**3. Techniques employed for fractionation of carbohydrates**

The fractionation of carbohydrates from dairy foods is carried out by using some kind of cleanup or extraction methods prior to their analysis. The general scheme for obtaining lactose and other sugars from dairy foods is to first precipitate fat and protein by different reagents (Carrez solution, Biggs-Szijarto solution, and 60% methanol), giving a clear serum adequate for carbohydrate analysis [7]. After precipitation, filtration or centrifugation step yields a clear solution. The ulterior analysis may require an additional step. In this section, the techniques employed for extraction and fractionation of carbohydrates are described.

#### **3.1. Pressurized liquid extraction**

oligosaccharides have a lactose unit at their reducing end to which specific neutral monosaccharides/oligosaccharides (*N*-acetylglucosamine or *N*-acetylgalactosamine, galactose or glucose, and fucose or 6-deoxyhexose) or acidic oligosaccharides [*N*-acetylneuraminic acid (NANA)] can be attached [3]. However, β-galactooligosaccharides (GOSs) are oligosaccharides composed primarily of galactose and often terminate with a glucose residue at the reducing end, and they occur naturally in the milk of many animals including humans and cows. But, GOSs are normally produced industrially by transgalactosylation of lactose using β-galactosidase.

Substances such as lactulose, lactitol, and lactobionic acid are derived from lactose and are not present in natural sources. When lactose in milk is subjected to moderate heating, its isomerization can occur with the lactulose (4-*O*-β-d-galactopyranosyl-d-fructofuranose) formation through Lobry de Bruyn-Alberda van Ekenstein reaction, through the intermediate compound 1,2-enediol [4]. Consequently, the quantity of lactulose is directly proportional to the intensity of the heat treatment applied and could be useful as indicators of the quality of processing of milk [5]. Lactobionic acid (4-*O*-β-d-galactopyranosyl-d-gluconic acid) is an aldonic acid, comprising of galactose and gluconic acid, obtained through lactose oxidation using a metal catalyst or by enzymatic/microbiological oxidation, while lactitol (4-*O*-β-d-galactopyranosyl-d-glucitol) is a sugar alcohol derived from lactose by catalytic hydrogenation. In this chapter, we describe the different techniques employed for the fractionation of milk carbohydrates, the methods of analysis of fractionated carbohydrates, and applications of fractionated carbohydrates in food and pharmaceutical industry.

Intensive biochemical characterization of the carbohydrate molecules and their various bioactivities may facilitate an understanding of their importance in human nutrition and may suggest individual carbohydrate structures to target for industrial production [6]. Recently, many studies reveal the biological significance of some carbohydrates and their potential role as nutraceuticals. However, detailed analysis is beneficial for the identification of the specific oligosaccharides responsible for such activities, as the activity may be attributed to one oligosaccharide or even a fraction of oligosaccharides within the entire milk oligosaccharide pool. Extraction and fractionation techniques represent useful tool in the analysis of such carbohydrates from food samples. Thus, it is important to investigate the structure of oligosaccharides

The fractionation of carbohydrates from dairy foods is carried out by using some kind of cleanup or extraction methods prior to their analysis. The general scheme for obtaining lactose and other sugars from dairy foods is to first precipitate fat and protein by different reagents (Carrez solution, Biggs-Szijarto solution, and 60% methanol), giving a clear serum adequate for carbohydrate analysis [7]. After precipitation, filtration or centrifugation step yields a clear solution. The ulterior analysis may require an additional step. In this section, the techniques

to understand the relationship between their structure and biological function.

**3. Techniques employed for fractionation of carbohydrates**

employed for extraction and fractionation of carbohydrates are described.

**2. Need of fractionation of carbohydrates**

128 Technological Approaches for Novel Applications in Dairy Processing

Pressurized liquid extraction (PLE) is based on the use of solvents at high temperatures (50– 200°C) and pressures (1450–2175 psi) to ensure the rapid extraction rate of compounds [8]. The high temperature enables higher solubility and higher rate of solute diffusion in the solvent, while the application of high pressure maintains the solvent below its boiling point, thereby allowing a high penetration of the solvent into the sample [9]. Recently, the extraction and purification of lactulose from a mixture with lactose have been carried out by using PLE (at 1500 psi) with ethanol/water (70:30, w/w) mixture at 40°C for 30 min, and the recovery of lactulose reached up to 84.4% with a purity of over 90% [10]. Despite the advantages over conventional extraction methods, this method is not found to be suitable for thermo-labile compounds as high temperature can have deleterious effects on their structural and functional activities [11].

