Determinacion De Lactosa.pdf

Accepted Manuscript Detection of lactose in products with low lactose content Doreen Gille, Barbara Walther, René Badertscher, Andreas Bosshart, Cédric Brügger, Maria Brühlhart, Roland Gauch, Priska Noth, Guy Vergères, Lotti Egger PII:

S0958-6946(18)30060-8

DOI:

10.1016/j.idairyj.2018.03.003

Reference:

INDA 4288

To appear in:

International Dairy Journal

Received Date: 20 February 2018 Accepted Date: 6 March 2018

Please cite this article as: Gille, D., Walther, B., Badertscher, R., Bosshart, A., Brügger, C., Brühlhart, M., Gauch, R., Noth, P., Vergères, G., Egger, L., Detection of lactose in products with low lactose content, International Dairy Journal (2018), doi: 10.1016/j.idairyj.2018.03.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Detection of lactose in products with low lactose content

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Doreen Gillea,b, Barbara Waltherb, René Badertscherb, Andreas Bosshartb, Cédric Brüggerb,

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Maria Brühlhartb, Roland Gauchb, Priska Nothb, Guy Vergèresb, Lotti Eggerb*

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Institute for Epidemiology, Biostatistics und Prevention, Hirschengraben 84, 8001 Zurich,

Switzerland b

Federal Department of Economic Affairs, Education and Research EAER, Agroscope,

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Schwarzenburgstr. 161, 3003 Bern, Switzerland

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*Corresponding author. Tel.: +41 58 463 81 65 E-mail address: [email protected] (L. Egger)

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ABSTRACT

14 Quantification of lactose in lactose-free products requires a sensitive method and is often

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hindered by high levels of glucose and galactose that are present after lactase treatment.

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The enzymatic method presented here includes an enzymatic glucose depletion step and

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enables sensitive analysis of low levels of lactose at a detection limit of 25 mg kg-1, with

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repeatability of 57 mg kg-1 and a recovery rate of 94%. This method was used to measure

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lactose levels in different cheese types and residual levels of lactose in lactose-free products.

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Lactose concentrations were below the detection limit in all ripened cheeses, below 0.1% in

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lactose-free products, and highly variable in fresh cheeses and other products.

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1.

Introduction

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Dairy products significantly contribute to a balanced diet because of their beneficial macro- and micronutrient composition, including high-quality proteins and high calcium

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content. As a result, adults’ recommended daily intake of dairy products is 1–3 portions in

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most European countries (FAO, 2016). However, lactose, the main carbohydrate present in

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dairy products, recently gained interest because the demand for lactose-free products by

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people with lactase intolerance and reduced lactose metabolism is increasing.

The lactose molecule comprises two monosaccharides, glucose and galactose, linked

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via a β-1,4 glycosidic bond that, in humans, can only be cleaved by lactase (β-

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galactosidase). In lactose-intolerant subjects, undigested lactose enters the large intestine,

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where it is metabolised by the gut microbiome. This fermentation process causes the

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formation of gaseous metabolites such as hydrogen or methane, which may lead to

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unwanted symptoms, including flatulence, diarrhoea, and abdominal pain (Mattar, de

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Campos Mazo, & Carrilho, 2012).

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Today, many different lactose-free dairy products are generated by lactase treatment technology, resulting in lactose concentrations below 0.1%, and are suitable for people

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suffering from symptoms caused by significantly reduced lactase activity. To guarantee a

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successful hydrolysis process, the level of residual lactose needs to be analysed regularly.

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However, quantification of low levels of lactose in these products is often hindered by the

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high levels of glucose and galactose released by lactase treatment.

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Most cheeses are naturally lactose-free or contain very low levels of lactose because,

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during fermentation by starter bacteria, the lactose is transformed to lactic acid in the initial

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stages of cheese ripening (McSweeney, 2004). However, in many cases, people with lactose

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malabsorption are reluctant to eat cheeses because the lactose levels of those products are

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not clearly declared.

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To overcome these issues, the main objectives of this study are to develop a

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sensitive and precise method for quantification of low levels of lactose in dairy products and 3

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to apply this method to a variety of dairy products to generate an up-to-date survey of lactose

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in dairy products.

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2.

Materials and methods

2.1.

Selection of dairy products

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In total, 121 dairy products were selected from the market. Lactose was quantified in all products and, depending on the amount, different methods were applied. All chemicals

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were purchased from Grogg (Stettlen, Bern, Switzerland) if not otherwise specified.

