Quantification of lactose and lactulose in hydrolysed-lactose UHT milk using capillary zone electrophoresis

International Dairy Journal 106 (2020) 104710

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Quantification of lactose and lactulose in hydrolysed-lactose UHT milk using capillary zone electrophoresis Leandra N. de Oliveira Neves, Marcone A. Leal de Oliveira* Department of Chemistry, Federal University of Juiz de Fora, Juiz de Fora, MG 36036-330, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 October 2019 Received in revised form 2 March 2020 Accepted 3 March 2020 Available online 2 April 2020

A new method for quantification of lactose and lactulose in hydrolysed-lactose UHT milk, using capillary zone electrophoresis with indirect detection in the ultraviolet region (CZE-UV), is proposed. A 33 Box eBehnken design for instrumental electrophoretic parameters optimisation was used, with the best conditions shown to be an injection pressure of 15 mbar 2 s, applied voltage of
20 kV, and cartridge temperature of 20  C. Extraction procedures were investigated and protein precipitation using trichloroacetic acid solution was shown to give the best results. Limits of quantification for lactose and lactulose were 0.024 g 100 mL
1 and 0.0094 g 100 mL
1, respectively. Recovery levels ranging from 86% to 102% were obtained. The method was successful applied to several hydrolysed-lactose and regular UHT milk samples from six commercial leading brands. Results showed that all hydrolysed-lactose samples are in accordance with the legal recommendations to be classified as “lactose-free” products. © 2020 Elsevier Ltd. All rights reserved.

1. Introduction Ultra-high temperature (UHT) processing (135e145  C for 2e4 s) is a common heat treatment used by the dairy industry; UHT milk has the great advantage of room temperature transportation and storage as well as extended shelf life. UHT processing technologies may be classified as direct or indirect according to the heat transfer mechanism. Indirect UHT heating is characterised by the use of tubular heat exchangers where the heat is transferred from the heating medium (generally water or steam) to the milk throughout the wall of the equipment. In direct UHT processing, steam is injected directly (injection or infusion systems) into the milk providing almost instantaneous heating, which imparts a lower thermal load on the product due to significantly faster heating and cooling rates (Kelleher et al., 2019; Walstra, Wouters, & Geurts, 2006b). Both direct and indirect heating may induce chemical modifications causing nutritional losses (e.g., lysine blockage during the Maillard reaction) and physical stability issues (mainly protein denaturation). To determine the intensity of these modifications, the dairy industry monitors chemical compounds formed or modified during heating, such as 5-hydroxymethylfurfural (HMF),

* Corresponding author. Tel.: þ55 32 21023310. E-mail address: [email protected] (M.A. Leal de Oliveira). https://doi.org/10.1016/j.idairyj.2020.104710 0958-6946/© 2020 Elsevier Ltd. All rights reserved.

furosine, whey protein nitrogen index (WPNI), b-lactoglobulin and lactulose (Walstra et al., 2006b). The latter compound, lactulose, is a lactose epimer with a chemical structure of 4-O-b-D-galactopyranosyl-D-fructofuranose; it is formed in heated milk through the alkaline isomerisation of lactose, which is characterised by the transformation of the glucose moiety of lactose to fructose. Many mechanisms of this transformation have been proposed but the most accepted is the Lobry de BruyneAlberda van Ekenstein (LA) reaction that consists of an enolisation step followed by a b-elimination (O'Brien, 2009). Lactulose formation during thermal treatment of dairy products depends on lactose concentration and time and temperature of heating, and follows pseudo-zero order kinetics with energy of activation of 90.2 kJ mol
1 (Claeys, Ludikhuyze, & Hendrickx, 2001). Lactulose has been considered the main chemical compound from heat damage in dairy products compared with other indicators such as HMF and furosine, and its quantification provides information about degree of heat application (Elliott, Dhakal, Datta, & Deeth, 2003; Olano & Calvo, 1989). Lactose is a disaccharide made up of glucose and galactose residues linked by a b-1,4 glycosidic linkage. It is the major carbohydrate in cows' milk, 3.8e5.3% depending on breed of cow and its metabolism and stage of lactation (Walstra, Wouters, & Geurts, 2006a). It is known that about 70% of the world population has a degree of lactose malabsorption, characterised by the inability to digest lactose due to a decrease in lactase expression. Consequently,

