**4. Methods of analysis of fractionated carbohydrates**

#### **4.1. Enzymatic methods**

samples obtained by ultrafiltration [18] and purified oligosaccharide-rich solutions from bovine colostrums [19], thus allowing proper oligosaccharide identification by mass spec-

Chromatographic techniques, usually set up in open columns with stationary phases based on anion exchange, adsorption, or gel-filtration/permeation mechanisms, are commonly used for the fractionation of carbohydrates. Brand-Miller et al. [20] used charcoal column chromatography for the separation of human milk oligosaccharides (HMOs) from the other constituents in milk. In this method, milk fat was first removed using centrifugation, and protein precipitated with organic solvents followed by enzymatically converting lactose to glucose and galactose to facilitate separation. The extract was filtered through a column packed with granular charcoal to separate the sugars. Glucose and galactose were eluted from the column initially with water and then with 2% v/v ethanol. The HMOs were then eluted from the column with 50% ethanol. In order to improve detection and characterization of less abundant oligosaccharides from bovine colostrums, fractionation of 2-aminobenzamide-labeled sample into neutral and acidic oligosaccharide fractions was performed by weak anionic exchange chromatography, and its separation ability is based on the combination of the charge and size of molecules [21]. Carbohydrates can be readily fractionated by gel-filtration chromatography on the basis of their relative sizes. To separate the HMO from lactose and salts, gel-filtration chromatography (G25 Sephadex column) has often been used [22–25]. When gel permeation chromatography is used for further separation of the different oligosaccharide fractions, lactose can be obtained separately in one of the fractions [Fractogel TSK HW 40 (S)] [26]. Various problems, however, have limited the development of gel-filtration methods for oligosaccharides. First, many of the commercially available gel-filtration matrices are themselves carbohydrates (e.g., Sephadex, Sepharose, etc.), shedding milligram quantities of heterodisperse carbohydrate polymers into the mobile phase. Second, nonspecific interactions with matrix materials are common because sugars are essentially amphipathic with a hydrophobic ring structure and hydrophilic functional groups [27]. Despite these problems, however, gel-filtration chromatography still remains an important option for the purification

Ultrafiltration (UF) and nanofiltration (NF) are increasingly used for the removal of lactose and other soluble components from milk, desalting and separation of interfering compounds; the resulting permeate has numerous applications including the production of lactose. The choice for selecting the UF and NF membrane is mainly based on the value of the molecular weight cutoff (MWCO), which is the molecular mass of the smallest compound retained to an extent larger than 90% [12]. Mehra et al. [28] employed the membrane filtration technology to produce powders enriched in bovine milk oligosaccharides (BMOs) using mother liquor (the liquid remaining after the separation of lactose crystals from whey UF permeate) as a starting raw material. The microfiltrate of mother liquor from the microfiltration step was utilized as the feed to the ultrafiltration (spirally wound membranes with a porosity of 1 kDa MWCO) for fractionation and enrichment of milk oligosaccharides from lactose and mineral salts. NF

trometry without the need of any further purification.

130 Technological Approaches for Novel Applications in Dairy Processing

**3.4. Chromatography-based methods**

of complex oligosaccharides.

**3.5. Membrane filtration**

An enzymatic method has been adopted by the IDF [29] for the determination of lactulose content of milk in the presence of much higher concentrations of lactose. Lactulose is often determined by the enzymatic methods using commercially available kits supplied by companies such as Boehringer-Mannheim and Merck. An enzymatic electrochemical method for the detection of lactulose content in milk samples was developed by Moscone et al. [30]. This method uses the enzyme β-galactosidase in solution to hydrolyze lactulose to galactose and fructose, and then the latter is oxidized by a fructose dehydrogenase enzyme reactor using potassium ferricyanide as mediator and platinum-based electrochemical transducer. The sensitivity of the procedure allowed pasteurized, UHT, and in-container sterilized milk can be distinguished. Lactulose content can also be determined by enzymatic method based on amperometric detection [31, 32].

