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Lactose is a natural disaccharide obtained from the milk of most mammals and a waste product of cheese and casein manufacturing. Over the past decades, lactose in whey has increasingly been promoted as an important resource, and an increasing number of significant advances have been made to investigate its healthy and functional properties. Lactose can be biotransformed into many kinds of derivatives, including galacto-oligosaccharides, epilactose, lactulose, lactosucrose, and D-tagatose. Biological efficiency and safety are critical for the enzymatic production of lactose derivatives from lactose. These lactose derivatives show a range of prominent physiological features and effects, such as prebiotic properties, indigestibility, and obesity prevention, which can be utilized in the pharmaceutical, health, and food industries. In this review, we present the properties and physiological effects of lactose derivatives, detailing their biological production by various enzymes and their applications in dairy products, especially directly in the milk industry.

Consumers are conscientiously changing their eating preferences toward healthier options, such as functional foods enriched with pre- and probiotics. Prebiotics are attractive bioactive compounds with multidimensional beneficial action on both human and animal health, namely on the gastrointestinal tract, cardiometabolism, bones or mental health. Conventionally, prebiotics are non-digestible carbohydrates which generally present favorable organoleptic properties, temperature and acidic stability, and are considered interesting food ingredients. However, according to the current definition of prebiotics, application categories other than food are accepted, as well as non-carbohydrate substrates and bioactivity at extra-intestinal sites. Regulatory issues are considered a major concern for prebiotics since a clear understanding and application of these compounds among the consumers, regulators, scientists, suppliers or manufacturers, health-care providers and standards or recommendation-setting organizations are of utmost importance. Prebiotics can be divided in several categories according to their development and regulatory status. Inulin, galactooligosaccharides, fructooligosaccharides and lactulose are generally classified as well established prebiotics. Xylooligosaccharides, isomaltooligosaccharides, chitooligosaccharides and lactosucrose are classified as "emerging" prebiotics, while raffinose, neoagaro-oligosaccharides and epilactose are "under development." Other substances, such as human milk oligosaccharides, polyphenols, polyunsaturated fatty acids, proteins, protein hydrolysates and peptides are considered "new candidates." This chapter will encompass actual information about the non-established prebiotics, mainly their physicochemical properties, market, legislation, biological activity and possible applications. Generally, there is a lack of clear demonstrations about the effective health benefits associated with all the non-established prebiotics. Overcoming this limitation will undoubtedly increase the demand for these compounds and their market size will follow the consumer's trend.

Lactulose and epilactose are nondigestible disaccharides, which can be interconverted from lactose directly by using cellobiose 2-epimerase as catalyst. The characteristics of cellobiose 2-epimerase and its application in enzymatic production of lactulose and epilactose are discussed in this chapter.

An LC–MS method was developed for routine determination of lactose in low-lactose and lactose-free dairy products. The lactose was separated on an amide column under an isocratic elution condition. Baseline separation of lactose from common interferents, including epilactose, lactulose, and allolactose were achieved under this LC conditions. The lactose chloride-35 adduct ions were detected by a single quadrupole MS in ESI- mode. 13C labeled lactose was used as an internal standard. The LOQ was estimated at 2.6 mg/100 g in a soy based infant formula. The recovery was found within the range of 90%–109% in the spiking study on commercial dairy products at spiking levels between 3.4 and 461 mg/100 g. The analysis of lactose concentration in the NIST SRM 1849a showed an accuracy of 109%. The RSD in the analysis of those commercial products were less than 4.3%. This method is sensitive and selective enough and fit for the routine determination of lactose in low-lactose and lactose-free dairy products.

The cellobiose 2-epimerases (CEs) from Caldicellulosiruptor saccharolyticus ( Cs CE) and Dictyoglomus turgidum ( Dt CE) are the two CEs best investigated which are able to catalyse an epimerisation and an isomerisation reaction on lactose. However, both enzymes are thermophilic and have, so far, only been characterised under their respective optimal conditions. The CE from Caldicellulosiruptor saccharolyticus has recently been applied for epilactose and lactulose production in milk at lower temperatures. The kinetic parameters under supposedly industrial relevant conditions and the direct comparability of Cs CE and Dt CE still remain unknown and were, therefore, the object of this study. In general, Cs CE and Dt CE displayed similar reaction progresses and similar kinetic parameters. Surprisingly, the Cs CE was observed to catalyse the isomerisation reaction of lactulose into lactose and epilactose twice as efficiently ( k cat / K m ) as Dt CE. For the first time, the reaction progress for lactose, epilactose and lactulose as substrates was observed for both Cs CE and Dt CE over 48 h at 50 °C, pH 7.5. These conversions led to the same final sugar concentrations of about 30.5% ts lactose, 14.5% ts epilactose and 55% ts lactulose with both CEs. During the course of the conversions, the interrelated ratios of lactose and epilactose remained steady at about 70:30, respectively. In conclusion, both CEs were of comparable aptitude for industrial application under the conditions tested and most probably catalyse the same course of reaction.