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demand for lactose-reduced products is constantly increasing and promotes the development of new lactose-free products by the dairy industry. One of the most frequently produced lactose-free dairy products is UHT milk, since it can be directly consumed or used in food recipes. Although there is no global consensus on regulatory requirements for lactose-free claims, some regulatory agencies are trying to standardise this growth sector of the dairy industry. The European Food Safety Agency defines that products labelled as “lactose-free” must have a lactose content lower than 0.1 g 100 mL
1 (EFSA, 2010), while United States Food Drug and Administration (FDA) suggests that they should not contain any lactose and that products claiming to be “lactose reduced” should have a meaningful reduction in lactose (FDA, 2009). Brazilian legal guidelines from the Health Surveillance Agency (ANVISA) sets maximum residual lactose content of 0.1 g 100 mL
1 for products labelled as “lactose-free” and a range of 0.1e1.0 g 100 mL
1 for “low-lactose” products (Brasil, 2017a). This regulatory agency also stipulates a mandatory declaration of the presence of lactose in foods containing more than 0.1 g 100 mL
1, which must include the statement “contains lactose” in a legible and easily identifiable form (Brasil, 2017b). Hydrolysed-lactose (LH) UHT milk involves lactose hydrolysis before or after heat treatment and is characterised by enzymatic degradation under specific and controlled conditions. During this process, the enzyme b-D-galactosidase, known as lactase, hydrolyses the b-1,4 linkage releasing glucose and galactose and thereby provides a sweeter flavour to the final product (Fox & McSweeney, 1998). If lactose hydrolysis occurs prior to thermal processing, the released glucose and galactose, as well as the residual lactose, will be available for chemical modifications such as isomerisation and the Maillard reaction during heating. Intense changes in colour and flavour are more likely to occur when hydrolysis is performed prior to heating, since reducing monosaccharides, e.g., glucose and galactose, are more reactive than reducing disaccharides such as lactose (BeMiller & Huber, 2008). Isomerisation reactions of glucose and galactose are also likely to occur, generating fructose and tagatose, respectively, which can be used as an innovative heat indicator in LH matrices (Ruiz-Matute et al., 2012). However, if lactose hydrolysis occurs after heating process these chemical modifications tend to be less significant if product is kept under milder temperature conditions (Harju, Kallioinen, & Tossavainen, 2012). Considering the increase in LH UHT milk production (Dekker, Koenders, & Bruins, 2019), control of both thermal and hydrolysis processes becomes critical. In this sense, a practical and quick analytical method to quantify both lactose and lactulose in milk containing different levels of lactose becomes necessary, but it is also a challenge due to chemical similarities of these isomers. Despite the presence of several articles in the literature regarding lactose or lactulose quantification in dairy products using different analytical techniques, such as spectrophotometry (Adhikari, Sahai, & Mathur, 1991; Zhang, Wang, Yang, & Jiang, 2010), gas chromatography with flame ionisation detection (Montilla, Moreno, & Olano, 2005), high performance liquid chromatography with
vez-Servín, Castellote, & refractive index detection (HPLC-RID) (Cha
pez-Sabater, 2004; ISO/IDF, 2007; Neves, Carvalho, Aguiar, & Lo Silva, 2017), capillary zone electrophoresis with indirect detection in ultraviolet region (CZE-UV) (Neves, Marques, Silva, & de Oliveira, 2018; Soga & Serwe, 2000), capillary microchips (Duarte-Junior et al., 2019), electrochemical biosensors (Conzuelo, Amella, Ampuzano, Uiz, & Eviejo, 2010), and enzymaticspectrophotometric methods (ISO/IDF, 2004; Marconi et al., 2004), only a few have been applied to determine both carbohydrates in LH milk matrices (Messia, Candigliota, & Marconi, 2007;

Ruiz-Matute et al., 2012; Schuster-Wolff-Bühring, Michel, & Hinrichs, 2011; Trani et al., 2017). Within this context, the aim of this work was to optimise the CZE-UV method, which we previously developed (Neves et al., 2018), for application in lactose and lactulose quantification in heated milk samples with different lactose content, mainly hydrolysed-lactose UHT milk. The present work also studied some classical procedures for milk sample preparation with the aim of improving the analytical frequency. 2. Materials and methods 2.1. Chemicals and solutions Deionised water (Milli-Q system, Millipore, Bedford, MA, USA) was used for all dilutions and solutions. Standards of lactose, lactulose, glucose and galactose, as well as 2,6-pyridinedicarboxylic acid (PDC), cetyltrimethylammonium bromide (CTAB) and sodium hydroxide (NaOH) were obtained from Sigma Aldrich (St. Louis, MO, USA). Bihydrated zinc acetate and glacial acetic acid (used for Carrez I solution), potassium hexacyanoferrate (used for Carrez II solution), trichloroacetic acid, phosphotungstic acid and ethanol were obtained from Vetec Fine Chemicals (Rio de Janeiro, RJ, Brazil); acetonitrile, chromatographic grade, was from JT Baker (Center Valley, PA, USA). These were used to test methodologies for sample preparation. Individual stock solutions of all standard carbohydrates were prepared in deionised water at 1 g 100 mL
1 and were kept at 4  C. Stock solution used for background electrolyte (BGE) containing 100 mmol L
1 PDC and 250 mmol L
1 NaOH was prepared and also stored at 4  C, while stock solution of CTAB at 10 mmol L
1 was stored at ambient temperature (approximately 25  C). 2.2. Capillary electrophoresis experimental procedures Experiments were performed using an Agilent 7100 capillary electrophoresis system (Agilent Technologies, Palo Alto, CA, USA) equipped with a diode array detector (DAD). Agilent ChemStation software (Rev. B.04.03) was used for data acquisition and processing. Polyimide-coated fused-silica (TSP) capillaries of 75 mm internal diameter (i.d.), 50.0 cm of effective length (LE) and 58.5 cm of total length (LT) (TSP 075150, Polymicro Technologies, Phoenix, AZ, USA) were used in all experiments. The signal was carried out at 350 nm, with a reference signal set at 275 nm. Frequency of data achievement was set at 2.5 Hz. The working BGE solution was prepared from dilution of BGE stock solution followed by CTAB addition, resulting in a solution composed by 20 mmol L
1 PDC, 50 mmol L
1 NaOH and 0.5 mmol L
1 CTAB, and final pH approximately 12.5. New capillaries were conditioned by rinsing sequentially with NaOH solution at 1.0 mol L
1 for 40 min, then deionised water and BGE for 15 min each. For ongoing analysis, capillaries were conditioned with 1 mol L
1 NaOH solution, deionised water and BGE for 15 min each, while between runs a flush composed by 1.0 mol L
1 NaOH solution for 3 min followed by deionised water and BGE during 2 min each was used. 2.3. Capillary zone electrophoresis parameter optimisation The CZE-UV method was based on our previous work (Neves et al., 2018) but a new electrophoretic parameter study was performed to determine the best conditions to be applied in UHT milk matrices containing different levels of lactose. A 33 BoxeBehnken design with a triplicate central point was used, with three different pressures of injection (10, 15 and 20 mbar), voltages applied

L.N. de Oliveira Neves, M.A. Leal de Oliveira / International Dairy Journal 106 (2020) 104710

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(
15,
17 and
20 kV) and cartridge temperatures (20, 22 and 25  C). Injection was performed in hydrodynamic mode using a constant injection time of 2 s. All factorial design assays were conducted using one regular and one hydrolysed-lactose UHT milk sample (called a sample-test), randomly. Both sample-tests were submitted to Carrez extraction
vez-Servín et al. (2004). as described by Cha

corresponding to sample signal. The same procedure was performed by adding 16 mL of lactose standard solution at 1 g 100 mL
1 plus 84 mL diluted extract, resulting in a total volume of 100 mL, and obtaining signal 2 (S2), corresponding to sample plus standard signal.

