Prebiotic skimmed UHT milk: advances in enzimatic conversion of lactose to galacto-oligosaccharides / Leite UHT numerado prebiotico: avanços na conversão enzimática de lactose a galacto-oligosacaridas

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Braz. J. of Develop., Curitiba, v. 6, n. 5, p. 25898-25908 may. 2020. ISSN 2525-8761

Prebiotic skimmed UHT milk: advances in enzimatic conversion of lactose to

galacto-oligosaccharides

Leite UHT numerado prebiotico: avanços na conversão enzimática de

lactose a galacto-oligosacaridas

DOI:10.34117/ bjdv6n5-155

Recebimento dos originais: 20/04/2020 Aceitação para publicação: 09/05/2020

Priscila Barbosa Bezerra Nunes

Mestra em Ciência e Tecnologia dos Alimentos Instituição: Universidade Federal Rural de Pernambuco

Endereço: Rua Dom Manuel de Medeiros, s/n - Dois Irmãos, Recife - PE, 52171-900 E-mail: priscilabbnunes@hotmail.com

Samara Alvachian Cardoso Andrade

Doutora em Nutrição pela Universidade Federal de Pernambuco Instituição: Universidade Federal de Pernambuco

Endereço: Rua Heitor Maia Filho 52/402 Madalena Recife -PE - Brasil, CEP: 50720-525 E-mail: samara.alvachian@gmail.com

Andrelina Maria Pinheiro Santos

Doutora em Engenharia de Alimentos pela Universidade Estadual de Campinas Instituição: Universidade Federal de Pernambuco

Endereço: Departamento Engenharia Química- Universidade Federal de Pernambuco. Av. dos Economistas, Cidade Universitária - Recife - PE, Brasil

E-mail: lia_pinheiro@yahoo.com

Tania Maria Sarmento da Silva

Doutora em Química pela Universidade Federal Rural de Pernambuco Instituição: Universidade Federal Rural de Pernambuco

Endereço: Rua Dom Manoel de Medeiros s/n, Dois Irmãos, Recife-PE-Brasil E-mail: sarmentosilva@gmail.com

Girliane Regina da Silva

Doutora em Desenvolvimento e Inovação Tecnológica em Medicamentos Instituição: Faculdade Santíssima Trindade – FAST

Endereço: Rua Profº. Américo Brandão, 46, Centro, Nazaré da Mata – PE, Brasil (institucional). E-mail: girlianeregina@gmail.com

Erilane de Castro Lima Machado

Doutora em Nutrição pela Universidade Federal de Pernambuco. Instituição: Universidade Federal de Pernambuco (UFPE). Endereço: Centro Acadêmico de Vitória – UFPE, Rua Alto do

Reservatório, s/n – Bela Vista, Cidade – Vitória de Santo Antão/PE, Brasil E-mail: erilanevet@hotmail.com

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Braz. J. of Develop., Curitiba, v. 6, n. 5, p. 25898-25908 may. 2020. ISSN 2525-8761 ABSTRACT

Galacto-oligosaccharides (GOS) are oligosaccharides with prebiotic properties derived from lactose. The milk has a favorable pH for the action of the β-galactosidase enzyme produced by Kluyveromyces

lactis and in this way may favor the hydrolysis of lactose and the synthesis of GOS. This work aimed to

investigate the efficiency of the enzymatic conversion of lactose to GOS in skimmed UHT milk to obtain a dairy raw material with the presence of GOS and reduced lactose content. The best conditions of lactose and enzyme concentrations, and temperature, by enzymatic conversion of lactose, through surface response methodology following a 2³ experimental design (data not published), were investigated in reaction systems during the period of time inferior to that previously performed to favor reduced lactose content and the formation of GOS in skim milk. The carbohydrate analysis was performed by HPLC-IR (High Performance Liquid Chromatography- Refractive Index Detector). A skimmed dairy product was obtained with reduced lactose content (0.66% w/v) and GOS (0.96% w/v). The process was executed in 1.5 hours, at 30° C, with 10% lactose (w/v), and 5 U.mL-1 of enzyme. Once the process was controlled, it was possible to develop dairy products with prebiotics that feature health benefits.

