathway in Lactobacillus casei group. Lactose/galactose utilization tests and ß-galactosidase assay suggest that PTSGal system, PTSLac system and T6P pathway are major contributors for lactose/galactose catabolism in this group of organisms. In addition, it was found than lactose catabolism by Lb. casei group accumulated very limited galactose in the MRS-lactose medium and in reconstituted skim milk, whereas Streptococcus thermophilus and Lb. delbrueckii subsp. bulgaricus (Lb. bulgaricus) strains secreted high amount of galactose extracellularly. Moreover, co-culturing Lb. casei group with Str. thermophilus showed significant reduction in galactose content, while co-culturing Lb. casei group with Lb. bulgaricus showed significant reduction in lactose content but significant increase in galactose content in milk. Overall, the present study highlighted the potential of Lb. casei group for reducing galactose accumulation in fermented milks due to its species-specific T6P pathway.