2.4. Samples and extraction methods

2.5.2. Lactulose quantification For lactulose quantification, both L-REG and LH samples extracts were injected without dilution. Thus, samples signal (S1) was obtained after CE vials (final volume 100 mL) preparation consisting in 84 mL extract plus 16 mL deionised water. While the sample plus standard signal (S2) was obtained by adding 16 mL of lactulose standard solution at 1 g 100 mL
1 plus 84 mL sample extract. Before injection, all vials were homogenised with a vortex for 10 s. Lactulose and lactose concentrations were calculated based on Equation (1):

A total of six commercial brands of UHT milk, with the largest market share in both regular and hydrolysed-lactose dairy sectors, were selected. One sample of conventional UHT milk and one sample of hydrolysed UHT milk of each commercial brand was collected, randomly, resulting in six samples of each matrix (conventional and hydrolysed) giving a total of 12 UHT milk samples analysed in authentic duplicates. The samples were identified according to the information about their lactose content given by the producer on the product label. Thus, conventional UHT milk with regular lactose content was identified as lactose regular (L-REG), while UHT milk labelled as “lactose-free”, “lactose reduced”, or similar expressions were identified as hydrolysed-lactose (LH). All commercial brands were also coded as B_01eB_06. Sample preparation or extraction methods tested were: E_1, precipitation with trichloroacetic acid solution at 40% (w/v) using equal volumes of milk and acid solution followed by centrifugation at 10,752  g for 10 min and filtration (Adhikari et al., 1991), adapted; E_2, precipitation using 5.5 mL of pretreatment solution (9.1% zinc acetate, 5.7% phosphotungstic acid, 5.8% glacial acetic acid), 15 mL of sample and deionised water to a final volume of 50 mL, and qualitative filtration after a stabilisation period of 1 h at 25  C (ISO/IDF, 2007); E_3, organic precipitation using a 20 mL solution composed of ethanol and water (1:1) added to 14 mL of milk sample followed by heating for 25 min at 60  C [a clarification step was then performed at 25  C, by addition of 500 mL of each Carrez I and II clarifier solutions; after stirring, 10 mL of acetonitrile was added, and final dilution up to a volume of 50 mL with ethanolewater (1:1) solution, followed by a stabilisation period of 1 h at 4  C, and filtration (Ch
avez-Servín et al., 2004)]; E_4, an E_3 method adaptation using a larger sample volume (25 mL to a final volume of 50 mL) and 10 mL of ethanol 95% instead of the diluent solution, followed by heating for 25 min at 60  C [acetonitrile (5 mL) was added, and clarifying solutions Carrez I and II (500 mL each) were added after cooling to 25  C and ethanol (95%) was added to give a final volume of 50 mL]; E_5, precipitation using 50 mL of glacial acetic acid for 5 mL of milk followed by centrifugation at 5376  g for 10 min and filtration (Meinhart, Ballus, Bruns, Lima, & Godoy, 2011); E_6, exclusive precipitation of the sample with ethanol (99%) at a ratio of 4:1 ethanol to sample (20 mL ethanol and 5 mL milk sample) followed by evaporation and suspension in 10 mL ethanol (Acquaro et al., 2013). All extracts were filtered using a 0.45 mm membrane (RC45/13) MachereyeNagel® (Bethlehem, PA, USA) before injection. 2.5. Single point standard addition quantification 2.5.1. Lactose quantification A dilution of the sample extracts was necessary for both L-REG and LH matrices to obtain a stable baseline and a well-defined peak. Dilution was performed by adding deionised water before the extract's filtration. The L-REG sample extracts were diluted using a 1:5 ratio (sample extract:final volume) while the LH extracts used a 1:2 dilution ratio. For quantification, 84 mL diluted extract plus 16 mL deionised water were added directly into CE vials, resulting in a final volume of 100 mL, and an analytical signal (S1) was obtained,

  Cx g 100 mL
1 ¼

S1 :Cs :Vs ðS2
S1 Þ:Vx

(1)

where: Cx is sample analyte concentration (g 100 mL
1); Cs is standard solution concentration (g 100 mL
1); Vs is standard volume added (mL); S1 is analytical signal of sample extract (mAU); S2 is analytical signal of sample extract plus standard (mAU); and Vx is aliquot of the sample extract used (mL). As single point standard addition (SPSA) quantification assumes that the slope (k) is constant (Cardone & Palermo, 1980), it is important to verify experimentally the k value for all samples, using Equation (2), where Vf is the final volume of vial injection (in mL). If the calculated global value for k shows low variability it indicates that the SPSA method can be safely used and there is no matrix effect during quantification.

 ðS
S Þ:V  2 1 f k mAU mL mg
1 ¼ Cs :Vs

(2)

2.6. Validation of CZE-UV method Limits of detection (LOD) and quantification (LOQ), calculated by means of the signal-to-noise ratio approach, were used to evaluate method sensitivity. LOD and LOQ were calculated for each commercial brand considering three different baseline regions, according to Equations (3) and (4) (Faria, De Souza, & De Oliveira, 2008).

 3:sd  noise :Cx LOD g 100 mL
1 ¼ Hmax
Hmin

(3)

 10:sd  noise :Cx LOQ g 100 mL
1 ¼ Hmax
Hmin

(4)

where: sdnoise is standard deviation of the electropherogram noise (mAU); Cx is sample analyte concentration (g 100 mL
1); Hmax is maximum height of the analyte peak (mAU); Hmin is minimum height of the analyte peak (mAU). Precision was assessed by injection of six replicates of both LREG and LH matrices for a single milk brand, randomly. Replicates were injected on the same working day (Ribani, Bottoli, Collins, Jardim, & Melo, 2004).

