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.

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1 Detection of lactose in products with low lactose content

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

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

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

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E-mail address: charlotte.egger@agroscope.admin.ch (L. Egger)

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12 _________________________________________________________________________

13 ABSTRACT

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

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

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

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

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

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

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

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

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26 1. Introduction

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28 Dairy products significantly contribute to a balanced diet because of their beneficial

29 macro- and micronutrient composition, including high-quality proteins and high calcium

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

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

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

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33 people with lactase intolerance and reduced lactose metabolism is increasing.

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

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

36 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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38 formation of gaseous metabolites such as hydrogen or methane, which may lead to

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

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40 Campos Mazo, & Carrilho, 2012).

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

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

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

45 However, quantification of low levels of lactose in these products is often hindered by the
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46 high levels of glucose and galactose released by lactase treatment.

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

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

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

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

51 not clearly declared.

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

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

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

55 in dairy products.

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57 2. Materials and methods

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59 2.1. Selection of dairy products

60

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61 In total, 121 dairy products were selected from the market. Lactose was quantified in

62 all products and, depending on the amount, different methods were applied. All chemicals

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

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65 2.2. Sample preparation

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

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68 shaking water bath for 20 min. They were clarified with Carrez solutions (Carrez I: 1.25 mL
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69 85 mmol L-1 potassium hexacyanoferrate(II) trihydrate; Carrez II: 1.25 mL 250 mmol L-1 zinc
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70 sulphate heptahydrate; Carrez III, 1.25 mL 0.2 mol L-1 NaOH) and diluted according to

71 product type: 1:100 for yoghurt, milk, curd cheese, and cream and 1:10 for hard-, semi-hard,
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72 and soft cheeses, butter, and cottage cheese. Samples were cooled at 2 °C for 20 min,

73 filtered through a Whatman filter (S&S 5892), and kept at −20 °C prior to analysis.
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75 2.3. Analytical methods for lactose quantification

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77 Depending on the lactose concentration and whether the product was liquid or solid,

78 different analytical methods were applied to quantify lactose.

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80 2.3.1. Sensitive analysis of lactose in dairy products with lactose levels < 0.2 g 100 g-1

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81 Free glucose was oxidised with glucose oxidase in the presence of catalase (Fig. 1a)

82 prior to lactose hydrolysis with β-galactosidase and transformation of glucose to glucose-6-

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

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

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

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

87 After addition of 10 mL H2O, samples were homogenised at 15,000 rpm for 60 s,

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88 followed by two more additions of 10 mL H2O and subsequent homogenisation (15,000 rpm

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

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

91 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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93 Sigma). Samples were incubated at 37°C overnight, filtered through Amicon ultra units (30

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

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95 galactosidase (1,500 U mL-1) was mixed with hydrolysis buffer (66 mmol L-1 phosphate
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96 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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97 shaking water bath at 37 °C for 90 min.

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

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

100 containing 10.14 mmol L-1 Mg2+), 0.05 mL 82.6 mmol L-1 ATP (Roche, Basel, Switzerland),
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101 and 0.05 mL 10 mg mL-1 NADP (Roche). Then, it was incubated at room temperature (RT)
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102 for 6 min before absorption A1 (340 nm) was measured. Next, 10 µL 3 mg mL-1

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

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

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

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

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108 2.3.2. Analysis of lactose in solid products with lactose levels > 0.2 g 100 g-1
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109 For solid samples, lactose was analysed with a less sensitive enzymatic method (limit

110 of quantification, LOQ, = 0.03 g 100g-1 and repeatability, r, = 0.033 g 100g-1). Samples were

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

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

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

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

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

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

117 610 nm after incubation at RT for 60 min in the dark. Lactose concentration was calculated

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

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

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121 2.3.3. Analysis of lactose in liquid dairy products with lactose levels > 0.2 g 100 g-1
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122 In liquid samples, analysis was performed with a Microlab EFA pH differential

123 instrument using the method provided by the manufacturer (Glucose/Lactose Kit MEA678,
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124 BioControl, Endotell, Basel, Switzerland), with a detection limit of 0.5 g 100g-1 and a
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125 repeatability of r = 0.7 g 100 g-1.

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127 2.4. Replication and representation of data

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129 The number (N) of individual samples analysed for each dairy product is indicated in
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130 Table 1. Samples were analysed in duplicate. Standard deviations (SDs) were determined

131 for all individual samples and replicates.

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133 3. Results and Discussion

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135 3.1. Development of a method for lactose quantification in lactose-free dairy products

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

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

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

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

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

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

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

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

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

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

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

148 upper (125%) limit of 3 * SD (Fig. 2). Moreover, the repeatability of the method was 0.057 g
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149 kg-1 based on 45 duplicate measurements of dairy products with lactose values above the

detection limit and below 2 g kg-1.

