Evolution of milk oligosaccharides and lactose: a hypothesis

Mammalian milk or colostrum contains up to 10% of carbohydrate, of which free lactose usually constitutes more than 80%. Lactose is synthesized within lactating mammary glands from uridine diphosphate galactose (UDP-Gal) and glucose by a transgalactosylation catalysed by a complex of β4-galactosyltransferase and α-lactalbumin (α-LA). α-LA is believed to have evolved from C-type lysozyme. Mammalian milk or colostrum usually contains a variety of oligosaccharides in addition to free lactose. Each oligosaccharide has a lactose unit at its reducing end; this unit acts as a precursor that is essential for its biosynthesis. It is generally believed that milk oligosaccharides act as prebiotics and also as receptor analogues that act as anti-infection factors. We propose the following hypothesis. The proto-lacteal secretions of the primitive mammary glands of the common ancestor of mammals contained fat and protein including lysozyme, but no lactose or oligosaccharides because of the absence of α-LA. When α-LA first appeared as a result of its evolution from lysozyme, its content within the lactating mammary glands was low and lactose was therefore synthesized at a slow rate. Because of the presence of glycosyltransferases, almost all of the nascent lactose was utilized for the biosynthesis of oligosaccharides. The predominant saccharides in the proto-lacteal secretions or primitive milk produced by this common ancestor were therefore oligosaccharides rather than free lactose. Subsequent to this initial period, the oligosaccharides began to serve as anti-infection factors. They were then recruited as a significant energy source for the neonate, which was achieved by an increase in the synthesis of α-LA. This produced a concomitant increase in the concentration of lactose in the milk, and lactose therefore became an important energy source for most eutherians, whereas oligosaccharides continued to serve mainly as anti-microbial agents. Lactose, in addition, began to act as an osmoregulatory molecule, controlling the milk volume. Studies on the chemical structures of the milk oligosaccharides of a variety of mammalian species suggest that human milk or colostrum is unique in that oligosaccharides containing lacto-N-biose I (LNB) (Gal(β1 → 3)GlcNAc, type I) predominate over those containing N-acetyllactosamine (Gal(β1 → 4)GlcNAc, type II), whereas in other species only type II oligosaccharides are found or else they predominate over type I oligosaccharides. It can be hypothesized that this feature may have a selective advantage in that it may promote the growth of beneficial colonic bacteria, Bifidobacteria, in the human infant colon.     

Evolution of milk oligosaccharides: Origin and selectivity of the ratio of milk oligosaccharides to lactose among mammals

BackgroundThe carbohydrate fraction of mammalian milk is constituted of lactose and oligosaccharides, most of which contain a lactose unit at their reducing ends. Although lactose is the predominant saccharide in the milk of most eutherians, oligosaccharides significantly predominate over lactose in the milk of monotremes and marsupials.Scope of reviewThis review describes the most likely process by which lactose and milk oligosaccharides were acquired during the evolution of mammals and the mechanisms by which these saccharides are digested and absorbed by the suckling neonates.Major conclusionsDuring the evolution of mammals, c-type lysozyme evolved to α-lactalbumin. This permitted the biosynthesis of lactose by modulating the substrate specificity of β4galactosyltransferase 1, thus enabling the concomitant biosynthesis of milk oligosaccharides through the activities of several glycosyltransferases using lactose as an acceptor. In most eutherian mammals the digestion of lactose to glucose and galactose is achieved through the action of intestinal lactase (β-galactosidase), which is located within the small intestinal brush border. This enzyme, however, is absent in neonatal monotremes and macropod marsupials. It has therefore been proposed that in these species the absorption of milk oligosaccharides is achieved by pinocytosis or endocytosis, after which digestion occurs through the actions of several lysosomal acid glycosidases. This process would enable the milk oligosaccharides of monotremes and marsupials to be utilized as a significant energy source for the suckling neonates.General significanceThe evolution and significance of milk oligosaccharides is discussed in relation to the evolution of mammals.     

Formation of oligosaccharides during enzymatic lactose: Part I: State of art

Enzymatic lactose hydrolysis by beta-galactosidase (lactase) was investigated with respect to the formation of oligosaccharides. An analysis of the formation of oligosaccharides and their control is important in the development of technical applications for enzymatic lactose hydrolysis. The available literature data on transfer reactions of lactase were reviewed, compared, and presented in a concise tabular form. Mechanisms and possible ways of modelling enzymatic lactose hydrolysis, including formation of oligosaccharides, are presented.     