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2.2.

Sample preparation

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All solid samples were homogenised with 25 mL H2O and incubated at 70 °C in a

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shaking water bath for 20 min. They were clarified with Carrez solutions (Carrez I: 1.25 mL

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85 mmol L-1 potassium hexacyanoferrate(II) trihydrate; Carrez II: 1.25 mL 250 mmol L-1 zinc

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sulphate heptahydrate; Carrez III, 1.25 mL 0.2 mol L-1 NaOH) and diluted according to

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product type: 1:100 for yoghurt, milk, curd cheese, and cream and 1:10 for hard-, semi-hard,

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and soft cheeses, butter, and cottage cheese. Samples were cooled at 2 °C for 20 min,

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filtered through a Whatman filter (S&S 5892), and kept at −20 °C prior to analysis.

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2.3.

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Analytical methods for lactose quantification

Depending on the lactose concentration and whether the product was liquid or solid, different analytical methods were applied to quantify lactose.

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2.3.1. Sensitive analysis of lactose in dairy products with lactose levels < 0.2 g 100 g-1

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Free glucose was oxidised with glucose oxidase in the presence of catalase (Fig. 1a) prior to lactose hydrolysis with β-galactosidase and transformation of glucose to glucose-6-

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phosphate with hexokinase in the presence of ATP. In the presence of glucose-6-phosphate-

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dehydrogenase, glucose-6-phosphate and NADP reacted stoichiometrically to NADPH,

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which was measured at 340 nm (Fig. 1b). The final dilution of samples in H2O was 1:2 for

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liquid samples; 1:5 for yoghurt, curd cheese, and butter; and 1:10 for cheeses.

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After addition of 10 mL H2O, samples were homogenised at 15,000 rpm for 60 s, followed by two more additions of 10 mL H2O and subsequent homogenisation (15,000 rpm

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for 60 s). Samples were then incubated in a shaking water bath at 70 °C for 20 min. For

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glucose oxidation in samples with a high concentration of glucose from lactase treatment, the

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samples were mixed with glucose oxidase (≥ 4,200 U mL-1, Sigma, Buchs, Switzerland),

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0.5% perhydrol (Merck, Buchs, Switzerland), NaOH (0.02 mol L-1), and catalase (300 U,

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Sigma). Samples were incubated at 37°C overnight, filtered through Amicon ultra units (30

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kDa, Millipore, Canton-Schaffhausen, Switzerland). Prior to lactose hydrolysis, β-

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galactosidase (1,500 U mL-1) was mixed with hydrolysis buffer (66 mmol L-1 phosphate

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buffer, pH 6.5, containing 1 mmol L-1 Mg2+), added to 0.2 mL of a sample, and incubated in a

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shaking water bath at 37 °C for 90 min.

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To determine the amount of released glucose, 1 mL of the hydrolysed sample was

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mixed with 0.5 mL tri-ethanolamine-HCl buffer (0.754 mol L-1 tri-ethanolamine-HCl, pH 7.6,

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containing 10.14 mmol L-1 Mg2+), 0.05 mL 82.6 mmol L-1 ATP (Roche, Basel, Switzerland),

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and 0.05 mL 10 mg mL-1 NADP (Roche). Then, it was incubated at room temperature (RT)

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for 6 min before absorption A1 (340 nm) was measured. Next, 10 µL 3 mg mL-1

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hexokinase/glucose-6-phosphate (Roche) solution was added to the sample and the mixture

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was incubated for 12 min prior to measurement of absorption A2 (340 nm). Free glucose was

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determined by analysing each sample without addition of β-galactosidase in parallel. As no

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glucose was expected to be present in the cheese samples, the oxidation step was omitted.

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2.3.2. Analysis of lactose in solid products with lactose levels > 0.2 g 100 g-1 5

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For solid samples, lactose was analysed with a less sensitive enzymatic method (limit of quantification, LOQ, = 0.03 g 100g-1 and repeatability, r, = 0.033 g 100g-1). Samples were

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incubated with β-galactosidase (15 U mL-1) in hydrolysis buffer (66.6 mmol L-1 phosphate

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buffer, pH 6.5, containing 1 mmol L-1 MgSO4) at RT for 30 min, followed by glucose oxidation

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to gluconate and H2O2 by glucose oxidase in GOD-Perid solution {6,900 U L-1 glucose

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oxidase, 0.9 g L-1 2.2’-azino-di-[3ethyl-benzthiazolin-sulphonic acid-(6)]-diammonium salt

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(ABTS), 500 U L-1 peroxidase in phosphate buffer, pH 7.0}. H2O2 reacted stoichiometrically

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with the ABTS chromogen, producing ABTSox and H2O. ABTSox absorption was measured at

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610 nm after incubation at RT for 60 min in the dark. Lactose concentration was calculated

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by using a glucose standard and multiplying the glucose results by 2. Free glucose was

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analysed in a sample run in the absence of β-galactosidase.