#### **4.2. Spectrophotometric methods**

High sensitivity, sufficient accuracy, simplicity, speed, and the necessity of less expensive apparatus make spectrophotometric method as an attractive method for the determination of lactose and lactulose in different dairy products. For the analysis of lactulose preparation, spectrophotometric-enzymatic methods were applied to sugar mixtures produced during isomerization of lactose [33]. A simple spectrophotometric method for lactulose detection was based on hydrolysis of lactulose under acidic conditions followed by reaction with resorcinol, giving absorption peaks at 398 and 480 nm [34]. There are several enzymatic methods based on spectrophotometric detection that have been reported for the determination of lactose or lactulose in milk based on hydrolysis of lactose or lactulose by β-galactosidase [35–37]. A rapid and nondestructive front-face fluorescence spectroscopic method to quantify furosine and lactulose in heat-treated milk has been reported by Kulmyrzaev and Dufour [38]. Zhang et al. [39] developed a sensitive and simple spectrophotometric method for the quantification of lactulose without interference from aldoses. The method was based on hydrolysis of lactulose under acidic conditions. The hydrolyzed product reacted with cysteine hydrochloridetryptophan reagent, giving an absorption peak at 518 nm.

## **4.3. Capillary electrophoresis**

Capillary electrophoresis (CE) is the technique of choice for the analysis of hydrophilic mono- and oligosaccharides, with an impressive number of different separation approaches and different detection modes, due to their simplicity, short analysis time, efficiency, and low sample consumption [40, 41]. Bao et al. [42] developed a method for the quantification of sialyl oligosaccharides by CE with detection at 205 nm. However, carbohydrates lack a light-absorbing chromophore, which makes direct UV detection impossible unless a derivatization procedure is involved prior to analysis. Baross et al. [43] determined the lactose in milk and milk products by CE with indirect UV detection. More recently, HMOs have been detected by single CE with laser-induced fluorescence (LIF) [42, 44] or additionally coupled online with MS [45–47]. Labeling of HMO with 8-aminopyrene-1,3,6-trisulfonic acid introduces a fluorophore for the LIF detection, simultaneously adding the negative charge needed for the separation. Albrecht et al. [17] also developed a method for qualitative and quantitative analysis of GOS in different food matrices using CE-LIF. A recent study by Kottler et al. [48] describes the use of multiplexed capillary gel electrophoresis with LIF as a high-throughput method for glycolanalysis, demonstrating the ability to identify and quantify approximately 17 oligosaccharide structures based on "fingerprint" electropherograms.

detectors coupled to HPLC, the refractive index detector (RID) is the most widely used for sugars because no fluorophore (fluorescence detector) or chromophore (UV detector) is necessary; in other words, no derivatization is required. The HPLC-RID was commonly used for the determination of lactose, lactulose, and some other monosaccharides in variety of dairy foods, for instance, milk [55–58], whey permeate [59], cheese [60], and also in some of the

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The evaporating light scattering detector (ELSD) is a universal, nonspecific detector, which detects all nonvolatile solutes after evaporation of the solvent of the column effluent by light dispersion on the solid analyte particles formed. Recently, Schuster-Wolff-Bühring et al. [64] developed a sensitive and reliable method for simultaneous detection and quantification of lactose and lactulose in milk-based products using HPLC with ELSD and also compared four analytical methods using different HPLC columns. A HPLC column with an amino-bonded polymeric matrix yielded better results compared to amino-bonded silica-phase resin or cation-exchange resin. The detection of nonchromophoric and nonvolatile compounds makes

High performance (high-pH) anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) takes advantage of the affinity between the ionized group of sugars at alkaline pH (pH 12) and a pellicular quaternary amine stationary phase. Therefore, high resolution and highly selective separation of nonderivatized mono-, di- and oligosaccharides can be achieved [65]. This very good match between liquid chromatography and electrochemical detection has allowed the determination of carbohydrates in various complex matrices including dairy foods. Cataldi et al. [66] gave a comprehensive overview of analytical applications in food for carbohydrate analysis by HPAEC-PAD. The sensitive and accurate quantification of lactulose in sterilized milk by HPAEC-PAD has been demonstrated, and it makes the differentiation between sterilized and pasteurized milks possible [67]. The separation of monosaccharides (glucose, galactose, and fructose) and disaccharides (lactose, lactulose, epilactose, sucrose, and maltose) in dairy products [68, 69] and infant formulas [70, 71] by HPAEC-PAD has been described. The quantitative determination of NANA present at the terminal position of many glycoprotein and glycolipid-oligosaccharides by HPAEC-PAD has been reported by Rohrer [72]. Underivatized neutral or acidic oligosaccharides from human milk have been resolved by HPAEC-PAD [73–79]. Sialyllactose isomers (3′- and 6′-sialyllac-