Keywords: β-galactosidase, Kluyveromyces lactis, Transgalactosilation. RESUMO

Galacto-oligossacarídeos (GOS) são oligossacarídeos com propriedades prebióticas derivadas da lactose. O leite possui um pH favorável à ação da enzima β-galactosidase produzida por Kluyveromyces lactis e, dessa forma, pode favorecer a hidrólise da lactose e a síntese de GOS. Este trabalho teve como objetivo investigar a eficiência da conversão enzimática de lactose em GOS em leite desnatado UHT para obter uma matéria-prima láctea com a presença de GOS e conteúdo reduzido de lactose. As melhores condições de concentração de lactose e enzimas, e temperatura, por conversão enzimática da lactose, por meio de metodologia de resposta de superfície seguindo um delineamento experimental de 2³ (dados não publicados), foram investigadas em sistemas de reação durante um período de tempo inferior ao anteriormente realizado para favorecer conteúdo de lactose reduzido e formação de GOS no leite desnatado. A análise de carboidratos foi realizada por HPLC-IR (Cromatografia Líquida de Alto Desempenho - Detetor de Índice de Refração). Um produto lácteo desnatado foi obtido com conteúdo de lactose reduzido (0,66% p / v) e GOS (0,96% p / v). O processo foi executado em 1,5 horas, a 30 ° C, com 10% de lactose (p / v) e 5 U.mL-1 de enzima. Uma vez controlado o processo, foi possível desenvolver produtos lácteos com prebióticos que trazem benefícios à saúde.

Palavras-chave: β-galactosidase, Kluyveromyces lactis, Transgalactosilação.

1 INTRODUCTION

A significant portion of the world's lactose intolerant populations suffers from symptoms such as gas, bloating, and diarrhea. In view of this, the increasing consumption of milk and/or lactose-free or lactose reduced-content products has become popular in many parts of the world, increasing the number of dairy industries that incorporated these items into their line of production (Adhikari, et al., 2010; Wooten, 2010).

β-galactosidases (EC 3.2.1.23) are enzymes that can hydrolyze lactose and are widely used in the dairy industry (Rodriguez-Colinas, et al., 2014). In addition, these enzymes can catalyze a

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transgalactosylation reaction and synthesize prebiotic compounds, GOS (Parh & Oh, 2010; Torres, et

al., 2010). In recent times, great attention has been placed on functional foods and prebiotics in the field

of food technology and the synthesis of galacto-oligosaccharides gained interest based on their prebiotic properties (González, et al., 2008). The enzyme source and reaction conditions (lactose concentration, water activity, temperature, pH, and etc.) influence the yield and composition of the synthesized GOS (Iqbal, et al., 2010; Urrutia, et al., 2013).

The milk’s pH (approximately 6.7) is adequate for the activity of many β-galactosidases. However, the formation of GOS during the treatment of milk with β-galactosidase is still poorly described (Puri, et al., 2010; Ruiz-Matute, et al., 2012; Colinas, et al., 2012; Rodriguez-Colinas, et al., 2014) likely because the lactose content in milk is about 5% (w/v), which is significantly lower compared to the typically reported lactose solutions (15-50%, w/v) used to promote the transgalactosylation reaction (Shen, et al., 2011; Padilla, et al., 2012; Rodriguez-Colinas, et al., 2012; Vera, et al., 2012; Guerrero, et al., 2013; Palai & Bhattacharya, 2013).

The present study verified the efficiency of enzymatic conversion of lactose into GOS in skimmed UHT milk, investigating the best conditions to obtain a dairy raw material containing GOS and reduced lactose content. The addition of lactose to milk, which has been little investigated, is one of the main points of observation in this study. The possibility of offering a dairy product with prebiotic features and reduced lactose content could be of interest in the dairy market in view of the development of products with these same functionalities and health benefits.

