Biotechnology Letters 24: 17371739, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands.

1737

A quick and simple biostrip technique for detection of lactose

Sandeep K. Sharma1 , Neeta Sehgal2 & Ashok Kumar1,
1 Centre

for Biochemical Technology, Delhi University Campus, Mall Road, Delhi110007, India of Zoology, Delhi University, Delhi110007, India Author for correspondence (Fax: +91-11-7667471; E-mail: ashokcbt@rediffmail.com)
2 Department

Received 18 June 2002; Revisions requested 15 July 2002; Revisions received 15 August 2002; Accepted 23 August 2002

Key words: -galactosidase, biostrip, lactase, lactose, lactose intolerance

Abstract A quick, simple and economical biostrip technology was developed for estimation of lactose by immobilizing galactosidase, galactose oxidase and horseradish peroxidase on to a polymeric support. The biostrip is dipped in milk or milk products and, from the colour that develops from an added chromogen, the concentration of lactose can be estimated from < 20 to 100+g l1 . The biostrips may be used in dairy industries, hospitals and remote areas where expensive instruments are not available.

Introduction The absence or decrease of lactase activity in humans leads to a clinical syndrome lactose intolerance. Lactose intolerance is the inability to digest lactose (Paige et al. 1975). It leads to various physical symptoms such as excessive intestinal gas, nausea, cramps and diarrhea (Suarez et al. 1995). There is no way to prevent the development of lactose intolerance. However, avoiding and restricting the milk products in the diet can eliminate the symptoms. Several types of biosensors involving different enzymes are available for the detection of lactose (Svorc et al. 1990, Eshkenazi et al. 2000, Tkac et al. 2000). -Galactosidase isolated from different thermostable sources is the key enzyme to detect lactose (Athes et al. 1997, Furlan et al. 2000). Different methods of immobilization have been attempted in our laboratory for the development of biostrips for various tests (Kumar et al. 2000, Tulsani et al. 2001). The inability of humans to digest lactose has enormous health consequences particularly among the poor population of the world where milk is often the only economical and reasonably available source of nutrition (Scrimshaw & Murray 1988). Therefore, our attention was drawn to develop a simple and economical biostrip for detection of lactose in food products.

Materials and methods Chemicals and reagents -Galactosidase, o-dianisidine, 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS] and glutaraldehyde were from Sigma-Aldrich (USA). Galactose oxidase and peroxidase were from Boehringer (Germany). Cresol Green, citric acid and sodium citrate were purchased from Qualigens (India). Nylon papers and Whatman lter papers were purchased from Whatman Co. (UK). Plastic sheets and adhesive were obtained from the local market. Preparation of enzymes -Galactosidase 40 U, galactose oxidase 40 U and horseradish peroxidase 200 U were dissolved together in 100 l 0.1 M citrate buffer, pH 6.2. The mixture was vigorously shaken and stored at 4 C. Preparation of chromogens The chromogen solution was made in 0.1 M citrate buffer, pH 6.2 by mixing o-dianisidine (10 mg ml1 ), ABTS (2 mg ml1 ) and Cresol Green (1 mg ml1 ). The chromogen solution (100 l) and enzyme mixture

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Fig. 1. Effect of lactase concentration on response time for development of colour gradient. Galactose oxidase (60 U), peroxidase (150 U) and chromogens (100 l) were dissolved in 100 mul 0.1 M citrate buffer, pH 6.2. The response time of the strips was recorded by developing the colour.

Fig. 2. Effect of galactose oxidase concentration on response time for development of colour gradient. Peroxidase (150 U), lactase (40 U) and chromogens (100 l) were dissolved in 100 l 0.1 M citrate buffer, pH 6.2. The response time of the strips was recorded by developing the colour.

(100 l) were mixed together for immobilization on to the matrices (nylon and Whatman papers). Preparation of buffers Citrate and phosphate buffers of different pH were made to standardize the method for development of biostrips. The response time of the strip was kept uniform throughout the standardization. Preparation of lactose biostrips The enzyme/chromogen solution was immobilized on Whatman lter paper using crosslinking reagent (0.2% glutaraldehyde) in a humidity-free chamber. After complete drying, the paper was cut into pieces of 5 mm width. PVC plastic sheet (1 mm thickness) was cut to 9 90 cm. The immobilized enzyme papers were pasted onto one edge of the plastic sheet using a non-reactive adhesive. The sheet was dried in a humidity-free chamber for 34 h. After complete drying, the sheet was cut into 0.5 9 cm pieces in such a manner that one end of the strip had an enzymatic pad and the other end was free for handling. The strips were packed in dark brown bottles containing silica bags as desiccant and stored at room temperature.
Fig. 3. Effect of peroxidase concentration on response time for development of colour gradient. Galactose oxidase (40 U), lactase (40 U) and chromogens (100 l) were dissolved in 100 l 0.1 M citrate buffer, pH 6.2. The response time of the strips was recorded by developing the colour.

galactose oxidase (40 U/200 l) and peroxidase (200 U/200 l) were found to be the optimum required for showing colour at different concentrations of lactose at xed response time (120 s). Different buffers were also tried to achieve better gradient of the
Table 1. Colour produced for lactose detection in milk and milk products using the biostrip. Lactose (g l1 ) 20 40 Colour Light blue Yellowish brown

Results and discussion To develop the biostrip, different parameters were standardized keeping others constant at xed response time 120 s (Figures 13). Lactase (40 U/200 l),

60 Light brown

80 Brown

100 Dark brown

1739 colour and citrate buffer (0.1 M, pH 6.2) was the most appropriate. Lactose content in milk and milk products is determined by various analytical methods (Svorc et al. 1990, Eshkenazi et al. 2000, Tkac et al. 2000). These methods require trained persons and sophisticated instruments such as a spectrophotometer, biosensor etc. while the test strip developed in our laboratory offers quick detection of lactose in samples. The biostrip is dipped in the milk or milk products and the colour developed on the strip can be compared with a colour chart. The strip gives different shades of colour from light blue to dark brown depending on the concentration of lactose present in the sample (Table 1). The test strip has the sensitivity of 20100 g lactose l1 . The colour of the strip does not show any signicant change with lactose concentration lower than 20 g lactose l1 . Lactose-free milk was used as control and different concentrations of lactose were made with this milk. The response time of the strip was found to be 120 s. In this lactose biostrip, co-immobilized enzymes (viz. -galactosidase, galactose oxidase and peroxidase) and the chromogens react with lactose present in the sample resulting in colour formation on the enzymatic pad of the strip. The intensity of the colour developed is proportional to the concentration of lactose. Different types of matrices (Whatman lter paper, Millipore lter paper and nylon paper) were tried for the immobilization of enzymes but Whatman paper was the most suitable since it did not react with the enzymes and chromogens and gave the best gradient of colours. The biostrips were stable at room temperature for one year when stored under dry conditions. The use of strip can prevent the development of lactose intolerance syndrome. Being a visual assessment technique, it does not require any specialized training or sophisticated instruments. The test is economical and can be used in dairies, hospitals and remote places where expensive instruments may not be available. Acknowledgements The authors are thankful to Prof S.K. Brahmachari, Director, Centre for Biochemical Technology and Prof S.V. Goswami, Department of Zoology, Delhi University for providing necessary facilities and valuable guidance to carry out the research work. This work was supported by the Council of Scientic and Industrial Research, New Delhi.

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