However, since most sugars have no significant UV absorbing and/or fluorophore groups, derivatization prior to separation usually results in higher sensitivity. For detection by UV, a derivatization reagent is required. Reagents such as 1-phenyl-3-methyl-5-pyrazolone (PMP) and p-aminobenzoic ethyl ester are the most popular labels that react with reducing carbohydrates under mild conditions [80, 81]. Common fluorescent tags used for labeling the monosaccharides prior to HPLC analysis are anthranilic acid, 2-aminobenzamide, 2-aminopyridine, phenyl isothiocyanate, 9-fluorenylmethoxycarbonylhydrazine, 7-amino-4-methylcoumarin, and 7-amino-1, 3-naphthalene-disulphonate [82]. Normal-phase [83] and reversed-phase HPLCs [83–92] have been used efficiently for the analysis of derivatized milk oligosaccharides. Retention and separation of the HMO on reversed-phase liquid chromatography depend thus mainly on the method of derivatization; some isomer separation was

milk-based infant formulae [61–63].

ELSD a suitable technique for the detection of sugars.

tose) from whey streams were also quantified by HPAEC-PAD [28].

#### **4.4. Infrared spectrometry methods**

Infrared (IR) spectroscopy works by expressing vibrational modes of covalent bonds in molecules and assists in the quantification of any component present in a sample that absorbs IR radiation. The spectral ranges from 750 to 2500 nm and 2500 to 25,000 nm, respectively, are the near infra-red (NIR) and mid-infra-red (MIR) regions. NIR spectra are a result of combination of complex overtones and high frequency, while MIR is due to fundamental stretching, bending, and rotating vibrations in the sample [49]. The use of short-wave NIR wavelengths from 700 to 1100 nm has a key role in the analysis of protein, moisture, fat, and lactose contents in a wide range of dairy products including liquid milk, milk powder, cream, and processed cheese. NIR also offers the possibility of on-line analysis, which avoids the need for batch sampling and minimizes sampling error by averaging of virtually instantaneous, continuous measurements [50]. More recent instrument based on Fourier transform infrared (FTIR) spectroscopy is a rapid, accurate, and nondestructive method that can detect a range of functional groups and is sensitive to change in molecular structure. FTIR provides information on the basis of chemical composition and physical state of the whole sample [51]. Lactulose content in freeze-dried heat-treated milk was determined using diffuse reflectance FTIR spectroscopy in the MIR spectral region of 1286–754 cm−1 without any chemical treatment of milk. The authors suggested a broader range of 1300–750 cm−1 to be an important region for the study of carbohydrates [52]. FTIR spectroscopy was also used to monitor the lactose and protein concentration, during the UF and NF of whey. A range of 1220–800 cm−1 provides details about the lactose content of the sample [53].

#### **4.5. Liquid chromatography**

The HPLC is one of the most extensively used techniques employed for the separation of a large variety of carbohydrates in foods [54], as it is particularly advantageous in terms of speed, simplicity of sample preparation (without a prior derivatization), and obtaining a high-resolved chromatogram in a short period of time. A considerable research has been carried out on the quantitative determination of lactose and lactulose by using HPLC. Over all detectors coupled to HPLC, the refractive index detector (RID) is the most widely used for sugars because no fluorophore (fluorescence detector) or chromophore (UV detector) is necessary; in other words, no derivatization is required. The HPLC-RID was commonly used for the determination of lactose, lactulose, and some other monosaccharides in variety of dairy foods, for instance, milk [55–58], whey permeate [59], cheese [60], and also in some of the milk-based infant formulae [61–63].

detection modes, due to their simplicity, short analysis time, efficiency, and low sample consumption [40, 41]. Bao et al. [42] developed a method for the quantification of sialyl oligosaccharides by CE with detection at 205 nm. However, carbohydrates lack a light-absorbing chromophore, which makes direct UV detection impossible unless a derivatization procedure is involved prior to analysis. Baross et al. [43] determined the lactose in milk and milk products by CE with indirect UV detection. More recently, HMOs have been detected by single CE with laser-induced fluorescence (LIF) [42, 44] or additionally coupled online with MS [45–47]. Labeling of HMO with 8-aminopyrene-1,3,6-trisulfonic acid introduces a fluorophore for the LIF detection, simultaneously adding the negative charge needed for the separation. Albrecht et al. [17] also developed a method for qualitative and quantitative analysis of GOS in different food matrices using CE-LIF. A recent study by Kottler et al. [48] describes the use of multiplexed capillary gel electrophoresis with LIF as a high-throughput method for glycolanalysis, demonstrating the ability to identify and quantify approximately 17 oligosaccharide