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3. Results and discussion 3.1. CZE-UV method optimisation The effective mobility (meff) study, Fig. 1, was performed using the pKa for glucose (12.28), galactose (12.35), lactose (11.98), and lactulose (10.28), and electrophoretic conditions applied. Sugars are very weak acids, ionising only in strongly alkaline medium (pH > 12). Therefore, it was important to keep BGE at high pH, ensuring that the carbohydrates yield negatively charged species during electrophoretic runs. The migration order given by the meff study was verified through a standard mixture injection containing all carbohydrates at 1.0 mg mL
1 and using a BGE at pH 12.5, described in section 2.2. Peaks were identified by injections of the mixture, with a spike for each analyte. Fig. 1 also shows the experimental migration order. For electrophoretic parameter optimisation, the analytical response monitored (Respi) was set as the ratio between the peak pair resolution and the migration time of the last analyte, according to Equation (5).

Respi



   min
1 ¼ Rs=t m

(5)

where: Rs is peak pair resolution, tm is migration time of the last analyte (min), and i is milk matrix type (L-REG or LH). Analytical response was designed to give good peak resolution between pairs of analytes, lactuloseelactose (in L-REG milk) and glucose-galactose (in LH milk) with shorter run time for both dairy matrices. It is important to highlight that in LH dairy products the major carbohydrates are glucose and galactose. According to the migration order given by Fig. 1, lactose (peak 3) appears between glucose and galactose (peaks 2 and 4, respectively), indicating that good peak resolution of these monosaccharides is mandatory. Therefore, analytical responses for L-REG and LH milk matrices were calculated for each factorial design experiment. The assays

with high analytical response values were evaluated to select the condition which better fit both matrices. Table 1 shows the coded 33 BoxeBehnken containing levels, factors and response obtained for each assay. It can be seen that the highest response value for L-REG milk was achieved by CE_10 (0.94) while the highest analytical response of the LH sample-test was obtained simultaneously by two assays (CE_10 and CE_12). Graphs A and B, depicted in Fig. 2, show the electrophoretic profile of L-REG and LH samples submitted to the experimental conditions of the CE_10 and CE_12 assays, respectively, and assisted in CE parameter selection. CE_10 and CE_12 assays differed from each other in cartridge temperature (20  C and 25  C, respectively). Comparing the

Table 1 Coded 33 BoxeBehnken matrix containing levels, factors and response (resolution peaks and time migration ratio) obtained for UHT milk samples-test with regular and hydrolysed-lactose content.a Assay

X1

X2

X3

RespL-REG

RespLH

CE_01 CE_02 CE_03 CE_04 CE_05 CE_06 CE_07 CE_08 CE_09 CE_10 CE_11 CE_12 CE_13 CE_14 CE_15


1 þ1
1 þ1
1 þ1
1 1 0 0 0 0 0 0 0


1
1 þ1 þ1 0 0 0 0
1 þ1
1 þ1 0 0 0

0 0 0 0
1
1 þ1 þ1
1
1 þ1 þ1 0 0 0

0.007 0.066 0.091 0.074 0.078 0.009 0.009 0.062 0.059 0.094 0.046 0.088 0.078 0.079 0.079

0.075 0.056 0.085 0.080 0.068 0.083 0.078 0.086 0.070 0105 0.076 0.105 0.076 0.076 0.077

a Factors and levels: X1, injection pressure of (mbar) 10 (
1), 15 (0) and 20 (1); X2, voltage of (kV)
15 (
1),
17, (0) and
20 (1); X3, cartridge temperature ( C) of 20 (
1), 22 (0) and 25 (1). Analytical response for: RespL-REG, regular lactose content matrix, and RespLH, hydrolysed-lactose matrix.

Fig. 1. Effective mobility (meff) study for lactulose ( ), glucose ( ), lactose ( ), and galactose ( ) showing the migration order over all the pH range. Insert confirms the migration order at pH 12.5, experimentally: 1, lactulose; 2, glucose; 3, lactose; 4, galactose. Experimental conditions were: injection (10 mbar 2 s), voltage (-15 kV), cartridge temperature (20 ºC), acquisition frequency (2.5 Hz), standards concentration (1.0 mg mL-1).

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Fig. 2. Electrophoretic profiles for regular lactose (L-REG) and hydrolysed-lactose (LH) UHT milk sample-test submitted to (A) CE_10 and (B) CE_12 assays according Table 1. Peaks are identified according migration order: 1, lactulose; 2, glucose; 3, lactose; 4, galactose. Sample extraction method: Carrez (method E_3).

electrophoretic profiles of both milk matrices obtained in each assay it was noted that an increase in temperature results in a migration time reduction and a slight reduction in peak resolution, compromising lactulose peak identification in the L-REG dairy matrix (graph B). For the LH sample-test, there was a reduction in both migration time and peak resolution when the CE_12 assay was applied, which coincidentally gave a ratio equal to that obtained by CE_10 (0.105). However, it is readily apparent in Fig. 2, and confirmed by results in Table 1, that electrophoretic conditions for the CE_10 assay provide a more suitable profile for analyte identification and quantification in both regular and hydrolysed dairy matrices. 3.2. Study of sample preparation methods An analytical method with sample preparation which can be applied to both dairy matrices, minimising analytical interferences, improving throughput, and easily performed, is always a challenge. Different extraction procedures were evaluated, considering the optimised electrophoretic parameters. The results are summarised in Fig. 3, which shows the resolution values for each peak pair, calculated at half height of the peaks (w1/2). Comparing methods E_1 and E_4 (graphs A and D), similar values were achieved for pair resolution of lactuloseelactose (peaks 1 and 3) in L-REG milk, and glucose-galactose (peaks 2 and 4) in LH milk. However, the lactulose peak was not identified in LH milk using E_4. Sample preparation methods E_2, E_3 and E_5

