DEVELOPMENT OF METHODS FOR THE DETECTION OF CELLULOSE IN MILK AND MILK PRODUCTS
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Date
2022
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Publisher
ICAR-NDRI, KARNAL
Abstract
Milk and milk products are the important source of nutrients like proteins, carbohydrate, fat,
vitamins and minerals for humans. However, the safety of milk has always been challenging
due to the use of adulterants by unscrupulous people for economic gain. Cellulose is one of the
such adulterants which is used to increase the solid-not-fat content of milk. The present study
was conducted to develop a rapid (strip-based & liquid-based) and instrument-based method
for the detection of cellulose in milk. The concept used in the study include conversion of
cellulose present in the adulterated milk sample to glucose by cellulase and detection of glucose
produced using appropriate detection reagent. Cellulase from two different source
(Trichoderma reesei and Aspergillus niger) was used and spiked cellulose (microcrystalline
cellulose and carboxymethyl cellulose) in milk or buffer was hydrolysed into glucose using
cellulase. Produced glucose was detected following the glucose oxidase- peroxidase coupled
assay using prepared glucose strip and liquid-based method. In both strip-based as well as
liquid-based method it was found that cellulase from Trichoderma reesei showed nil activity
towards cellulose hydrolysis to produce glucose. During the experiments with cellulase from
Aspergillus niger, it was found that glucose strip showed colour change in control samples also
containing only cellulase. After conducing many experiments, it was concluded that cellulase
itself contained glucose. So, efforts were made to remove glucose from cellulase using
ultrafiltration/diafiltration and treatment of cellulase with glucose oxidase-peroxidase. The
cellulase treated with glucose oxidase-peroxidase was used in the further study. Cellulose in
MCC or CMC spiked buffer or milk samples was detected by both strip-based and liquid-based
method. The optimized conditions include treatment of milk samples with treated cellulase at
50°C at pH 5.0 for 4 h. Limit of detection (LOD) of the method was 0.5% for MCC and CMC
in milk. Cellulose detection strip was prepared but it did not work and this may be due to fact
that cellulase action requires at least 4 h to hydrolyse cellulose into glucose. Instrument-based
method of cellulose detection was developed using normal phase- high performance liquid
chromatography. MCC/CMC were spiked (0.5%) in the pure raw milk (pH 5.0) followed by
cellulose hydrolysis with treated cellulase for different incubation period at 50°C. An extra peak
of glucose was observed in the milk samples spiked with MCC/CMC and hydrolysed with
treated cellulase at the retention time of 4.699 ± 0.131 min followed by peak of lactose having
retention time of 5.561 ± 0.32 min. It is proposed that the presence of extra peak of glucose as
per the developed protocol may be used as test of presence of cellulose in milk.