Infrared (IR) spectroscopy works by expressing vibrational modes of covalent bonds in molecules and assists in the quantification of any component present in a sample that absorbs IR radiation. The spectral ranges from 750 to 2500 nm and 2500 to 25,000 nm, respectively, are the near infra-red (NIR) and mid-infra-red (MIR) regions. NIR spectra are a result of combination of complex overtones and high frequency, while MIR is due to fundamental stretching, bending, and rotating vibrations in the sample [49]. The use of short-wave NIR wavelengths from 700 to 1100 nm has a key role in the analysis of protein, moisture, fat, and lactose contents in a wide range of dairy products including liquid milk, milk powder, cream, and processed cheese. NIR also offers the possibility of on-line analysis, which avoids the need for batch sampling and minimizes sampling error by averaging of virtually instantaneous, continuous measurements [50]. More recent instrument based on Fourier transform infrared (FTIR) spectroscopy is a rapid, accurate, and nondestructive method that can detect a range of functional groups and is sensitive to change in molecular structure. FTIR provides information on the basis of chemical composition and physical state of the whole sample [51]. Lactulose content in freeze-dried heat-treated milk was determined using diffuse reflectance FTIR spectroscopy in the MIR spectral region of 1286–754 cm−1 without any chemical treatment of milk. The authors suggested a broader range of 1300–750 cm−1 to be an important region for the study of carbohydrates [52]. FTIR spectroscopy was also used to monitor the lactose and protein concentration, during the UF and NF of whey. A range of 1220–800 cm−1 provides details about

The HPLC is one of the most extensively used techniques employed for the separation of a large variety of carbohydrates in foods [54], as it is particularly advantageous in terms of speed, simplicity of sample preparation (without a prior derivatization), and obtaining a high-resolved chromatogram in a short period of time. A considerable research has been carried out on the quantitative determination of lactose and lactulose by using HPLC. Over all

structures based on "fingerprint" electropherograms.

132 Technological Approaches for Novel Applications in Dairy Processing

**4.4. Infrared spectrometry methods**

the lactose content of the sample [53].

**4.5. Liquid chromatography**

The evaporating light scattering detector (ELSD) is a universal, nonspecific detector, which detects all nonvolatile solutes after evaporation of the solvent of the column effluent by light dispersion on the solid analyte particles formed. Recently, Schuster-Wolff-Bühring et al. [64] developed a sensitive and reliable method for simultaneous detection and quantification of lactose and lactulose in milk-based products using HPLC with ELSD and also compared four analytical methods using different HPLC columns. A HPLC column with an amino-bonded polymeric matrix yielded better results compared to amino-bonded silica-phase resin or cation-exchange resin. The detection of nonchromophoric and nonvolatile compounds makes ELSD a suitable technique for the detection of sugars.

High performance (high-pH) anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) takes advantage of the affinity between the ionized group of sugars at alkaline pH (pH 12) and a pellicular quaternary amine stationary phase. Therefore, high resolution and highly selective separation of nonderivatized mono-, di- and oligosaccharides can be achieved [65]. This very good match between liquid chromatography and electrochemical detection has allowed the determination of carbohydrates in various complex matrices including dairy foods. Cataldi et al. [66] gave a comprehensive overview of analytical applications in food for carbohydrate analysis by HPAEC-PAD. The sensitive and accurate quantification of lactulose in sterilized milk by HPAEC-PAD has been demonstrated, and it makes the differentiation between sterilized and pasteurized milks possible [67]. The separation of monosaccharides (glucose, galactose, and fructose) and disaccharides (lactose, lactulose, epilactose, sucrose, and maltose) in dairy products [68, 69] and infant formulas [70, 71] by HPAEC-PAD has been described. The quantitative determination of NANA present at the terminal position of many glycoprotein and glycolipid-oligosaccharides by HPAEC-PAD has been reported by Rohrer [72]. Underivatized neutral or acidic oligosaccharides from human milk have been resolved by HPAEC-PAD [73–79]. Sialyllactose isomers (3′- and 6′-sialyllactose) from whey streams were also quantified by HPAEC-PAD [28].