Properties of immobilized β-d-galactosidase from Bacillus circulans

Partially purified β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans showed high activity towards both pure lactose and lactose in skim milk, and a better thermal stability than the enzyme from yeast or Escherichia coli. During the course of hydrolysis of lactose catalysed by the enzyme, considerable amounts of oligosaccharides were produced. β-d-Galactosidase from B. circulans was immobilized onto Duolite ES-762, Dowex MWA-1 and sintered alumina by adsorption with glutaraldehyde treatment. The highest activity for hydrolysis of lactose was obtained with immobilization onto Duolite ES-762. During a continuous hydrolysis of lactose, the immobilized enzyme was reversibly inactivated, probably due to oligosaccharides accumulating in the gel. The inactivation was reduced when a continuous reaction was operated at a high percent conversion of lactose in a continuous stirred tank reactor (CSTR). The half-life of the immobilized enzyme was estimated to be 50 and 15 days at 50 and 55°C, respectively, when the reaction was carried out in a CSTR with a percent conversion of lactose >70%.     

Changes in alpha-lactalbumin, total lactose, UDP-galactose hydrolase and other factors in tammar wallaby (Macropus eugenii) milk during lactation

alpha-Lactalbumin was isolated from milk of M. eugenii and its concentration in milk samples taken at various times during lactation (0-40 weeks post partum) was determined by single radial immunodiffusion using rabbit antiserum to the purified protein. The alpha-lactalbumin concentration remained almost constant throughout lactation even though the concentration of total lactose (free lactose plus lactose contained in oligosaccharides) fell to zero after 34 weeks post partum. This fall in lactose was accompanied by a rise in the free galactose and glucose concentrations and marked increases in UDP-galactose hydrolase, nucleotide pyrophosphatase, alkaline phosphatase and acid beta-galactosidase activities. It is suggested that the in vitro hydrolysis of UDP-galactose was due to nucleotide pyrophosphatase and that this enzyme may also play a role in vivo late in lactation by making UDP-galactose unavailable for the synthesis of lactose. Alternatively, lactose and lactose-containing oligosaccharides might be degraded by the acid beta-galactosidase during or after secretion.     

Separation from protein kinase C--a calcium-independent TPA-activated phosphorylating system

Peptide:N-glycosidase F removed both the asparagine-linked oligosaccharide chains of ricin B-chain in the absence of lactose. In the presence of lactose, which binds specifically to the B-chain, only one oligosaccharide chain was removed. Lactose also protected Ricinus communis agglutinin B-chain against the removal of one of the two susceptible oligosaccharides present in each B-chain subunit.     

生物制造高附加值乳糖衍生物的研究进展

乳糖来自于乳清,是奶酪行业的低附加值副产物.由于亚洲人普遍存在乳糖不耐受性,因此乳糖缺乏有效的经济增值途径.随着我国乳制品行业的不断发展,利用乳糖制备高附加值产品具有重要的经济价值和产业升级意义.本文就近年来发展的利用生物技术转化乳糖为稀少糖或益生寡糖的方法进行了综述,以期对该领域研究提供借鉴.     

A novel approach to the recovery of biologically active oligosaccharides from milk using a combination of enzymatic treatment and nanofiltration

A new easily scalable approach to the recovery of biologically active oligosaccharides from milk has been developed which relies on the combination of enzymatic treatment of defatted milk using beta-galactosidase and nanofiltration. It was shown that enzymatic hydrolysis of lactose significantly improves the efficiency and selectivity of membrane-based separations. With the best membrane, as much as 6.7 g of oligosaccharides (containing very little contaminating lactose) could be obtained from one liter of defatted human milk in just four nanofiltration cycles. The human milk oligosaccharides recovered by this method were shown to inhibit binding of intimin, an adhesion molecule of enteropathogenic Escherichia coli, to epithelial cells in vitro. No significant difference in the oligosaccharide profile between samples prepared by this method and conventional gel-permeation chromatography was found. The developed approach is also suitable for the recovery of substantial quantities of tri- and tetra-saccharides from caprine milk.     