2 MATERIAL AND METHODS

The β-galactosidase Lactozyme 2600L G3665 enzyme, from Kluyveromyces lactis, was purchased from Sigma-Aldrich® (São Paulo, SP, Brazil). The o-nitrophenyl-β-galactopyranoside (o-NPG) substrate was purchased from AMRESCO LLC (Solon, OH, USA). The monohydrate lactose used was from Himedia© (Curitiba, PR, Brazil). The HPLC-IR standards used for glucose, galactose, and lactose were from Sigma-Aldrich® (São Paulo, SP, Brazil) and those for 6-galactobiose and 6'-galactosyl-lactose were from Carbosynth, San Diego, CA, USA).

2.1 DETERMINATION OF ENZIME ACTIVITY

The β-galactosidase activity was determined using the o-NPG substrate hydrolysis method with adaptation to the reaction time (Lima, et al., 2013). Briefly, 100 μL of the diluted enzyme was added to test tubes containing 4 mL of 1.25 mM o-NPG solution in 50 mM potassium phosphate buffer solution and maintained at 37 °C for one minute. The reaction was stopped by immersing the tubes in boiling

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water for 5 minutes and then in an ice bath for 5 minutes. The concentration of o-NP was determined in a spectrophotometer (Shimadzu Europe 1650PC) at 420 nm. One β-galactosidase unit is defined as the amount of enzyme needed to releases 1 μmol of o-NP per minute under the assay’s conditions. The enzyme used in this study had an average activity of 18.26 U.mL-1_.

2.2 DETERMINATION OF THE COMPOSITION AND PHYSICAL-CHEMICAL CHARACTERISTICS OF THE SKIMMED UHT MILK

The composition and some physicochemical characteristics of the skimmed UHT milk including fat, total solids, solid-not-fats and lactose content were determined using the Master Mini - Milk Analyzer ultrasonic instruments from Akso Electronic Products (São Leopoldo, RS, Brazil) duly calibrated. The pH was determined by a previously calibrated digital pH meter. These analyses were undertaken before the enzymatic reaction to determine the amount of lactose to be added to the milk, according to the proposed planning.

2.3 LACTOSE HYDROLYSIS AND SYNTHESIS OF GOS

The reaction system was composed of skimmed UHT milk, lactose powder, and enzyme, initially following the experimental design (23 factorial) in the preliminary step with eleven assays (data not published). Based on the statistical analysis of the results obtained in the preliminary step, a deeper investigation of the enzymatic GOS synthesis was repeated using three best assays, in the conditions that previously presented the highest levels of GOS synthesis performed by different conditions of lactose concentration (p/v), enzyme concentration (U.mL-1_{) and temperature (ºC), but at time intervals}

of 30 min during 2 hours of reaction. The flasks were placed in a rotating incubator, kept at each testing constant temperature, and under agitation (200 rpm). Then, 4 mL aliquots were taken of each reaction system and incubated in a thermal bath at 100 °C for 5 minutes to inactivate the enzyme, and subsequently placed in an ice bath for 5 minutes.

2.4 CARBOHYDRATE QUANTIFICATION BY HPLC-IR

The analyses of lactose, glucose, galactose, 6'-galactosyl lactose, and 6-galactobiose were performed by HPLC-IR. A Shimadzu Prominence LC-20AT liquid chromatography (Shimadzu, Kyoto, Japan) was used. It consisted of an LC-20AT quaternary pump, DGU-20 as degasser, CTO-20AC column oven, RID-10A refractive index detector, auto injector (SIL-20A), and CBM-20A communication module, controlled by the LcSolution software. A Phenomenex Rezex RCM - Monosaccharide Ca2+ column (8%) (300 mm x 7.8 mm x 8 μm id) was used at 85 °C. The mobile phase

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used was ultra-pure water with 5% (v/v) methanol at a flow rate was 0.6 mL.min-1. The injected volume was of 10 μL for standards and samples.