However, since most sugars have no significant UV absorbing and/or fluorophore groups, derivatization prior to separation usually results in higher sensitivity. For detection by UV, a derivatization reagent is required. Reagents such as 1-phenyl-3-methyl-5-pyrazolone (PMP) and p-aminobenzoic ethyl ester are the most popular labels that react with reducing carbohydrates under mild conditions [80, 81]. Common fluorescent tags used for labeling the monosaccharides prior to HPLC analysis are anthranilic acid, 2-aminobenzamide, 2-aminopyridine, phenyl isothiocyanate, 9-fluorenylmethoxycarbonylhydrazine, 7-amino-4-methylcoumarin, and 7-amino-1, 3-naphthalene-disulphonate [82]. Normal-phase [83] and reversed-phase HPLCs [83–92] have been used efficiently for the analysis of derivatized milk oligosaccharides. Retention and separation of the HMO on reversed-phase liquid chromatography depend thus mainly on the method of derivatization; some isomer separation was obtained so far, but no method has emerged that provides comprehensive isomer separation [93]. Underivatized acidic oligosaccharides or derivatized neutral oligosaccharides have also been resolved by ion-exchange HPLC with detection at 200 nm absorbance [94]. However, the lack of structural discrimination by UV detection can make identification of peaks ambiguous.

Porous graphitized carbon (PGC) has also been recognized as a valuable stationary phase for the analysis of native oligosaccharides including neutral [100] and sialylated oligosaccharides [101], mostly because of the extensive separation of isomers that may be obtained. PGC-LC-MS was recently described as the stationary phase of choice for N-glycan analysis [103]. Bao et al. [104] developed a quantitative method for the analysis of HMO using PGC as the stationary phase and single-quadrupole MS detector. This analytical technique provides sensitive, precise, accurate quantification for each of 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different

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Beside LC, GC has also been extensively applied for the determination of carbohydrates in dairy foods. Compared to LC, the main advantage of GC is much higher separation power. However, derivatization is crucial for nonvolatile carbohydrates converted to volatile derivatives amenable to GC analysis. Most of the GC methods are based on the previous trimethylsilylation of sugars. Mainly hexamethyldisilazine, trimethylchlorosilane, *N*,*O*-bis(trimethylsilyl) trifluoroacetamide, trimethylsilylimidazole, and *N*,*O*-bis(trimethylsilyl)acetamide have been used as silylation reagents, and the good volatility and stability characteristics of the derivatives formed make trimethylsilyl (TMS) ethers the most popular derivatives applied to GC analysis of saccharides. Due to the relatively low volatility of carbohydrates, GC analysis is limited to derivatized sugars of low molecular weight, mainly mono-, di-, and trisaccharides [105]. In general, GC methods with flame ionization detection (FID) or MS detection provide a good separation of sugars and a good sensitivity. Recently, the use of MS as detector has increased over the FIDs for the identification of carbohydrates and the determination of molecular mass. Analysis of free carbohydrates in milk and milk products by GC-MS has been described by Reineccius et al. [106]. In this study, free carbohydrates (galactose, glucose, and lactose) in milk products, i.e., milk, cream, and cheeses, were isolated by dialysis, converted to trimethylsilyl ethers, and then analyzed by GC-MS. Troyano et al. [107, 108] developed a GC method for the analysis of free monosaccharides in raw and market milk samples. Valero et al. [109] determined the amount of lactulose formed in pasteurized milk by GC of the TMS derivatives of the free sugar, besides monosaccharides were also determined. Lactulose content was also determined by GC analysis of their corresponding TMS ethers separated on a 50% diphenyl/50% dimethylsiloxane capillary column [110]. Using this method, it was possible to quantify lactulose on different treated milks (UHT, sterilized, pasteurized, condensed, and powder milk). Although GC is a sensitive method for sugar analysis, the sample preparation is laborious and not very practical in routine analysis. Besides in the GC procedure, the anomeric composition of α- and β-anomers is obtained, which means more than one peak

Mass spectrometry provides a sensitive technique to assist in the determination of the structure of carbohydrates, but it is essential that the samples are pure to achieve maximum ionization.

stages of lactation.

**4.6. Gas chromatography (GC)**

area for each compound.

**4.7. Mass spectrometry methods**

Labeling with chromophoric active tags such as 1-phenyl-3-methyl-5-pyrazolone, 2-aminopyridine, and 2-aminobenzoicacid as well as perbenzoylation has been applied for the analysis of oligosaccharides [93]. Recently, hydrophilic interaction liquid chromatography (HILIC)- HPLC has been used for separation and characterization of 2-aminobenzamide-labeled milk oligosaccharides (N- and O-glycans) from bovine colostrum and has proven efficient and robust method for oligosaccharide analysis [21]. Using exoglycosidases, the structures of 37 bovine milk oligosaccharides could be confirmed, which revealed the separation of several structural isomers.