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

Formation of Oligosaccharides during Enzymic Hydrolysis of Milk Whey Permeates

Lactose present in whey UF-permeates was hydrolysed by an immobilised enzyme reactor and the formation of monosaccharides (glucose + galactose) and oligosaccharides was monitored. The enzyme used was β-galactosidase from A. oryzaeimmobilised in a porous film. The reactor, run in the flow-through mode, allowed large conversions at short residence times (60% conversion in 1 min). The conversion to oligosaccharides as a function of the reaction time (or degree of conversion) reaches a maximum and then declined as oligosaccharides were converted back to mono- and disaccharides. The higher the initial lactose concentration the higher the conversion to oligosaccharides and these maxima appear at higher degrees of conversion. Some trials were carried out on the concentration of the oligosaccharides present in the hydrolysates by means of membrane filtration (nanofiltration) .     

Incorporation of orally applied (13)C-galactose into milk lactose and oligosaccharides

Biosynthesis and functions of human milk oligosaccharides (HMO) are not well known. A typical housekeeping enzyme, beta1,4-galactosyltransferase, links galactose to glucose to form lactose which is then used as backbone for the assembly of HMO. We investigated whether milk lactose and HMO may be labeled in vivo by an orally given (13)C-galactose bolus. Eleven exclusively breastfeeding mothers were given a (13)C-galactose bolus at the end of their breakfast. Milk and urine samples from each nursing up to 36 h were analyzed for carbohydrate composition by high-performance thin-layer chromatography, high-pH anion-exchange chromatography, and fast atom bombardment mass spectrometry. (13)C enrichment of milk fractions, urinary carbohydrates, lactose, and oligosaccharides as well as of breath CO(2) was determined by isotope ratio mass spectrometry. Up to 10% of the orally given galactose bolus was directly transported to the mammary gland and incorporated into milk components. Characteristic for most milk samples was the appearance of two (13)C-peaks, the first immediately after the (13)C-bolus was taken and the second on the next morning. The highest (13)C enrichment was found in lactose followed by neutral and acidic oligosaccharides. In breath samples, the (13)C-excretion followed the same pattern as in milk. (13)C nuclear magnetic resonance of isolated lactose revealed (13)C only at C(1)-atom of galactose and C(1)-atom of glucose. This label was without any exception at the same position as the (13)C-label of the orally applied galactose. Neutral and acidic HMO can easily be (13)C-labeled in vivo which facilitates investigations of their metabolic fate in infants.     

Release of carbohydrates from sphingoglycolipid by osmium-catalyzed periodate oxidation followed by treatment with mild alkali

The carbohydrate moiety of sphingoglycolipid, after preliminary acetylation, can be released by periodate oxidation catalyzed by a trace amount of osmium tetroxide, followed by alkaline treatment. Cerebroside, lactosyl ceramide, hematoside, globoside, and gangliosides were degraded to yield, respectively, galactose, lactose, sialyl lactose, a tetrasaccharide, and various oligosaccharides containing sialic acid. Oligosaccharides were separated by paper chromatography and paper electrophoresis. The procedure is useful for characterizing micromolar amounts of sphingoglycolipids.     

Isolation and physico-chemical properties of oligosaccharides of human milk]

From a non-dialysable fraction of human milk the authors have isolated 30 oligosaccharides by combining anion exchange and paper chromatography. These oligosaccharides (MW 998 to 3113) contain D (+)-galactose, L (-)-fucose, N-acetyl-D (+)-glucosamine, N-acetyl-D (-)-neuraminic acid in variable proportions and one single D (+)-glucose residue in terminal reducing position. The characteristics of the oligosaccharides are those of the so-called "Polonovski and Lespagnol gynolactose". These sugars do not originate from glycoproteins nor glycolipids. The authors suggest that the biosynthesis of human milk oligosaccharides is due to an activation of glycosyltransferases related to blood group substances, induced by lactose. They base this hypothesis on the fact that, as demonstrated by Strecker and Montreuil, spontaneous or glucose, galactose and lactose induced meliturias are accompagnied by an important urinary excretion of blood group substance related oligosaccharides.     

Characterization of oligosaccharides in milk of a mink, Mustela vison

Carbohydrates were extracted from a sample of milk from a mink, Mustela vison (Family Mustelidae). Free neutral and acidic oligosaccharides were isolated from the carbohydrate fraction and their chemical structures were compared with those of white-nosed coati (Nasua narica, Procyonidae) and harbour seal (Phoca vitulina, Phocidae) that we had studied previously. The ratio of free lactose to milk oligosaccharides was similar to that in milk of the white-nosed coati; in both species, this ratio was much lower than that in the milk of most eutherians. The neutral oligosaccharides of mink milk had alpha(1-3)-linked Gal or alpha(1-2)-linked Fuc residues at their non-reducing ends, as in the neutral oligosaccharides of white-nosed coati milk. Some of the neutral and acidic oligosaccharides, determined here, had been found also in harbour seal milk, but the harbour seal oligosaccharides did not contain alpha(1-3)-linked Gal residues.     

