FIELD OF INVENTION
This invention relates to highly sensitive method for detection of maltodextrin and starch in milk and milk products suitable for food and dairy industry.
BACKGROUND OF INVENTION
Milk adulteration is one of the most common type of food sophistication. Milk vendors have mastered the trick of milk - adulteration. As a result milk is deliberately mixed with toxic substances like urea, preservatives, hydrogen peroxide and caustic soda. Numerous ingredients of origin other than milk, like cheap edible oil, sugar and starch and starch hydrolysis products like maltodextrin are also added to increase the fat and total solid contents so as to fetch higher profits. Besides gaining higher profit margins, these additives are used to mask, undesirable taste contributed by adultrants.
Maltodextrin ((C6H10O5)n, is a type of dextrin, a partial hydrolysis product of starch. It is a nonsweet nutritive saccharide polymer that consists of D-glucose units linked primarily by a -1-4 bonds and that has a dextrose equivalent (D.E.) of less than 20. Dextrose equivalent (DE), is a quantitative measure of the degree of starch-polymer hydrolysis. It measures reducing power compared to a dextrose standard of 100. In general, food formulators add maltodextrin for bulking and binding without adding sweetness.
Milk plays important role in our diets, especially vegetarians, as a significant source of good quality proteins. Adulteration of milk with maltodextrin lowers the protein content making it poor constituent of diet in terms of protein content. Maltodextrin, if added to milk, not only impacts the nutritional composition and sensory attributes of milk but also makes milk a poor raw material for products like dahi, paneer, chhana etc. Starch (C6H10O5)n is a polysaccharide carbohydrate consisting of a large number of glucose monosaccharide units joined together by glycosidic bonds.

For detection, starch and starch products are first degraded in to glucose using amyloglucosidase and then glucose so formed is detected with glucose testing strip. Glucose and hydrogen peroxide do not require any degradation for detection. Amyloglucosidase is an exoglucosidase enzyme derived from a selected strain of fungus, Aspergillus niqer, which hydrolyses a-D 1,6 branch points and a-D 1,4 glycosidic linkages.
Presently dextrins are being detected with iodine solution, giving a red coloration with green spots. The routine iodine based test, being practiced in dairy industry give erratic results as colour intensity varies with the different type of maltodextrin added and samples under test. More over iodine solution itself also imparts yellowish red tinge to solution under test so it becomes difficult to conclude the adulteration, particularly at low levels. Presence of other adulterants in milk further complicates the detection. More over till date no method is available for screening of milk and milk products for all these adulterants. Hence the study was initiated to develop a sensitive method for qualitative detection of maltodextrin in milk, milk powder and milk concentrate. We could arrive at a sensitive method for detection of maltodextrin as well as other adulterants which are very commonly encountered in incoming/raw milk, which can serve as an important tool for Quality control.
OBJECTIVE OF INVENTION
The principal object of the present invention is to provide a method for detection of maltodextrin and starch in milk and milk products.
Another object of the present invention is to provide a method which detects adulteration at lower levels.

SUMMARY OF THE INVENTION
The present invention relates to a method for the detection of maltodextrin and starch in milk and milk product sample comprising:
- contacting glucose strip with the milk or milk product sample,
- adding 1-10% of acid to the milk or milk product sample to maintain pH at 4.0-4.5,
- adding enzyme solution to the milk or milk product sample e in step (iii),
- incubating the solution in step (iii) for 5-30 mins at 60-65°C,
- cooling the solution to room temperature
- contacting the solution with glucose strips to detect change
in colour.
DESCRIPTION
Detection of maltodextrin, glucose, starch, and hydrogen peroxide in milk and milk products is based on sequential enzymatic reactions. Starch and intermediate products of hydrolysis including maltodextrin in the sample are further hydrolyzed by externally dosing with Amyloglucosidase under controlled conditions to yield glucose units. Glucose so formed and/or hydrogen peroxide present in the test solution is detected by reagent strip which incorporates two enzymes (glucose oxidase and peroxidase) and a chromogen. Glucose oxidase, catalyzes the formation of gluconic acid and hydrogen peroxide from the oxidation of glucose. Another enzyme, peroxidase, catalyzes the reaction of hydrogen peroxide with a potassium iodide (chromogen) to oxidize it to the colours ranging from green to brown.

A method was standardized for maltodextrin detection at very low levels even up to 0.1% in milk and milk products. Besides maltodextrin this method simultaneously detects presence of very commonly encountered adulterants namely glucose, hydrogen peroxide and starch at low levels. Presence of these adulterants glucose, maltodextrin and starch can be detected at low levels of 0.1%, when present singly. Hydrogen peroxide can be detected at even lower level of 0.0015%.
The method for detection of maltodextrin and starch in milk and milk product sample involves the use of the following:
Testing strip: Glucose testing strips which incorporates a reagent area that changes colour (from green to brown) during the test. Results are obtained directly from comparison of test strip with the colour chart (the color of the strip) usually provided on bottle. Alternately an original strip can be used for comparison.
Amyloglucosidase Solutio: 0.1 - 1 g of enzyme is dissolved in 100 ml water and stored under refrigeration (2-8°C). This solution should not be used for more than 15 days as loss of enzyme activity may result in misleading results.
Dilute acid solution: Dilute (strength ranging from 1 -10%) solution of organic/inorganic acid like Nitric/hydrochloric/sulphuric/acetic/lactic/citric is prepared with distilled water for adjusting pH of solution.
Sample Preparation
In order to carry out the detection, a sample of milk is prepared by taking 20 ml homogenous milk sample in a 100 ml beaker. In case of concentrated milk and other dairy products including skimmed milk powder, whole milk powder, partially skimmed milk powder, khoya, paneer, curd, butter milk and chhana , appropriate dilution is made so as to adjust total solids in a range of 10 - 15 %. Pestle and mortar can be used to obtain a homogenous test sample wherever