Ultra performance liquid chromatography (UPLC) has also shown improved resolution of *N*-glycans released from glycoproteins [95], with reduced run times and solvent consumption, and it could provide a further improvement in the detection and separation of larger isomeric oligosaccharides.

All the analytical techniques described so far are based on separation alone; however, structural confirmation can in such cases only be obtained based on standards. These standards are expensive and not available for all types of oligosaccharides. For better identification, coupling of the separation with mass spectrometry has proven to be effective. Several applications are reported where sugars were analyzed as their sodium adduct, acetate adduct, or chloride adduct. Indeed, the combination of liquid chromatography and time of flight (TOF) mass spectrometry by the HPLC-chip technology has been extensively and successfully used for oligosaccharide profiling in human milk [96, 97] and bovine milk [19, 98]. Nano-liquid chromatography porous graphitic carbon TOF MS in the positive mode has also been applied in the analysis of HMO [99–101]. Both neutral and sialylated compounds may be separated in one run, and using a library containing retention time, mass, and fragmentation information, immediate identification is possible [100, 101]. Using this method, >200 HMO structures can be separated. More recently, oligosaccharides in bovine colostrum were investigated by Aldredge et al. [102], who employing nano-liquid chromatography tandem mass spectrometry, identified five fucosylated oligosaccharides that were found to be in common with human milk. Nano-LC chip/TOF MS offers an orthogonal dimension of retention time and accurate mass, making it possible to separate isomeric HMOs, with and without sialic acids, in a single-chromatographic separation. Ultra high performance chromatography coupled to mass spectrometry or tandem mass spectrometer (UHPLC-MS or MS/MS) is highly specific and sensitive if single- or multi-reaction monitoring is used. Trani et al. [58] carried out a very interesting comparison of HPLC-RI, LC/MS-MS, and enzymatic assays for the analysis of residual lactose in lactose-free milk. The enzymatic methods as well as HPLC coupled to RI detector are not suitable for quantitative determination of residual lactose in lactose-free milk. But, LC-MS/MS method based on the use of lactose formate adduct was proved to be very sensitive and offered highly reproducible results even at the lowest lactose concentrations.

Porous graphitized carbon (PGC) has also been recognized as a valuable stationary phase for the analysis of native oligosaccharides including neutral [100] and sialylated oligosaccharides [101], mostly because of the extensive separation of isomers that may be obtained. PGC-LC-MS was recently described as the stationary phase of choice for N-glycan analysis [103]. Bao et al. [104] developed a quantitative method for the analysis of HMO using PGC as the stationary phase and single-quadrupole MS detector. This analytical technique provides sensitive, precise, accurate quantification for each of 11 milk oligosaccharides and allows measurement of differences in milk oligosaccharide patterns between individuals and at different stages of lactation.

#### **4.6. Gas chromatography (GC)**

obtained so far, but no method has emerged that provides comprehensive isomer separation [93]. Underivatized acidic oligosaccharides or derivatized neutral oligosaccharides have also been resolved by ion-exchange HPLC with detection at 200 nm absorbance [94]. However, the lack of structural discrimination by UV detection can make identification of

Labeling with chromophoric active tags such as 1-phenyl-3-methyl-5-pyrazolone, 2-aminopyridine, and 2-aminobenzoicacid as well as perbenzoylation has been applied for the analysis of oligosaccharides [93]. Recently, hydrophilic interaction liquid chromatography (HILIC)- HPLC has been used for separation and characterization of 2-aminobenzamide-labeled milk oligosaccharides (N- and O-glycans) from bovine colostrum and has proven efficient and robust method for oligosaccharide analysis [21]. Using exoglycosidases, the structures of 37 bovine milk oligosaccharides could be confirmed, which revealed the separation of several

Ultra performance liquid chromatography (UPLC) has also shown improved resolution of *N*-glycans released from glycoproteins [95], with reduced run times and solvent consumption, and it could provide a further improvement in the detection and separation of larger