Biochemical characterization of a thermophilic cellobiose 2-epimerase from a thermohalophilic bacterium, Rhodothermus marinus JCM9785

Cellobiose 2-epimerase (CE) reversibly converts glucose residue to mannose residue at the reducing end of β-1,4-linked oligosaccharides. It efficiently produces epilactose carrying prebiotic properties from lactose, but the utilization of known CEs is limited due to thermolability. We focused on thermoholophilic Rhodothermus marinus JCM9785 as a CE producer, since a CE-like gene was found in the genome of R. marinus DSM4252. CE activity was detected in the cell extract of R. marinus JCM9785. The deduced amino acid sequence of the CE gene from R. marinus JCM9785 (RmCE) was 94.2% identical to that from R. marinus DSM4252. The N-terminal amino acid sequence and tryptic peptide masses of the native enzyme matched those of RmCE. The recombinant RmCE was most active at 80 °C at pH 6.3, and stable in a range of pH 3.2-10.8 and below 80 °C. In contrast to other CEs, RmCE demonstrated higher preference for lactose over cellobiose.     

Synthesis and fermentation properties of novel galacto-oligosaccharides by beta-galactosidases from Bifidobacterium species

beta-Galactosidase enzymes were extracted from pure cultures of Bifidobacterium angulatum, B. bifidum BB-12, B. adolescentis ANB-7, B. infantis DSM-20088, and B. pseudolongum DSM-20099 and used in glycosyl transfer reactions to synthesize oligosaccharides from lactose. At a lactose concentration of 30% (wt/wt) oligosaccharide yields of 24.7 to 47.6% occurred within 7 h. Examination of the products by thin-layer chromatography and methylation analysis revealed distinct product derived spectra from each enzyme. These were found to be different to that of Oligomate 55, a commercial prebiotic galacto-oligosaccharide. Fermentation testing of the oligosaccharides showed an increase in growth rate, compared to Oligomate 55, with products derived from B. angulatum, B. bifidum, B. infantis, and B. pseudolongum. However B. adolescentis had a lower growth rates on its oligosaccharide compared with Oligomate 55. Mixed culture testing of the B. bifidum BS-4 oligosaccharide showed that the overall prebiotic effect was equivalent to that of Oligomate 55.     

Structural characterization of multibranched oligosaccharides from seal milk by a combination of off-line high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and sequential exoglycosidase digestion

A complex mixture of diverse oligosaccharides related to the carbohydrates in glycoconjugates involved in various biological events is found in animal milk/colostrum and has been challenging targets for separation and structural studies. In the current study, we isolated oligosaccharides having high molecular masses (MW ∼ 3800) from the milk samples of bearded and hooded seals and analyzed their structures by off-line normal-phase-high-performance liquid chromatography-matrix-assisted laser desorption/ionization-time-of-flight (NP-HPLC-MALDI-TOF) mass spectrometry (MS) by combination with sequential exoglycosidase digestion. Initially, a mixture of oligosaccharides from the seal milk was reductively aminated with 2-aminobenzoic acid and analyzed by a combination of HPLC and MALDI-TOF MS. From MS data, these oligosaccharides contained different numbers of lactosamine units attached to the nonreducing lactose (Galβ1-4Glc) and fucose residue. The isolated oligosaccharides were sequentially digested with exoglycosidases and characterized by MALDI-TOF MS. The data revealed that oligosaccharides from both seal species were composed from lacto-N-neohexaose (LNnH, Galβ1-4GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc) as the common core structure, and most of them contained Fucα1-2 residues at the nonreducing ends. Furthermore, the oligosaccharides from both samples contained multibranched oligosaccharides having two Galβ1-4GlcNAc (N-acetyllactosamine, LacNAc) residues on the Galβ1-4GlcNAcβ1-3 branch or both branches of LNnH. Elongation of the chains was observed at 3-OH positions of Gal residues, but most of the internal Gal residues were also substituted with an N-acetyllactosamine at the 6-OH position.     