Prior to HPLC analysis the samples were extracted according to Silveira et al. (2015). Briefly, 2 mL of the sample were placed in Eppendorf tubes containing 120 μL of Carrez I solution (15.2 g of potassium ferrocyanide trihydrate in 100 mL of ultrapure water) and 120 μL of Carrez II solution (29.9 g zinc sulfate heptahydrate in 100 mL of ultrapure water); these tubes were centrifuged at 12,000 rpm for 30 minutes and the collected supernatant was diluted (1:4) and filtered in 0.45 μm nylon filters.

The conversion of lactose and synthesis of GOS was calculated by Equation 1 and 2 as proposed by Hsu, Lee and Chou (2007) where Laci = initial lactose concentration; Lact = final lactose

concentration; GOSt = final GOS concentration; Glit = final glucose concentration; Galt = final galactose

concentration:

(Equation 1)

2.5 STATISTICAL ANALYSIS

The results were submitted to analysis of variance (ANOVA) and the means were compared using the Duncan test at 5% probability. The computational program Statistic for Windows 7.0 (STATSOFT, 2004) was used.

3 RESULTS AND DISCUSSION

3.1 ANALYSIS OF THE BEST GOS SYNTHESIS CONDITIONS

Figure 1 shows the best GOS synthesis results from assays 1, 2, and 3, the lactose hydrolysis behavior, and the GOS synthesis by β-galactosidase in skimmed milk, at time intervals of 30 minutes to 2 hours of reaction. A maximum of 0.96% (w/v) of total GOS was achieved in skimmed milk in 1.5 hours of reaction under the conditions of assay 1 with 93.30% of lactose conversion. The conditions in assay 3 allowed reaching a total GOS concentration of 0.80% (w/v) in the same reaction time and with 95.42% lactose reduction. After 1.5 hours of reaction time, there was a tendency to reduced GOS concentration, indicating the predominance of hydrolysis by β-galactosidase (Figure 1).

Conversion of lactose (%) = Laci – Lact x 100 Laci

Rendimento de GOS (%) = GOSt x 100 Glit + Galt + Lact + GOSt

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The maximum GOS synthesis (0.83%, w/v) occurred in a shorter time (1 hour) in assay 2, with the same lactose concentration used in the other assays (10%, w/v), higher enzymatic concentration (10 U.mL-1_{), at 30 °C, and with 0.57% (w/v) of lactose present in the skim milk. After this time, a}

progressive reduction in total GOS concentration was observed (Figure 1).

The higher concentration of total GOS achieved in assay 2, obtained in a shorter time compared to the yield in assay 3, implies the influence of enzyme concentration and temperature on the synthesis and decomposition of GOS over the reaction time. The temperature of 40 °C (assay 3) appeared to disadvantage the transgalactosylation reaction in view of the lower GOS yield obtained, relative to that achieved at 30 °C (assay 2).

Figure 1 - Lactose concentrations and total GOS (%, w/v) in UHT skimmed milk samples by CLAE-IR. Assays 1 (5% (p/v),

10 U.mL-1_{, 30 ºC), 2 (10% (p/v), 10 U.mL}-1_{, 30 ºC), and 3 (5% (p/v), 10 U.mL}-1_{, 40 ºC), in the different reaction times (0.5,}

1, 1.5, and 2 hours). Lactose concentration = (%, p/v); Enzyme concentration = (U.mL-1_{); Temperature = ºC.}

Guerrero et al. (2015) explain that the enzymatic concentration in milk may interfere with the onset and progress of the secondary hydrolysis in the reaction mixture. The highest enzyme concentration in assay 2 (10 U.mL-1_{) appeared to favor GOS synthesis at shorter reaction times,}

consequently leading to the anticipated degradation of these oligomers. This fact was observed by González-Delgado et al. (2016), who pointed to the concentration of β-galactosidase as a factor of strong influence on the evolution of GOS throughout the reaction time. It was observed that at lower enzyme

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concentrations a longer reaction time was required to achieve adequate GOS yields while increased β-galactosidase concentration led to increased GOS yield, appearing at progressively shorter reaction times.