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2.3.3. Analysis of lactose in liquid dairy products with lactose levels > 0.2 g 100 g-1

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In liquid samples, analysis was performed with a Microlab EFA pH differential instrument using the method provided by the manufacturer (Glucose/Lactose Kit MEA678,

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BioControl, Endotell, Basel, Switzerland), with a detection limit of 0.5 g 100g-1 and a

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repeatability of r = 0.7 g 100 g-1.

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2.4.

Replication and representation of data

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The number (N) of individual samples analysed for each dairy product is indicated in

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Table 1. Samples were analysed in duplicate. Standard deviations (SDs) were determined

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for all individual samples and replicates.

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Results and Discussion

3.1.

Development of a method for lactose quantification in lactose-free dairy products

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ACCEPTED MANUSCRIPT Lactose quantification in dairy products with low levels of lactose requires a sensitive

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and specific method enabling lactose detection below 0.1%. Enzymatic methods most often

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measure liberated glucose after hydrolysis with β-galactosidase. Such a method is often

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unsuitable for lactose-free products because of the high levels of monosaccharides released

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by lactase during the manufacturing process (Li et al., 2013). This problem was overcome in

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the present method by removing glucose via oxidation to gluconate with glucose oxidase

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(Fig. 1a). Subsequently, glucose released after hydrolysis by β-galactose (Fig. 1b) was

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quantified with high sensitivity, resulting in a detection limit of 0.024 g kg-1. The sensitivity

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and specificity of the method were investigated in recovery experiments with over 70 different

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lactose-free dairy products ranging from 0.06 to 0.7 g kg-1 of added lactose. The average

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recovery was 94% across all included products, and all results were within a lower (64%) and

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upper (125%) limit of 3 * SD (Fig. 2). Moreover, the repeatability of the method was 0.057 g

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kg-1 based on 45 duplicate measurements of dairy products with lactose values above the

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detection limit and below 2 g kg-1.

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Quantification of lactose in dairy products

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Lactose was quantified in several different dairy products. The values (g kg-1) were

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determined by duplicate analysis and SD (Table 1). The most common types of ripened

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cheese had lactose concentrations below the detection limit of 24 mg kg-1 and can be

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considered naturally lactose-free due to bacterial fermentation processes (Table 1). Only one

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cheese (Saint-Paulin) had detectable lactose content (0.03 g kg-1), which was still below the

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level detected in lactose-free products (Table 1). The levels of lactose in fresh cheeses,

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yogurt, and non-fermented products are presented in Table 1. In parallel, macronutrient

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concentrations were measured in all products and are summarised in Supplementary

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material Table S1.

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The newly developed enzymatic method is well suited for detection of low-levels of lactose in different dairy products. Moreover, this survey confirmed that all ripened cheeses

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are naturally lactose free and the products declared as lactose-free that were included in this

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study had levels below 0.1%, with one exception having a lactose concentration of 1.4 g kg-1.

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Acknowledgement

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The authors would like to acknowledge the assistance of Agroscope Laboratories for the analytical work.

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References

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FAO. (2016). Food-based dietary guidelines. Rome, Italy: Food and Agriculture Organisation of the United Nations.

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Li, Wang, Z., Li, S., Donelan, W., Wang, X., Cui, T., & Tang, D. (2013). Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from

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Pyrococcus furiosus. BMC Biotechnology, 13, Article 73.

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Lortal, S. (2004). Cheeses made with thermophilic lactic starters. In Y. H. Hui, L. MeunierGoddik, J. Josephsen, W.-K. Nip & P. S. Stanfield (Eds.), Handbook of food and

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beverage fermentation technology (Vol. 1, pp. 298–299). Boca Raton, FL, USA: CRC

Press.