(represented by graphs B, C and E) were disregarded due to the low lactuloseelactose peak resolution (values <1.5) for the L-REG milk matrix. Graphs B and E also showed unstable baselines and the presence of shoulders compromised analyte quantification and might be associated with the ineffectiveness of the extraction procedures. The E_6 method (graph F) was not considered as the solvent evaporation step became a problem due to possible lactose degradation by heating, which could compromise reliable quantification of its isomer in thermally treated milk samples. Additionally, graph F showed an unstable baseline, probably due to high levels of organic solvent. Thus the E_1 method was selected as the sample treatment method to be applied in lactose and lactulose determinations of L-REG and LH milk samples due its good peak resolution for both matrices, as well its fast performance and low residue generation compared with methods such as E_2 and E_3. 3.3. CZE-UV method validation Sensitivity and precision data are presented in Table 2. The AOAC International (2002) has established different variability levels for precision, according to analyte concentration in the sample. Thus, a relative standard deviation (RSD) ranging from 3% to 4% is acceptable for analytes in concentrations between 0.01% and 0.1% (e.g., lactulose in both L-REG and LH UHT milk matrices, and lactose in LH matrices). When analyte is above 1% in a sample, the RSD may vary from 1.5% to 2.0% (e.g., lactose concentration in L-REG milk). In this sense, according to data in Table 2, the method precision is

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Fig. 3. Electropherograms for all sample extraction methods applied in regular lactose (L-REG) and hydrolysed-lactose (LH) UHT milk sample-test using electrophoretic optimised parameters: pressure injection (15 mbar 2 s), voltage (
20 kV), acquisition frequency (2.5 Hz), cartridge temperature (20  C). Analytes are identified as: 1, lactulose; 2, glucose; 3, lactose; 4, galactose.

Table 2 Sensibility and precision evaluation for lactulose and lactose determination in UHT milk samples containing different levels of lactose.a Figures of merit Sensitivity Precision L-REG Precision LH

LOD LOQ Repeatability (n ¼ 6) %RSD Repeatability (n ¼ 6) %RSD

Lactulose (g 100 mL
1)

Lactose (g 100 mL
1)

0.0028 0.0094 0.0196 1.02 0.0199 1.01

0.007 (±0.001)** 0.024 (±0.004)** 4.80 (±0.04) 0.83 0.111 (±0.004) 3.60

(±0.0006)* (±0.0021)* (±0.0002) (±0.0002)

a Abbreviations are: L-REG, regular lactose content matrix; LH, hydrolysed-lactose matrix. Standard deviation given in parentheses; a single asterisk (*) indicates n ¼ 30, a double asterisk (**) indicates n ¼ 18.

acceptable (RSDlactulose approximately 1% for both milk matrices, and RSDlactose ¼ 0.83% and 3.6% for L-REG and LH milk, respectively). The accuracy was given by a recovery test using three different levels of fortification, using 1, 1.5 and 2 times the maximum allowed or suggested limit for the analyte of interest (European Commission, 2002). The maximum limits considered were 0.100 g 100 mL
1 for lactose and 0.0600 g 100 mL
1 for lactulose; according to ANVISA and IDF specifications, respectively. Recovery tests for each analyte were performed considering the matrix of

interest. Thus, recovery levels for lactose were performed using LH UHT milk while lactulose recovery was evaluated in L-REG UHT milk. All data are presented in Table 3. Regarding the recovery indexes, AOAC (2002) reports a range from 85% to 108% for analytes at sample concentrations between 0.01 and 0.1%. In this case, both recovery levels of lactulose and lactose in L-REG and LH milk are in accordance with these analytical specifications. According to the literature, the official enzymatic method proposed by IDF provides an LOD of about 0.0010 g 100 mL
1 for

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Table 3 Accuracy evaluation by recovery test for lactulose and lactose determination in UHT milk.a Lactulose

Lactose

Fortification level (g 100 mL
1)

% Recovery

Fortification level (g 100 mL
1)

% Recovery

0.0600 0.0900 0.120

86.25 (±1.25) 101.04 (±1.91) 98.54 (±4.03)

0.10 0.15 0.20

102.08 (±11.81) 96.25 (±4.88) 101.15 (±0.79)

a Recovery tests realised considering lactulose in a regular lactose content matrix, and lactose in a hydrolysed-lactose matrix. Standard deviation given in parentheses (n ¼ 4).

lactulose quantification in milk samples (De Block, Merchiers, Van Renterghem, & Moermans, 1996; Pereda, Ferragut, Quevedo, Guamis, & Trujillo, 2009). The enzymatic method is based on the spectrophotometric determination of the amount of fructose potentially released by the action of b-D-galactosidase during hydrolysis of carbohydrates present in milk. Despite the IDF enzymatic method allowing numerous samples to be measured simultaneously, it is characterised by the use of six different enzymes and by a total time for analysis of about 13 h, including sample preparation step (ISO/IDF, 2004). The official HPLC-RID method, also proposed by IDF, shows a LOD of 0.0040 g 100 mL
1 for lactulose quantification in milk samples, using an exclusive column separation and a pre-column, with a complete chromatographic run of 25 min (Elliott et al., 2003). Additionally, the IDF HPLC method guidelines indicate a sample preparation step (also evaluated in the present study and coded as E_2 assay; Section 2.4.) characterised by use of many different reagents, and a stabilisation time of 1 h before injection. A period of 1 h between injections of consecutive samples is also recommended to stabilise the chromatographic system (ISO/IDF, 2007). Consequently, length of time required by these methods, need of large amounts of reagents, extensive reactions, and high cost of enzymes are drawbacks which may negatively affect their use for routine evaluation. It is worth emphasising that both IDF methods do not mention their application for lactose quantification in hydrolysed-lactose dairy products. Furthermore, according to our knowledge, there is no data in the literature indicating use of these official methods for lactose determinations in LH milk samples. A comparison between the two separation methods, the IDF HPLC and the proposed CZE-UV, was performed to highlight the improvements achieved through the optimised steps. The first advantage of the CZE-UV method is its lower LOD for lactulose determination (0.0028 g 100 mL
1) compared with the official chromatographic method (0.0040 g 100 mL
1). Analytical frequency was also evaluated and, in this case, although sample treatment steps of the two methods allows for preparation of more than one sample at the same time, note that the proposed and optimised step is faster and easier, since it uses of a simple acid solution, followed by centrifugation and filtration (E_1 assay, described in Section 2.4.). Therefore, to evaluate the analytical frequency, we analysed only one milk sample at this point. The CZEUV method showed a reduction in analytical running time of about 13 min, since a chromatographic run is accomplished in 25 min while the electrophoretic run requires about 12 min. Using the official HPLC, with external modelling for quantification, it is possible to quantify lactulose content of a milk sample in 105 min (80 min for the sample preparation step plus 25 min for the chromatographic run). The same sample's lactulose content is determined in 44 min by the optimised CZE-UV method (20 min for sample preparation plus 24 min for the electrophoretic run, assuming two injections per sample since a SPSA quantification method is used). In addition, the CZE-UV method can also quantify the lactose content of the milk sample, both lactose and lactulose concentrations are determined