Using the Biolactase MB-878 enzyme (2740 U.mL-1_{) from Bacillus circulans at a concentration}

of 1.5 U.mL-1_{in skim milk (4.6% w/v lactose), Rodriguez-Colinas et al. (2012) reached a total GOS}

concentration of 0.71% (w/v) after 2 hours of reaction at 40 °C. The results obtained in the present study are similar, however, with higher concentrations of lactose (10%, w/v) and enzyme (10 U.mL-1) in skim milk.

Ruiz-Matute et al. (2012) analyzed the GOS formation in UHT milk at 30 °C with the Lactozym Pure enzyme (6500 U.mL-1) from K. lactis at a concentration of 0.3 U.mL-1. These authors observed a maximum concentration total GOS of about 1% (w/v), in approximately 5 hours of reaction, when 75 to 90% of the lactose content was hydrolyzed, with subsequent gradual decrease to values below 0.5% (w/v) of total GOS when more than 99% of the lactose was hydrolyzed. In the present study, a maximum total GOS concentration of 0.96% (w/v) was reached at 30 °C and 10% (w/v) of lactose in skimmed milk, which according to those authors, was sufficient to feature a beneficial effect on health, with 0.66% (w/v) of residual lactose. This high residual value may be due to the high initial lactose concentration in the milk (10%, w/v).

Rodriguez-Colinas et al. (2014) achieved a maximum concentration of 0.7% (w/v) of total GOS in skimmed milk in 1 hour of reaction using the K. lactis enzyme (Lactozym Pure 6500 L) at 40 °C, with the natural lactose concentration in the milk (4.4-4.6%, w/v). In this study, a similar total GOS (0.80%, w/v) result was achieved in 1.5 hours of reaction, with 10% (w/v) of lactose in skim milk and at 40 ºC.

Two types of GOS, characteristic of K. lactis β-galactosidase, were identified in the analysis for the determination of GOS (Figure 2). The enzyme used here produced one type of trisaccharide (6'-galactosyl lactose) and one type of disaccharide (6-galactobiose). Torres et al. (2010) reported that these β-1,6-linked oligosaccharides are characteristic of enzymes derived from K. lactis. These types of GOS from K. lactis have also been reported by other authors (Padilla, et al., 2012; Ruiz-Matute, et al., 2012; Rodriguez-Colinas, et al., 2014; Frenzel, et al., 2015).

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Figure 2 – HPLC-IR of the skimmed UHT milk sample after 1.5 hours of reaction at 30 ºC, with an enzymatic concentration

of 5 U.mL-1_{and 10% (w/v) of lactose. The peaks correspond to: (1) 6'-galactosyl lactose, (2) lactose, (3) 6-galactobiose, (4)}

glucose, and (5) galactose.

In this study, it was possible to obtain skim UHT milk in a short processing time (1.5 hours) using a low enzymatic concentration (5 U.mL-1_{) and adding lactose to the milk, yielding a total GOS}

concentration of 0.96% (w/v) and low lactose residual content (0.66%, w/v). This level of GOS content is similar to that present in human milk, with the oligosaccharides being basically 6'-galactobiose, allolactose, and 6-o-β-galactosyl-lactose, at concentrations between 0.5 and 1.5% (w/v) (Rodriguez-Colinas, et al., 2014).

Torres et al. (2010) emphasize that milk and infant formulas enriched with GOS alone, or with FOS, can replicate the recognized bifidogenic effect of human milk, contributing to improve the frequency of bowel movements, decrease fecal pH, and stimulate the growth of intestinal bifidobacteria and lactobacilli (Ben, et al., 2008). Different research groups have reported several health benefits that these carbohydrates can offer, and thus, the current main focus among prebiotics is on their production and use as a component in functional foods (Sangwan, et al., 2011).