All the analytical techniques described so far are based on separation alone; however, structural confirmation can in such cases only be obtained based on standards. These standards are expensive and not available for all types of oligosaccharides. For better identification, coupling of the separation with mass spectrometry has proven to be effective. Several applications are reported where sugars were analyzed as their sodium adduct, acetate adduct, or chloride adduct. Indeed, the combination of liquid chromatography and time of flight (TOF) mass spectrometry by the HPLC-chip technology has been extensively and successfully used for oligosaccharide profiling in human milk [96, 97] and bovine milk [19, 98]. Nano-liquid chromatography porous graphitic carbon TOF MS in the positive mode has also been applied in the analysis of HMO [99–101]. Both neutral and sialylated compounds may be separated in one run, and using a library containing retention time, mass, and fragmentation information, immediate identification is possible [100, 101]. Using this method, >200 HMO structures can be separated. More recently, oligosaccharides in bovine colostrum were investigated by Aldredge et al. [102], who employing nano-liquid chromatography tandem mass spectrometry, identified five fucosylated oligosaccharides that were found to be in common with human milk. Nano-LC chip/TOF MS offers an orthogonal dimension of retention time and accurate mass, making it possible to separate isomeric HMOs, with and without sialic acids, in a single-chromatographic separation. Ultra high performance chromatography coupled to mass spectrometry or tandem mass spectrometer (UHPLC-MS or MS/MS) is highly specific and sensitive if single- or multi-reaction monitoring is used. Trani et al. [58] carried out a very interesting comparison of HPLC-RI, LC/MS-MS, and enzymatic assays for the analysis of residual lactose in lactose-free milk. The enzymatic methods as well as HPLC coupled to RI detector are not suitable for quantitative determination of residual lactose in lactose-free milk. But, LC-MS/MS method based on the use of lactose formate adduct was proved to be very sensitive and offered highly reproducible results even at the lowest lactose concentrations.

peaks ambiguous.

134 Technological Approaches for Novel Applications in Dairy Processing

structural isomers.

isomeric oligosaccharides.

Beside LC, GC has also been extensively applied for the determination of carbohydrates in dairy foods. Compared to LC, the main advantage of GC is much higher separation power. However, derivatization is crucial for nonvolatile carbohydrates converted to volatile derivatives amenable to GC analysis. Most of the GC methods are based on the previous trimethylsilylation of sugars. Mainly hexamethyldisilazine, trimethylchlorosilane, *N*,*O*-bis(trimethylsilyl) trifluoroacetamide, trimethylsilylimidazole, and *N*,*O*-bis(trimethylsilyl)acetamide have been used as silylation reagents, and the good volatility and stability characteristics of the derivatives formed make trimethylsilyl (TMS) ethers the most popular derivatives applied to GC analysis of saccharides. Due to the relatively low volatility of carbohydrates, GC analysis is limited to derivatized sugars of low molecular weight, mainly mono-, di-, and trisaccharides [105]. In general, GC methods with flame ionization detection (FID) or MS detection provide a good separation of sugars and a good sensitivity. Recently, the use of MS as detector has increased over the FIDs for the identification of carbohydrates and the determination of molecular mass. Analysis of free carbohydrates in milk and milk products by GC-MS has been described by Reineccius et al. [106]. In this study, free carbohydrates (galactose, glucose, and lactose) in milk products, i.e., milk, cream, and cheeses, were isolated by dialysis, converted to trimethylsilyl ethers, and then analyzed by GC-MS. Troyano et al. [107, 108] developed a GC method for the analysis of free monosaccharides in raw and market milk samples. Valero et al. [109] determined the amount of lactulose formed in pasteurized milk by GC of the TMS derivatives of the free sugar, besides monosaccharides were also determined. Lactulose content was also determined by GC analysis of their corresponding TMS ethers separated on a 50% diphenyl/50% dimethylsiloxane capillary column [110]. Using this method, it was possible to quantify lactulose on different treated milks (UHT, sterilized, pasteurized, condensed, and powder milk). Although GC is a sensitive method for sugar analysis, the sample preparation is laborious and not very practical in routine analysis. Besides in the GC procedure, the anomeric composition of α- and β-anomers is obtained, which means more than one peak area for each compound.

#### **4.7. Mass spectrometry methods**

Mass spectrometry provides a sensitive technique to assist in the determination of the structure of carbohydrates, but it is essential that the samples are pure to achieve maximum ionization. The most widely used ionization methods for oligosaccharides are matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI), coupled with tandem techniques such as collision-induced dissociation (CID). Such advances have improved carbohydrate research with mass spectrometry. The tandem mass spectrometry (MS/MS) has been used for detailed structural analysis of oligosaccharide molecules in which a molecular ion is selected by a first stage of MS, which undergoes activation and fragmentation, and the product ions are analyzed to provide information about the sequence monosaccharide compositions, linkages, and locations of various modifications [111].