Epilactose (4-O-β-d-galactopyranosyl-d-mannopyranose) is a rare disaccharide derived from lactose (4-O-β-d-galactopyranosyl-d-glucopyranose) and possesses prebiotic properties. Epilactose is needed for biological studies in high amounts and purified form. The purification of epilactose is challenging because it behaves similarly to lactose, the starting substance of the epilactose production process. Epilactose can be produced both chemically and enzymatically, using cellobiose 2-epimerases for the latter. In this study, a process for semi-preparative epilactose purification is presented. Firstly, epilactose was produced in a buffered system using recombinant cellobiose 2-epimerase from Flavobacterium johnsoniae. After the enzymatic conversion, some of the residual lactose was removed by crystallisation at 6 °C for 72 h. Subsequently, the lactose still remaining in the conversion mixture was separated from epilactose by ligand-exchange chromatography. The conditions for the most effective chromatographic separation of lactose and epilactose were determined using a design of experiments approach with surface response methodology. The best conditions led to an epilactose purity of 99 % with a total yield of 51 %.

Lactulose and epilactose are potentially prebiotic isomers of the milk sugar lactose. Enzymatically they can be produced using cellobiose 2-epimerases. These sugars are notoriously hard to quantify due to their chemical similarity, which results in incomplete separation. With a new commercial stationary phase we achieved baseline separation on a basic HPLC system. The method allows the parallel quantification of lactose, epilactose and lactulose within a wide range of concentrations befitting most food samples.

Cellobiose 2-epimerase (CE), found mainly in anaerobes, reversibly converts D-glucose residues at the reducing end of β-1,4-linked oligosaccharides to D-mannose residues. In this study, we characterized CE-like proteins from various aerobes (Flavobacterium johnsoniae NBRC 14942, Pedobacter heparinus NBRC 12017, Dyadobacter fermentans ATCC 700827, Herpetosiphon aurantiacus ATCC 23779, Saccharophagus degradans ATCC 43961, Spirosoma linguale ATCC 33905, and Teredinibacter turnerae ATCC 39867), because aerobes, more easily cultured on a large scale than anaerobes, are applicable in industrial processes. The recombinant CE-like proteins produced in Escherichia coli catalyzed epimerization at the C2 position of cellobiose, lactose, epilactose, and β-1,4-mannobiose, whereas N-acetyl-D-glucosamine, N-acetyl-D-mannosamine, D-glucose, and D-mannose were inert as substrates. All the CEs, except for P. heparinus CE, the optimum pH of which was 6.3, showed highest activity at weakly alkaline pH. CEs from D. fermentans, H. aurantiacus, and S. linguale showed higher optimum temperatures and thermostability than the other enzymes analyzed. The enzymes from D. fermentans, S. linguale, and T. turnerae showed significantly high k(cat) and K(m) values towards cellobiose and lactose. Especially, T. turnerae CE showed a very high k(cat) value towards lactose, an attractive property for the industrial production of epilactose, which is carried out at high substrate concentrations.

Epilactose (4-O-β-D-galactopyranosyl-D-mannose), an epimer of lactose, is a rare disaccharide existing extremely small quantities in heat-treated milk, in which epilactose is produced by non-enzymatic catalysis from lactose. This disaccharide is a kind of non-digestible carbohydrate, has a good prebiotic effect, and promotes intestinal mineral absorption. This article presents a review of recent studies on epilactose formation in food system, qualitative and quantitative analysis, and its physiological functions. In addition, the biochemical properties and kinetic parameters of the epilactose-producing enzyme, cellobiose 2-epimerase, are compared, and the biotechnological production of epilactose from lactose is reviewed.

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)6-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.

Health benefits associated with the consumption of nondigestible oligosaccharides (NDOs) are well documented, and being also functional food components, they are now in a privileged position within the functional food business. Lactose-derived NDOs have some salient features for being derived from industrial outputs and have some unique properties, like their similar functionality to human milk oligosaccharides. This chapter reviews the available technologies for their production (chemical or enzymatic), identifying the more relevant products derived from lactose (galacto-oligosaccharides, lactulose, lactosucrose, lactitol, lactobionic acid, tagatose, epilactose, and fructosyl-galacto-oligosaccharides). Applications for each of them are reviewed, analyzing its present status and technological perspectives. An updated market outlook is presented for each of them, being a central issue in this chapter. Galacto-oligosaccharides and lactulose, as well-documented prebiotics, outstand; the case of tagatose as a noncaloric sweetener with health-promoting attributes is gaining a salient position in the functional food business.