Chemical characterization of oligosaccharides in the milk of six species of New and Old world monkeys

Human and great ape milks contain a diverse array of milk oligosaccharides, but little is known about the milk oligosaccharides of other primates, and how they differ among taxa. Neutral and acidic oligosaccharides were isolated from the milk of three species of Old World or catarrhine monkeys (Cercopithecidae: rhesus macaque (Macaca mulatta), toque macaque (Macaca sinica) and Hamadryas baboon (Papio hamadryas)) and three of New World or platyrrhine monkeys (Cebidae: tufted capuchin (Cebus apella) and Bolivian squirrel monkey (Saimiri boliviensis); Atelidae: mantled howler (Alouatta palliata)). The milks of these species contained 6-8% total sugar, most of which was lactose: the estimated ratio of oligosaccharides to lactose in Old World monkeys (1:4 to 1:6) was greater than in New World monkeys (1:12 to 1:23). The chemical structures of the oligosaccharides were determined mainly by (1)H-NMR spectroscopy. Oligosaccharides containing the type II unit (Gal(β1-4)GlcNAc) were found in the milk of the rhesus macaque, toque macaque, Hamadryas baboon and tufted capuchin, but oligosaccharides containing the type I unit (Gal(β1-3)GlcNAc), which have been found in human and many great ape milks, were absent from the milk of all species studied. Oligosaccharides containing Lewis x (Gal(β1-4)[Fuc(α1-3)]GlcNAc) and 3-fucosyl lactose (3-FL, Gal(β1-4)[Fuc(α1-3)]Glc) were found in the milk of the three cercopithecid monkey species, while 2-fucosyl lactose (5'-FL, Fuc(α1-2)Gal(β1-4)Glc) was absent from all species studied. All of these milks contained acidic oligosaccharides that had N-acetylneuraminic acid as part of their structures, but did not contain oligosaccharides that had N-glycolylneuraminic acid, in contrast to the milk or colostrum of great apes which contain both types of acidic oligosaccharides. Two GalNAc-containing oligosaccharides, lactose 3'-O-sulfate and lacto-N-novopentaose I (Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc) were found only in the milk of rhesus macaque, hamadryas baboon and tufted capuchin, respectively. Further research is needed to determine the extent to which the milk oligosaccharide patterns observed among these taxa represent wider phylogenetic trends among primates and how much variation occurs among individuals or species.     

Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides

The aim of this research is to quantify the effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Quantification of these effects is important because temperature and enzyme origin are important process parameters. A kinetic model was used to describe the concentrations in time. The kinetic parameters were determined by using data obtained in batch experiments at various temperatures (20, 30, 40, and 50 degrees C) and by using beta-galactosidases from Bacillus circulans, Aspergillus oryzae, Kluyveromyces lactis, and Kluyveromyces fragilis. The effect of temperature on the kinetic parameters could be described with the Arrhenius equation, except for the inhibition parameter. Slightly higher oligosaccharide yields were found at higher temperatures. However, the influence of the initial lactose concentration was much larger. The higher yield at higher temperatures is an additional advantage when operating at high initial lactose concentrations and consequently elevated temperatures. Clear differences between the beta-galactosidases were found concerning amount, size, and type of oligosaccharides produced. The beta-galactosidase from B. circulans produced the most abundant amount, the most different, and largest-sized oligosaccharides. The beta-galactosidases from Kluyveromyces spp. produced mainly trisaccharides. The kinetic parameters for the different enzymes were determined and differences were discussed.     

Identification and synthesis of a trisaccharide produced from lactose by transgalactosylation

Enzymatic transgalactosylation of lactose by means of Streptococcus thermophilus, subspecies DN-001065, led to a mixture of D-galactose (approximately 4%), D-glucose (approximately 15%), lactose (approximately 51%), minor disaccharides (6%), trisaccharides (approximately 20%) and tetrasaccharides (3%). The major trisaccharide (approximately 16%) was identified by NMR spectroscopy and chemical synthesis as being the known beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranosyl-(1-->4)-D-glucos e (3'-beta-D-galactopyranosyl-lactose). It was purified from a mixture of peracetylated oligosaccharides by column chromatography followed by deacetylation. For the first time, 3'-beta-D-galactopyranosyl-lactose has been obtained on the 1 g scale, by resorting to simple techniques and equipment. NMR spectra have been unambiguously assigned.