#### **3.2. Supercritical fluid extraction**

The use of supercritical fluid extraction consists of the separation of the analyte from the matrix using supercritical fluids as the extracting solvent. Carbon dioxide (probably the most used supercritical fluid) is nontoxic, nonflammable, can act at low temperatures, and is relatively cheap; unfortunately, the solubility of carbohydrates in the supercritical phase of this fluid is low [12]. Some of the advantages of supercritical fluid extraction are solvating powers similar to liquid organic solvents, high solute diffusivities, lower viscosity, lower surface tension, and the possibility of adjusting the solvating power by changing pressure or temperature [9]. Carbon dioxide is sometimes modified by co-solvents such as ethanol that change its polarity. This technique is useful for the separation of lactulose and tagatose from their isomeric aldoses (i.e., lactose and galactose, respectively) [13] and GOS from monosaccharides in a commercial sample using CO2 with ethanol/water as co-solvent (at 150 bar and 80°C) [14].

#### **3.3. Solid phase extraction**

Solid phase extraction (SPE) is the very popular technique currently available for rapid and selective sample preparation. The versatility of SPE allows the use of this technique for several purposes, such as purification, trace enrichment, desalting, derivatization, and class fractionation. The selection of an appropriate SPE extraction sorbent depends on understanding the mechanism of interaction between sorbent and analyte of interest [15]. Reverse-phase (RP) cartridges are commonly used for the purification of carbohydrates. Octyl (C<sup>8</sup> ) and octadecyl (C18) silica phases are the most common RP cartridges used for carbohydrate cleanup. These sorbents show high affinity for hydrophobic compounds but less affinity for hydrophilic solutes such as oligosaccharides [16]. Moreover, C18 cartridges are useful for the fractionation of (1–4)-α-glucans depending on their degree of polymerization. Ion-exchange SPEs are used for desalting oligosaccharides mixtures, but care should be taken to avoid the loss of charged sugars during their purification [12]. Solid-phase extraction on graphitized carbon material upon enzymatic amyloglucosidase pre-treatment enabled a good recovery and a selective purification of the different GOS structures from the exceeding amounts of particularly lactose- and maltodextrin-rich preparations [17]. These cartridges are also used effectively to remove salts and residual contaminants (traces of protein and lipids) from whey permeate samples obtained by ultrafiltration [18] and purified oligosaccharide-rich solutions from bovine colostrums [19], thus allowing proper oligosaccharide identification by mass spectrometry without the need of any further purification.

is an attractive method for HMO isolation due to the speed with which separations can be performed, and it does not require the use of organic solvents. An easily scalable approach to the recovery of HMO from milk has been developed by Sarney et al. [23], which relay on the combination of enzymatic treatment of defatted and deproteinated milk using β-galactosidase and NF and compared the resulting HMO produced with gel filtration. The authors obtained a yield of 6.7 g of HMO from 1 L of milk in just four NF cycles, yet residual lactose appeared in the oligosaccharide fraction produced with NF but not in that prepared using gel filtration.

Advances in Fractionation and Analysis of Milk Carbohydrates

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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].

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 hydrochloride-

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

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

**4.1. Enzymatic methods**

**4.2. Spectrophotometric methods**

**4.3. Capillary electrophoresis**

tryptophan reagent, giving an absorption peak at 518 nm.

#### **3.4. Chromatography-based methods**

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 of complex oligosaccharides.

#### **3.5. Membrane filtration**

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 is an attractive method for HMO isolation due to the speed with which separations can be performed, and it does not require the use of organic solvents. An easily scalable approach to the recovery of HMO from milk has been developed by Sarney et al. [23], which relay on the combination of enzymatic treatment of defatted and deproteinated milk using β-galactosidase and NF and compared the resulting HMO produced with gel filtration. The authors obtained a yield of 6.7 g of HMO from 1 L of milk in just four NF cycles, yet residual lactose appeared in the oligosaccharide fraction produced with NF but not in that prepared using gel filtration.