Mattar, R., de Campos Mazo, D. F., & Carrilho, F. J. (2012). Lactose intolerance: diagnosis, genetic, and clinical factors. Clinical and Experimental Gastroenterology, 5, 113–121. McSweeney, P. L. (2004). Biochemistry of cheese ripening. International Journal of Dairy Technology, 57, 127–144.

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Steffen, C. (1975). Enzymatische Bestimmungsmethoden zur Erfassung der

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Gärungsvorgänge in der milchwirtschaftlichen Technologie. Lebensmittel-

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Wissenschaft und Technologie, 8, 6. Suchy, F. J., Brannon, P. M., Carpenter, T. O., Fernandez, J. R., Gilsanz, V., Gould, J. B., et

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al. (2010). National Institutes of Health Consensus Development Conference: lactose

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intolerance and health. Annals of Internal Medicine, 152, 792–796.

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Figure legends

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Fig. 1. Treatment of products with high free glucose content with glucose oxidase and

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catalase (A) prior to lactose hydrolysis with β-galactosidase (B) after lactase treatment.

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Fig. 2. Lactose recovery rates of different dairy products with low concentrations of lactose (<

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800 mg kg-1):

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, fresh cheeses;

, soft cheeses;

, diverse dairy products;

, yoghurt;

, semi-hard and hard cheeses. Middle line: average

recovery of 94%; upper and lower limits of 3 * SD, 125% and 64% of recovery.

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, milk, milk powder, lactose free-milk;

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Table 1

Lactose concentrations in dairy products. Product (N)

Lactose

Product (N)

Lactose

(g kg-1) Lactose-free (LF) products LOD LOD LOD LOD LOD

Soft cheese Vacherin Mont-D'Or DOP (2) Limburger (2) Tomme (3) Brie (45% fat) (2) Brie (60% fat) (1) Camembert (60% fat) (2) Camembert (45% fat) (2) Feta type cheese (Switzerland) (1)

LOD LOD LOD LOD LOD LOD LOD LOD

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18.24±5.95 7.43±1.71 46.40±8.59 44.25±11.3 36.18±2.61

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Fresh cheese Cottage cheese (6) Mozzarella (2) Skim curd cheese (3) Semi-skim curd cheese (2) Cream curd cheese (3)

0.37±0.14 0.51±0.25 0.18±0.03 0.40±0.17 0.25±0.00 1.41±0.03 0.55±0.03 0.30±0.11 0.26±0.02 0.01±0.02 LOD

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LOD LOD 0.3±0.1 LOD LOD LOD LOD LOD LOD

Yoghurt

Yoghurt, low fat (0.1% fat) (2) Yoghurt, semi skim (1.5 fat) (3) Yoghurt, whole milk (3.5% fat) (3) Yoghurt Greek style (10% fat) (3) Bifidus yoghurt semi skim (1.5% fat) (1) Bifidus yoghurt (3.5% fat) (2)

33.83±1.65 28.97±3.99 33.10±4.60 30.27±3.74 42.35±0.21 32.08±0.46

Milk, cream, butter

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Semi-hard cheese Appenzeller (2) Vacherin Fribourgeois DOP (2) St. Paulin (2) Tête de Moine DOP (2) Tilsiter Switzerland-Past (2) Tilsiter Switzerland red (1) Tilsiter Switzerland surchoix (1) Raclette (2) Cream cheese (2)

LF Cottage cheese (3) LF Skim curd cheese (0.1% fat) (2) LF Yoghurt (3.5% fat) (2) LF Whole milk (3.5% fat) (2) LF Semi skim milk (1.5% fat) (1) LF Drink milk (2.5% fat) (1) LF Coffee cream (15% fat) (2) LF Half cream (25% fat) (2) LF Cream (35% fat) (2) LF Butter (1) LF Butter, reconstituted (1)

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Hard cheese

(g kg-1)

Skim milk (0.1% fat) (3) Semi skim milk (1.5% fat) (2) Drink milk (2.5% fat) (5) Whole milk (3.5% fat) (8) Coffee cream (15% fat) (3) Half cream (25% fat) (3) Cream (35% fat) (3) Butter (2)

49.65±0.21 48.93±0.40 48.35±0.21 47.67±0.31 41.25±1.33 37.32±0.83 33.00±1.51 7.50±1.27

Abbreviations are: N, number of samples tested; LF, product declared as lactose-free. LOD: value below the limit of detection of 0.024 g kg-1. Values are the average ± SD.

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