Cellobiose 2-epimerase (CE) efficiently forms epilactose which has several beneficial biological functions. A thermostable CE from Rhodothermus marinus was immobilized on Duolite A568 and packed into a column. Lactose (100 g/L) was supplied to the reactor, kept at 50 °C at a space velocity of 8 h(-1). The epilactose concentration of the resulting eluate was 30 g/L, and this was maintained for 13 d.

Many kinds of lactose derivatives can be obtained using various methods, including epimerization, oxidation and reduction. Investigations of the beneficial functions of lactose derivatives developed have resulted in them being put into practical use in numerous fields. In particular, lactulose, galacto-oligosaccharide and lactitol are used in foodstuffs and pharmaceuticals, and new lactose derivatives such as epilactose and tagatose have also recently received attention. Various physiological effects that lactulose exerts have been studied, including the beneficial promotion of bifidobacteria proliferation in the intestine.

The conditions for maximum lactulose production from lactose, as a single substrate, by a thermostable recombinant cellobiose-2-epimerase from Caldicellulosiruptor saccharolyticus were determined to be pH 7.5, 80 °C, 700 g l −1 lactose, and 150 U ml −1 of enzyme. Under the conditions, the enzyme produced the two bifidus factors lactulose at 408 g l −1 and epilactose at 107 g l −1 after 2 h. The yields of lactulose and epilactose from lactose and the productivities of lactulose and epilactose were 58%, 15%, 204 g l −1 h −1 , and 54 g l −1 h −1 , respectively. The yield and productivity of both lactulose and epilactose from lactose were 74% and 258 g l −1 h −1 , respectively. The yield, concentration, and productivity of lactulose in the present study are the highest among enzymatic syntheses. This is the first trial of enzymatic synthesis of lactulose using the single substrate lactose.

The enzymatic production of lactulose was described recently through conversion of lactose by a thermophilic cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE). In the current study, we examined the application of CsCE for lactulose and epilactose production in milk (1.5% fat). The bioconversions were carried out in stirred reaction vessels at 2 different temperatures (50 and 8°C) at a scale of 25 mL volume. At 50°C, 2 highly different CsCE amounts were investigated for the time course of formation of lactulose and epilactose. The conversion of milk lactose (initial lactose content of 48.5 ± 2.1 g/L) resulted in a final yield of 57.7% (28.0 g/L) lactulose and 15.5% (7.49 g/L) epilactose in the case of the approximately 9.5-fold higher CsCE amount (39.5 µkat epilactose, 50°C) after 24 h. Another enzymatic lactose conversion was carried out at low 8°C, an industrially relevant temperature for milk processing. Although the CsCE originated from a thermophilic microorganism, it was still applicable at 8°C. This enzymatic lactose conversion resulted in 56.7% (27.5 g/L) lactulose and 13.6% (6.57 g/L) epilactose from initial milk lactose after 72 h. The time courses of lactose conversion by CsCE suggested that first epilactose formed and afterward lactulose via epilactose. To the best of our knowledge, this is the first time that an enzyme has produced lactulose directly in milk in situ at industrially relevant temperatures.

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.

Strong base catalysts, prepared by substituting a part of the Mg2+ located at the borders of the channels of sepiolite with alkaline ions (Li+, Na+, K+ and Cs+), were investigated as catalysts for the isomerization of lactose to lactulose and epilactose. The activities exhibited by alkaline-exchanged sepiolites were significantly higher than that of natural sepiolite. The influence of temperature, time of the reaction and catalyst loading were also evaluated. A 20% conversion was obtained at 90°C at a catalyst loading of 15 g litre-1. Copyright (C) 1999 Elsevier Science Ltd.

The kinetics of formation of lactulose, galactose and epilactose during heating of milk were examined over a wide temperature/time range (). Formation of the studied carbohydrates was best described with apparent first order reactions. The Arrhenius plot of the logarithm of the rate constant as a function of the reciprocal of the temperature (°K) enabled calculation of the activation energies (125 kJ/mol for lactulose, 131 kJ/mol for epilactose and 139 kJ/mol for galactose). By using the kinetics parameters for calculating lines of equal degrees of formation in a plot of log-time versus reciprocal of absolute temperature it was possible to predict the effect of different heat-treatments on the formation of individual carbohydrates., The authors wish to acknowledge financial support for the research project No. 158/88 provided by the Comisi6n Interministerial de Ciencia y Tecnologia.

Carbohydrate composition of commercial pasteurized, UHT, sterilized and dried milk samples, and laboratory heat-treated milks was determined by gas-liquid chromatography. Glucose, galactose and lactulose were present in all types of commercial milk samples. The concentrations of galactose and lactulose increase with the severity of the heating process. The amount of glucose present in commercial milks was similar to that found in unheated milk. Epilactose was only detected in sterilized milk samples. UHT and sterilized milks can be differentiated by their lactulose, galactose or epilactose content.