4 CONCLUSIONS

The enzyme used (5 U.mL-1) was efficient to produce GOS in skimmed UHT milk with 10% (w/v) of lactose in a short reaction time (1 to 2 hours), at 30 °C. This process allowed obtaining a milk-based raw material with prebiotics and reduced lactose content. The experiments performed demonstrated that it is possible to obtain a dairy raw material with GOS and reduced lactose contents for the elaboration of new dairy products displaying prebiotic characteristics. Such products could offer health benefits to the population, especially to those with lactose intolerance.

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Braz. J. of Develop., Curitiba, v. 6, n. 5, p. 25898-25908 may. 2020. ISSN 2525-8761 ACKNOWLEDGEMENTS

This work was financially supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES).

REFERENCES

Adhikari, K.; Dooley, L. M.; Chambers, E.; Bhumiratana, N. (2010). Sensory characteristics of commercial lactose-free milks manufactured in the United States. LWT - Food Science and Technology,

43 (1), 113–118.

Ben, X.-M.; Li, J.; Feng, Z.-T.; Shi, S.-Y.; Lu, Y.-D.; Chen, R.; Zhou, X.-Y.; Xm, B.; Li, J.; Zt, F.; et al. (2008). Low level of galacto-oligosaccharide in infant formula stimulates growth of intestinal Bifidobacteria and Lactobacilli. World Journal of Gastroenterology, 14 (42), 6564–6568.

Frenzel, M.; Zerge, K.; Clawin-Rädecker, I.; Lorenzen, P. C. (2015). Comparison of the galacto-oligosaccharide forming activity of different β-galactosidases. LWT - Food Science and Technololy, 60, 1068–1071.

González, R.; Klaassens, E. S.; Malinen, E.; de Vos, W. M.; Vaughan, E. E. (2008). Differential transcriptional response of Bifidobacterium longum to human milk, formula milk, and galactooligosaccharide. Applied and Environmental Microbioogy, 74 (15), 4686–4694.

González-Delgado, I.; López-Muñoz, M.-J.; Morales, G.; Segura, Y. (2016). Optimisation of the synthesis of high galacto-oligosaccharides (GOS) from lactose with β-galactosidase from Kluyveromyces lactis. International Dairy Journal, 61, 211–219.

Guerrero, C.; Vera, C.; Acevedo, F.; Illanes, A. (2015). Simultaneous synthesis of mixtures of lactulose and galacto-oligosaccharides and their selective fermentation. Journal of Biotechnology, 209, 31–40. Guerrero, C.; Vera, C.; Illanes, A. (2013). Optimisation of synthesis of oligosaccharides derived from lactulose (fructosyl-galacto-oligosaccharides) with β-galactosidases of different origin. Food Chemistry, 138 (4), 2225–2232.

Hsu, C. A.; Lee, S. L.; Chou, C. . C. Enzymatic production of galactooligosaccharides by β-galactosidase from Bifidobacterium longum BCRC 15708. (2007). Journal of Agricultural and Food Chemistry, 55 (6), 2225–2230.

Iqbal, S.; Nguyen, T.-H.; Nguyen, T. T.; Maischberger, T.; Haltrich, D. (2010). β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides. Carbohydrate Research, 345 (10), 1408–1416.

(10)

L. R. B. (2013). Comparative biochemical characterization of soluble and chitosan immobilized β-galactosidase from Kluyveromyces lactis NRRL Y1564. Process Biochemistry, 48 (3), 443–452. Padilla, B.; Ruiz-Matute, A. I.; Belloch, C.; Cardelle-Cobas, A.; Corzo, N.; Manzanares, P. (2012). Evaluation of oligosaccharide synthesis from lactose and lactulose using β-galactosidases from kluyveromyces isolated from artisanal cheeses. Journal of Agricultural and Food Chemistry, 60, 5134– 5141.