Most recently, lactulose has been proposed as a treatment for the *Salmonella* carrier state, a preventative and treatment for high cholesterol, a preventative for gall stones, and an adjunctive treatment for colorectal cancers [124]. Moreover, lactulose is used in pharmaceuticals as a

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The main characteristics of lactobionic acid include moisturizing, antioxidant, stabilizing, and acidifying capacities. Lactobionic acid appears to be a less commonly used prebiotic for both *Lactobacilli* and *Bifidobacteria*. However, lactobionic acid has been suggested to improve gut health [125]. Other applications of lactobionic acid include as a calcium supplement (calcium lactobionate) in pharmaceutical solutions for stabilizing the organs during transplant [126] and in the cosmetic industry [127]. Lactitol is widely used in chocolates, biscuits, sweets, ice cream, and confectionary manufacture as an alternative to other sugar alcohols, such as mannitol, sorbitol, and xylitol. It is used in low-energy, low-fat, and glycemic foods for diabetics

It is widely accepted that milk oligosaccharides play several important protective, physiological, and biological roles including selective growth stimulation for beneficial gut microbiota, inhibition of pathogen adhesion, and immunoregulation [88, 129]. The use of free oligosaccharides and their conjugated derivatives in anti-adhesion therapy may provide an effective therapeutic approach to prevent diseases [21]. GOSs are widely used in infant milk formula, infant foods, follow-on formula, beverages, and fermented milks and in confectionary and bread making, of their functional properties as well as health-enhancing properties. The use of GOS in the livestock feed and a pet food industry is also increasing, especially in the poultry, pig, and aqua-culture [130].

There are several fractionations and analytical techniques currently being used for the determination of carbohydrates in different milk and milk products. A problem with studying carbohydrate bioactivities is the limited access to well-defined oligosaccharides. Purification of oligosaccharides from natural sources is laborious, and it is difficult to obtain preparation free of contaminants. In addition, the nonlinear nature of oligosaccharides creates a high structural diversity and their overall complexity makes it difficult for a single analytical method to characterize them. Numerous health benefits of milk oligosaccharides and lactose derivatives are well

\*, Battula Surendra Nath<sup>3</sup>

1 College of Dairy Science and Technology, GADVASU, Ludhiana, Punjab, India

3 ICAR-National Dairy Research Institute, Bengaluru, Karnataka, India

2 Dairy Technology Division, ICAR-National Dairy Research Institute, Karnal, Haryana,

and Ashish Kumar Singh<sup>2</sup>

mild laxative and to treat hyperammonemia.

and is also recognized as not causing dental caries [128].

established, including the pharmaceutical and food applications.

, Neelam Upadhyay<sup>2</sup>

\*Address all correspondence to: neelam\_2912@yahoo.co.in

**6. Conclusion**

**Author details**

Veena Nagaraj1

India

Milk oligosaccharides have been analyzed using tandem mass spectrometry such as collisioninduced dissociation (CID) and infrared multiphoton dissociation (IRMPD) [112]. A comprehensive label-free procedure to identify and quantify milk oligosaccharides and other glycans using nano-liquid chromatography-time of flight MS was recently developed [113]. During this study, MALDI-Fourier transform ion cyclotron resonance (FTICR) was used for accurate mass measurement compositional analysis. FTICR MS provides exceptional mass accuracy at even few parts per million with extremely high mass resolution and has been extensively and successfully used in complex samples. Oligosaccharides in bovine cheese whey permeate were characterized by a combination of nanoelectrospray (nESI)-FTICR-MS and MALDI-FTICR-MS, and this method was possible to identify 15 (8 were neutral and 7 were acidic) milk oligosaccharides [18]. Recently, Mehra et al. [28] identified 25 oligosaccharides including 6 high molecular weight fucosylated oligosaccharides in a mother liquor of dairy streams using MALDI-FTICR mass spectrometer, and the researchers also confirmed the fucosylated oligosaccharides composition and putative structure by using accurate tandem mass spectrometry.

#### **4.8. Nuclear magnetic resonance (NMR)**

Many different analytical techniques have been used to elucidate the free glycan profile present in milk samples. Compared with LC-MS and GC-MS, one of the advantages of NMR spectroscopy is the direct and quantitative relationship between the molar concentration and the intensity of the NMRs. For instance, NMR has played an important role in the structural characterization of free oligosaccharides in human milk [114–117]. Monakhova et al. [118] suggest the use of NMR spectroscopy as screening tool to validate nutrition labeling of lactose-free milk, reporting a detection limit of 30 mg/L.