in about 68 min (20 min for mutual sample preparation plus 24 min for lactulose electrophoretic runs plus 24 min for lactose electrophoretic runs). 3.4. UHT milk sample analysis L-REG samples exhibited very similar electrophoretic profiles as well as the LH samples. Fig. 4 shows electropherograms of L-REG and LH samples of one brand (Brand 01, randomly selected). Since quantification of each isomer was done separately, due to the need for a dilution step for lactose quantification, Fig. 4 indicates the analytical signals obtained for (i) lactulose quantification and (ii) for lactose quantification each milk (L-REG and LH). According to Section 2.5, the final standard concentration in the injected sample extracts was 0.16 g 100 mL
1 for each isomer. The electroosmotic flow (EOF) is characterised by a sudden decay of the analytical signal at the end of the electrophoretic run, and can be seen for all electropherograms (Fig. 4) obtained for (i) lactulose and (ii) lactose quantifications of L-REG and LH samples. From Fig. 4, it is also possible to verify slight shifts in migration times (tm) of the lactulose and lactose isomers occurring under two different conditions: (a) between the different types of matrices, during both lactose and lactulose quantifications [comparing Fig. 4A(i) and 4B(i)]; and (b) intra matrix, during lactose quantification, due to the need for sample extract dilution. Differences in the composition of L-REG and LH matrices can influence the medium ionic force, contributing to the occurrence of these small signal shifts. On the other hand, the dilution step required for lactose quantification in both matrices reduces the mass injected, promoting an easier migration of the analytes mainly due to viscosity reduction (Tavares, 1997). This behaviour was verified for all milk samples evaluated. However, as SPSA is applied, these signal shifts do not compromise the quantification step once this phenomenon occurs equally in both injections (sample and sample plus standard). All tm of the analytes and EOF are shown in Table 4 for each L-REG and LH milk brand. Variability in tm of lactulose and lactose (2.33% and 2.07%, respectively) is close to the EOF tm variation (1.20%) obtained during lactulose determinations, as well as the variations found for the tm of lactose and EOF during lactose determination (3.25% and 3.11%, respectively) in L-REG brands. The same behaviour can be verified for lactulose (tm lactulose ¼ 2.53%; tm EOF ¼ 1.17%) and lactose (tm lactose ¼ 0.58%; tm EOF ¼ 0.40%) determinations in all LH brands. Table 4 also shows the effect of the matrix on tm of the analytes. In this case, similar variations were found between analyte and EOF signals for both lactulose (tm lactulose ¼ 1.62% and tm EOF ¼ 0.81%) and lactose (tm lactose ¼ 2.02% and tm EOF ¼ 1.77%) determinations for all L-REG and LH brands. This indicates that matrix effect acts similarly on analytes and EOF electrophoretic mobilities. Very small deviations were also found when tm of lactose and EOF were evaluated, considering the dilution step used for lactose determinations in L-REG brands (2.32% and 2.07%, for tm lactose and tm EOF, respectively; which considers the tm of lactose and EOF during the lactulose determinations, without dilution, and

8

L.N. de Oliveira Neves, M.A. Leal de Oliveira / International Dairy Journal 106 (2020) 104710

Fig. 4. Electrophoretic profiles for lactulose quantification (i) and lactose quantification (ii) of B_01 samples with regular lactose (A) and hydrolysed-lactose (B). Full line indicates the sample extract signal (S1), and dotted line represents the sample extract with standard addition signal (S2). Analytes are identified as: 1, lactulose; 2, glucose; 3, lactose; 4, galactose. Electrophoretic parameters are as given in Fig. 3. Sample extraction method: method E_1. SPSA method: final standard concentration added in the injected sample extracts was 0.16 g 100 mL
1 for each isomer.

Table 4 Lactulose, lactose and electroosmotic flow (EOF) migration times (tm) of all milk brands.a Sample

L-REG

LH

Dilution Matrix type

Code

B_01 B_02 B_03 B_04 B_05 B_06 Mean ± RSD (%) B_01 B_02 B_03 B_04 B_05 B_06 Mean ± RSD (%) Mean ± RSD (%) Mean ± RSD (%)

Lactulose determinations

sd

sd sd sd

Lactose determinations

tm Lactulose (min)

tm Lactose (min)

tm EOF (min)

tm Lactose (min)

tm EOF (min)

9.75 9.23 9.13 9.44 9.53 9.44 9.42 ± 0.22 2.33 8.88 9.02 9.38 9.38 8.88 9.06 9.10 ± 0.23 2.53 n.a. n.a. 9.28 ± 0.15 1.62

9.98 9.50 9.40 9.66 9.73 9.66 9.66 ± 0.20 2.07 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.a. n.a. n.a. n.a.