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152 3.2. Quantification of lactose in dairy products

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154 Lactose was quantified in several different dairy products. The values (g kg-1) were
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155 determined by duplicate analysis and SD (Table 1). The most common types of ripened

156 cheese had lactose concentrations below the detection limit of 24 mg kg-1 and can be
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157 considered naturally lactose-free due to bacterial fermentation processes (Table 1). Only one
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158 cheese (Saint-Paulin) had detectable lactose content (0.03 g kg-1), which was still below the

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

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

161 concentrations were measured in all products and are summarised in Supplementary

162 material Table S1.

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164 Conclusion

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166 The newly developed enzymatic method is well suited for detection of low-levels of

167 lactose in different dairy products. Moreover, this survey confirmed that all ripened cheeses

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

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

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170

171 Acknowledgement

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173 The authors would like to acknowledge the assistance of Agroscope Laboratories for

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174 the analytical work.

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

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

182 Pyrococcus furiosus. BMC Biotechnology, 13, Article 73.

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183 Lortal, S. (2004). Cheeses made with thermophilic lactic starters. In Y. H. Hui, L. Meunier-

184 Goddik, J. Josephsen, W.-K. Nip & P. S. Stanfield (Eds.), Handbook of food and
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185 beverage fermentation technology (Vol. 1, pp. 298–299). Boca Raton, FL, USA: CRC
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186 Press.

187 Mattar, R., de Campos Mazo, D. F., & Carrilho, F. J. (2012). Lactose intolerance: diagnosis,

188 genetic, and clinical factors. Clinical and Experimental Gastroenterology, 5, 113–121.

189 McSweeney, P. L. (2004). Biochemistry of cheese ripening. International Journal of Dairy

190 Technology, 57, 127–144.

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

192 Gärungsvorgänge in der milchwirtschaftlichen Technologie. Lebensmittel-

193 Wissenschaft und Technologie, 8, 6.

194 Suchy, F. J., Brannon, P. M., Carpenter, T. O., Fernandez, J. R., Gilsanz, V., Gould, J. B., et

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

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

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1 Figure legends

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

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

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

7 800 mg kg-1): , milk, milk powder, lactose free-milk; , diverse dairy products; , yoghurt;

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8 , fresh cheeses; , soft cheeses; , semi-hard and hard cheeses. Middle line: average

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

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Table 1

Lactose concentrations in dairy products.

Product (N) Lactose Product (N) Lactose

(g kg-1) (g kg-1)
Hard cheese Lactose-free (LF) products
Emmentaler Switzerland DOP (4) LOD LF Cottage cheese (3) 0.37±0.14
Gruyère DOP (2) LOD LF Skim curd cheese (0.1% fat) (2) 0.51±0.25
Sbrinz DOP (2) LOD LF Yoghurt (3.5% fat) (2) 0.18±0.03
Switzerland Swiss (2) LOD LF Whole milk (3.5% fat) (2) 0.40±0.17

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Mountain cheese (2) LOD LF Semi skim milk (1.5% fat) (1) 0.25±0.00
LF Drink milk (2.5% fat) (1) 1.41±0.03
Semi-hard cheese LF Coffee cream (15% fat) (2) 0.55±0.03

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Appenzeller (2) LOD LF Half cream (25% fat) (2) 0.30±0.11
Vacherin Fribourgeois DOP (2) LOD LF Cream (35% fat) (2) 0.26±0.02
St. Paulin (2) 0.3±0.1 LF Butter (1) 0.01±0.02

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Tête de Moine DOP (2) LOD LF Butter, reconstituted (1) LOD
Tilsiter Switzerland-Past (2) LOD
Tilsiter Switzerland red (1) LOD Yoghurt
Tilsiter Switzerland surchoix (1) LOD Yoghurt, low fat (0.1% fat) (2) 33.83±1.65

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Raclette (2) LOD Yoghurt, semi skim (1.5 fat) (3) 28.97±3.99
Cream cheese (2) LOD Yoghurt, whole milk (3.5% fat) (3) 33.10±4.60
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Yoghurt Greek style (10% fat) (3) 30.27±3.74
Soft cheese Bifidus yoghurt semi skim (1.5% fat) (1) 42.35±0.21
Vacherin Mont-D'Or DOP (2) LOD Bifidus yoghurt (3.5% fat) (2) 32.08±0.46
Limburger (2) LOD
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Tomme (3) LOD Milk, cream, butter

Brie (45% fat) (2) LOD Skim milk (0.1% fat) (3) 49.65±0.21
Brie (60% fat) (1) LOD Semi skim milk (1.5% fat) (2) 48.93±0.40
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Camembert (60% fat) (2) LOD Drink milk (2.5% fat) (5) 48.35±0.21
Camembert (45% fat) (2) LOD Whole milk (3.5% fat) (8) 47.67±0.31
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Feta type cheese (Switzerland) (1) LOD Coffee cream (15% fat) (3) 41.25±1.33
Half cream (25% fat) (3) 37.32±0.83
Fresh cheese Cream (35% fat) (3) 33.00±1.51
18.24±5.95 7.50±1.27
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Cottage cheese (6) Butter (2)

Mozzarella (2) 7.43±1.71
Skim curd cheese (3) 46.40±8.59
Semi-skim curd cheese (2) 44.25±11.3
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Cream curd cheese (3) 36.18±2.61

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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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