Palai, T.; Bhattacharya, P. K. (2013). Kinetics of lactose conversion to galacto-oligosaccharides by β-galactosidase immobilized on PVDF membrane. Journal of Bioscience and Bioengineering, 115 (6), 668–673.

Park, A.R.; Oh, D.K. (2010). Galacto-oligosaccharide production using microbial $β$-galactosidase: current state and perspectives. Appied. Microbioogy and. Biotechnology, 85 (5), 1279–1286.

Puri, M.; Gupta, S.; Pahuja, P.; Kaur, A.; Kanwar, J. R.; Kennedy, J. F. (2010). Cell Disruption Optimization and Covalent Immobilization of $β$-D-Galactosidase from Kluyveromyces marxianus YW-1 for Lactose Hydrolysis in Milk. Appied Biochemistry and Biotechnology, 160 (1), 98–108. Rodriguez-Colinas, B.; Fernandez-Arrojo, L.; Ballesteros, A. O.; Plou, F. J. (2014). Galactooligosaccharides formation during enzymatic hydrolysis of lactose: Towards a prebiotic-enriched milk. Food Chemistry, 145, 388–394.

Rodriguez-Colinas, B.; Poveda, A.; Jimenez-Barbero, J.; Ballesteros, A. O.; Plou, F. J. (2012). Galacto-oligosaccharide Synthesis from Lactose Solution or Skim Milk Using the β-Galactosidase from Bacillus circulans. Journal of Agricultural and Food Chemistry, 60 (25), 6391–6398.

Ruiz-Matute, A. I.; Corzo-Martínez, M.; Montilla, A.; Olano, A.; Copovi, P.; Corzo, N. (2012). Presence of mono-, di- and galactooligosaccharides in commercial lactose-free UHT dairy products. Journal of

Food Composition and Analysis, 28, 164–169.

Sangwan, V.; Tomar, S. K.; Singh, R. R. B.; Singh, A. K.; Ali, B. (2011). Galactooligosaccharides: Novel Components of Designer Foods. Journal of Food Science, 76 (4), R103-11.

Shen, Q.; Yang, R.; Hua, X.; Ye, F.; Zhang, W.; Zhao, W. (2011). Gelatin-templated biomimetic calcification for β-galactosidase immobilization. Process Biochemistry, 46 (8), 1565–1571.

Silveira, M. F.; Masson, L. M. P.; Martins, J. F. P.; Alvares, T. D. S.; Paschoalin, Virginia Margaret Flosi, L. D. L. T.; Conte-Junior, C. A. (2015). Simultaneous determination of lactulose and lactose in conserved milk by HPLC-RID. Journal of Chemistry, 6p.

StatSoft Inc. StatSoft Inc., Statistica: Data Analysis Software System, version 7. (2004).

Torres, D. P. M.; Gonçalves, M. do P. F.; Teixeira, J. A.; Rodrigues, L. R. (2010). Galacto-Oligosaccharides: Production, properties, applications, and significance as prebiotics. Comprehensive

(11)

Reviews in Food Science and Food Safety, 9, 438-454.

Urrutia, P.; Rodriguez-Colinas, B.; Fernandez-Arrojo, L.; Ballesteros, A. O.; Wilson, L.; Illanes, A.; Plou, F. J. (2013). Detailed analysis of galactooligosaccharides synthesis with β-galactosidase from Aspergillus oryzae. Journal of Agricultural and Food Chemistry, 61 (5), 1081–1087.

Vera, C.; Guerrero, C.; Conejeros, R.; Illanes, A. (2012). Synthesis of galacto-oligosaccharides by β-galactosidase from Aspergillus oryzae using partially dissolved and supersaturated solution of lactose.

Enzyme and Microbial Technology, 50 (3), 188–194.

Wooten, W. J (2010). Lactose Intolerance and Ethnic Prevalence. In Lactose Intolerance and Health (p. 49–52). National Institutes of Health: Kensington.