10.94 10.82 10.96 10.71 10.65 10.71 10.80 ± 0.13 1.20 10.60 10.62 10.83 10.88 10.59 10.69 10.70 ± 0.12 1.17 n.a. n.a. 10.75 ± 0.09 0.81

10.06 9.50 9.65 9.30 9.08 9.64 9.54 ± 3.25 8.73 8.65 8.64 8.65 8.65 8.75 8.68 ± 0.58 9.60 ± 2.32 9.11 ± 2.02

11.82 11.30 11.56 10.93 10.98 11.56 11.36 3.11 10.44 10.34 10.37 10.36 10.36 10.43 10.38 0.40 11.08 2.07 10.87 1.77

0.31

0.05 0.22 0.18

± 0.35

± 0.04 ± 0.23 ± 0.19

a Mean values, relative standard deviation (RSD) are also reported (n.d., not detected; n.a., not applicable). Samples are as according to product label, where L-REG is for milk matrix with regular lactose content, and LH is for milk matrix with hydrolysed lactose. Dilution is the effect of the sample extract dilution lactose determinations in L-REG milk samples, which considers the tm of lactose and EOF during the lactulose determinations (without dilution), and during lactose determinations (with dilution). Matrix type is the effect of the type of matrix (L-REG or LH) in the tm of lactose, lactulose and EOF for both analyte determinations.

during lactose determinations, with dilution). These findings indicate that both lactose and EOF electrophoretic mobilities are similarly affected when dilution occurs. The effect of the dilution step was verified only for L-REG samples because the lactose peak was not clearly detected during lactulose determinations of LH samples. The lactulose peak was not detected in any sample during lactose determination, probably due to the dilution of the sample

extract. It is important to highlight that, a tm variation in the range of 0.40%e3.25% appears acceptable when dealing with different matrices of high chemical complexity. As the analytical shift occurs in both analytes and EOF, and the SPSA method is used, the quantification step is not compromised since the added standard will be submitted to the same matrix conditions (and so to the shift) of that sample.

L.N. de Oliveira Neves, M.A. Leal de Oliveira / International Dairy Journal 106 (2020) 104710

Lactulose and lactose concentrations calculated for each L-REG and LH milk brand are presented in Table 5. High lactulose quantities in dairy products can be related to over exposure to heat, which makes lactulose an important chemical marker for thermal treatment evaluation (Walstra et al., 2006b). Studies by Cattaneo, Masotti, and Rosi (2008), Elliott, Datta, Amenu, ^pre, and and Deeth (2005), Feinberg, Dupont, Efstathiou, Loua
nez-Pe
rez (2000) Guyonnet (2006), and Morales, Romero, and Jime found lactulose values of 0.0298 and 0.0606 g 100 mL
1, 0.0125 and 0.0466 g 100 mL
1, 0.0144 and 0.0400 g 100 mL
1, and 0.0120 and 0.0353 g 100 mL
1, respectively, for direct and indirect UHT milk. Considering only the L-REG milk samples, our results are in accordance with literature data, exhibiting a range of 0.0194e0.0557 g 100 mL
1. Although UHT process type was not mentioned on the selected product's label, the wide range of lactulose content exhibited by these samples (RSD approximately 41%), can be associated with different combinations of temperature and time exposure to heat, and with different UHT processes (direct and indirect). Nevertheless, all brands showed lactulose levels within the range recommended by IDF 0.0100e0.0600 g 100 mL
1 to be classified as a “UHT product”, suggesting that there was no additional heating during thermal processing. Regarding lactulose content of LH milk samples, it is possible to see in Table 5 that all brands exhibited lower concentrations of this isomer, when they were compared with L-REG samples of the same brand (L-REG B_i versus LH B_i, where i ¼ 1, 2, 3, 4, 5, and 6). At first, the low lactulose content values, ranging from 0.0097 to 0.0197 g 100 mL
1, could indicate the use of a milder heat treatment, taking into account the lactulose range for UHT processing suggested by the IDF. Also, some reports in the literature have also demonstrated low levels of lactulose formation in LH milk compared with L-REG milk samples, when both were submitted to the same heating conditions (Messia et al., 2007; RuizMatute et al., 2012; Sakkas, Moutafi, Moschopoulou, & Moatsou, 2014). These findings indicate that the classical lactulose threshold for L-REG UHT milk is not appropriate for LH UHT milk. It is worth pointing out that, despite the lower lactulose values in LH samples compared with those of the L-REG samples, when the percentage of lactulose formation is evaluated with regard to the lactose content available, the LH samples exhibit higher percentages of lactulose than the L-REG samples. This means that the average lactulose concentration of 0.0356 g 100 mL
1 and 0.0130 g 100 mL
1 found in L-REG and LH samples, respectively, corresponds to approximately 0.7% and

9

10% of the available lactose (considering the mean concentration of 4.74 g 100 mL
1 and 0.1070 g 100 mL
1, respectively) in each matrix. A study by Messia et al. (2007) evaluated 14 commercial samples of LH UHT milk for their lactose, lactulose, glucose and galactose contents. Three of the fourteen samples, with the lowest lactose values (0.4, 0.3 and 0.3 g 100 mL
1, respectively), had lactulose amounts of 0.0401, 0.0309, and 0.0265 g 100 mL
1, showing the same proportion of lactulose:lactose as achieved by our LH milk samples. The authors attributed these results to the activity of b-D-galactosidase, added after the thermal treatment, as mentioned by the producer in the product's label, since the highest levels of galactose and glucose were also identified in these samples, thus confirming that such monosaccharides were not exposed to heating. If the milk heating step occurs before the lactose hydrolysis, the lactulose formed is hydrolysed by the action of the added microbial b-D-galactosidase. On the other hand, if the product is heated after the lactose hydrolysis, lactulose cannot be formed in appreciable amounts (Mendoza, Olano, & Villamiel, 2005; Ruiz-Matute et al., 2012). In both cases, use of lactulose as a thermal indicator becomes inconclusive, since its formation will not only be related to the time and temperature of exposure but also to the degree of lactose hydrolysis achieved and, therefore, it is not recommended to infer information about heat damage in LH dairy matrices. Additionally, considerable increases in nutritional loss have been identified in LH milk when compared with L-REG, due to the high reactivity of monosaccharides released during the hydrolysis process, which indicates that heating prior to hydrolysis is most appropriate (Mendoza et al., 2005; Messia et al., 2007). As an alternative, other thermal indicators have been suggested for LH milk and dairy products. Glucose isomerisation to fructose in milk samples was confirmed by an increase of more than 0.050 g 100 mL
1 in fructose concentration when glucose was added to pasteurised milk subsequently submitted to sterilisation (Messia et al., 2007). The same study identified a slight increase in fructose when thermal treatment occurred before lactose hydrolysis, as a result of b-D-galactosidase action since it also hydrolyses the lactulose formed during the previous heating step. However, when lactose hydrolysis was performed before the heating treatment, higher quantities of fructose were obtained (0.42 mg 100 mL
1 versus 7.61 mg 100 mL
1 of fructose in milk hydrolysed after heating and milk hydrolysed before heating, respectively).

Table 5 Lactulose and lactose concentrations with the respective k values for all regular and hydrolysed-lactose UHT milk samples.a Matrix type

L-REG

LH

Code

B_01 B_02 B_03 B_04 B_05 B_06 Mean (±sd) RSD (%) B_01 B_02 B_03 B_04 B_05 B_06 Mean (±sd) RSD (%)

Lactulose

Lactose

(g 100 mL
1)

k (mAU mL mg
1)

(g 100 mL
1)

k (mAU mL mg
1)

0.0341 0.0201 0.0557 0.0364 0.0194 0.0477 0.0356 40.73 n.q. 0.0097 0.0124 0.0197 n.q. 0.0104 0.0130 35.38

21.34 24.17 24.64 23.08 23.72 23.66 23.44 4.91 n.q. 24.30 24.25 26.64 n.q. 25.20 25.10 4.46

4.78 (±0.02) 4.80 (±0.00) 4.63 (±0.01) 4.70 (±0.02) 4.71 (±0.00) 4.80 (±0.01) 4.74 (±0.07) 1.47 0.1177 (±0.0003) 0.0967 (±0.0003) 0.1174 (±0.0002) 0.1065 (±0.0004) 0.0964 (±0.0002) 0.1071 (±0.0001) 0.1070 (±0.0090) 8.41

26.80 24.74 23.91 25.77 26.31 26.58 25.69 4.44 23.18 23.09 23.96 23.80 20.61 23.47 23.02 5.34

(±0.0006) (±0.0000) (±0.0031) (±0.0002) (±0.0001) (±0.0010) (±0.0145)

(±0.0000) (±0.0008) (±0.0002) (±0.0002) (±0.0046)

(±0.26) (±0.05) (±0.16) (±0.53) (±0.52) (±1.61) (±1.15)

(±0.98) (±1.47) (±1.38) (±0.25) (±1.12)

(±0.62) (±0.02) (±3.80) (±0.04) (±0.08) (±1.93) (±1.14) (±0.56) (±1.10) (±0.46) (±0.53) (±1.48) (±1.40) (±1.23)

a Matrix type is as according to product label, where: L-REG is for milk matrix with regular lactose content, and LH is for milk matrix with hydrolysed lactose. For lactose and lactulose determinations, standard deviation in parenthesis (n ¼ 2, authentic duplicates; n.q., not quantified). Means and RSD are global values considering all analysed samples (n ¼ 12) of each matrix.

10

L.N. de Oliveira Neves, M.A. Leal de Oliveira / International Dairy Journal 106 (2020) 104710

Ruiz-Matute et al. (2012) also used isomerisation of glucose to fructose and that of galactose to tagatose to infer about the heat severity of lactose-free UHT milks, where appreciable amounts of such isomers were found (fructose ranging from 7.6 to 18.5 mg 100 mL
1 and tagatose varying from 3.6 to 9.8 mg 100 mL
1). According to the authors, the presence of tagatose in lactose-free products indicates that the hydrolysis process occurred prior to thermal treatment, since there are no reports on the incidence of tagatose in L-REG UHT milk. Despite fructose and tagatose appearing to be good alternative indicators for LH milk, their formation is always associated with the degree of lactose hydrolysis which denotes the necessity to fix appropriate thresholds for such indicators and even to assess other classes of markers, e.g., whey protein nitrogen index (WPNI) or b-lactoglobulin, to reliably understand heat damage in such dairy products. Analytical quantification of lactose is used for product characterisation and quality control and critical for lactose-free dairy products. The mean lactose concentration found in LH milk samples was 0.1070 g 100 mL
1 (Table 5), corresponding to a lactose hydrolysis level of about 97%, assuming an average of 4.75 g 100 mL
1 of lactose generally found in cows' milk. Since regulatory agencies established a tolerance of ±20% (Brasil, 2003), a range of 0.08e0.12 g 100 mL
1 of lactose, in the final product is allowed. Thus, all LH milk samples were classified as “lactose-free”. Lactose variability in these samples was approximately 9% (Table 5), which is possibly associated with variations in hydrolysis process, such as enzyme activity, temperature and time of hydrolysis, and the analytical control of this process by the dairy industry. With regard to the lactose content of L-REG milk samples, a mean value of 4.74 g 100 mL
1 was found. Our results indicated low variability in this disaccharide content (approximately 1.5%), which was expected since lactose is the cows' milk constituent less modification during its biochemical production (Fox & McSweeney, 1998). The slope (k) values experimentally obtained for the quantification of both disaccharides showed a maximum variability of approximately 5% (RSDlactulose ¼ 4.91% and 4.46%; RSDlactose ¼ 4.44% and 5.34% for L-REG and LH matrices, respectively). These low variations in k indicate that the SPSA quantification method is reliable and emphasises the absence of matrix effect interference in analyte quantification. 4. Conclusion The proposed and optimised CZE-UV method can be used to make inferences about the thermal processing applied to regular milk, through determination of its lactulose amounts, or to verify its lactose content, helping in its characterisation and classification according to content of this disaccharide. Additionally, the proposed method can be used for lactose content monitoring during the hydrolysis in LH milk production due to its high analytical frequency and low LOD. The CZE-UV method is also an analytical alternative for regulatory agencies to monitor lactose content of LH dairy products sold as “lactose-free”, to protect consumers and product quality. Thus, the method in this work presents inherent advantages, in comparison with traditional methods, such as ecofriendly behaviour, a comprehensive portfolio of dairy product applications (L-REG and LH milk), high throughput, and no need for a specific separation column. Acknowledgements The authors thank to the Conselho Nacional de Desenvolvi
gico (CNPq), Instituto Nacional de mento Científico e Tecnolo ^ncia e Tecnologia de Bioanalítica (INCTBio e Fundaça ~o de Cie Amparo  a Pesquisa do Estado de S~ ao Paulo (FAPESP) grant No. 2014/

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