required. For milk powder, 10g of skim milk or 13 g of whole milk powder is dissolved in 100 ml water (40°C) with the help of stirrer for proper reconstitution.
Procedure
The sample is first checked for glucose and hydrogen peroxide using reagent strip. Take one test strip, hold the plastic end of the strip and do not touch the green colored reagent area. Dip the reagent area of strip in to the test solution/sample and remove immediately. Remove excess of liquid on strip by single jerk. Observe the color (S1) of the reagent area exactly after 30 seconds of wetting. In such case sample may be rejected and further test need not to be performed. If there is no change in the color of strip, then proceed further for dextrin and starch test. Thereafter pH of sample is adjusted to 4 - 4.5 with the help of dilute acid solution. It is then dosed with 1 ml of enzyme solution and incubated the contents at 60- 65°C for 5 - 30 minutes. Contents are cooled at room temperature, again checked with glucose strip and change in color of strip (S2) is recorded.
If sample shows change in strip color before enzyme treatment (S1), it is indicative of presence of glucose and/or hydrogen peroxide in sample. Change in color of glucose strip after enzyme treatment (S2), indicates presence of maltodextrin and/or starch.

Schematic diagram of steps involved in detection of maltodextrin, glucose, hydrogen peroxide and starch detection in milk and milk (Figure Removed)
products.

EXAMPLES
This experiment was designed to establish that method claimed detects adulterants namely glucose, hydrogen peroxide, maltodextrin and starch in milk and milk products at low levels. Milk free of any adulterant was obtained from Patparganj Locality of Delhi (milking done in presence). Six different samples were spiked with adulterants singly or in combination and coded as detailed below.

(Table Removed)
Procedure:
A 20 ml of each sample is pipetted out in small beaker and test is performed as per the aforementioned protocol. The sample is first checked for glucose and hydrogen peroxide using reagent strip (Diastix from Diagnostics India Ltd.) Reagent area of strip is dipped in to the test solution/sample and removed immediately. Excess of liquid on strip is removed by single jerk and color (S1) of the reagent area is observed exactly after 30 seconds of wetting. Samples showing change in colour are rejected and further test is not performed. In other samples, pH of is adjusted to 4 - 4.5 with the help of dilute acid solution. Acid solution for pH adjustment is prepared by diluting 10 ml of concentrated lactic acid (88 - 92%; SD Fine Chem, India) to 100 ml with distilled water. It is

then dosed with 1 ml of Amyloglucosidase solution (0.2%) and contents are incubated at 62±2°C for 5 minutes. Contents were cooled at room temperature, again checked with glucose strip and change in color of strip (S2) was recorded.
Along with the spiked samples a reagent blank (distilled water) and a sample Blank (milk sample free of adulterant) is treated in same manner and tested for presence of adulterants. Substances used as adulterants were procured from following sources.
Adulterants Manufacturer/suppliers
Glucose From Heinz India Pvt. Ltd. Mumbai
H2O2 30% solution, purified, from Merk Specialties Pvt Ltd. Mumbai
Maltodextrin Devson Impex Pvt Ltd., Mumbai
Starch SD Fine Chem Ltd, India
Observations on change of color of reagent strip*

(Table Removed)

Original color of reagent area of the glucose strip is blue
Out come of the experiment conducted is presented in Table 2. No change in colour of glucose strip upon testing after as well as before enzyme treatment in case of sample blank and reagent blank reveals that no constituent of sample matrix and reagents suggested do not cause any change in color and thus do not

pose any interference in the test. Change in color of reagent strip from blue to shades ranging from green to brown in samples containing glucose, hydrogen peroxide and combination thereof indicates effectiveness of sample to detect these adulterants. Testing of samples containing maltodextrin, starch and combination of two after treating with enzyme under controlled conditions exhibited the color change from blue (original colour) to green, suggests suitability of method for detection of these adulterants. Observations of the study also reveal that presence of these adulterants glucose, maltodextrin and starch can be detected at low levels of 0.1%, when present singly. Hydrogen peroxide can be detected at even lower level of 0.0015%. Presence of combination of adulterants glucose + hydrogen peroxide and maltodextrin + starch can also be detected employing this protocol.
This test can be used to detect the presence of maltodextrin and other adulterants including starch, hydrogen peroxide and glucose in milk and milk products like skim milk powder, milk concentrates etc in following cases
■ Dairy and food manufacturer so as to screen incoming raw material and assure final product quality.
■ Milk collection centers/societies.
■ Regulatory authorities like PFA to stop the malpractice of adulteration of milk and milk products.

We Claim,
1. A method for the detection of maltodextrin and starch in milk and milk
product sample comprising:
i. contacting glucose strip with the milk or milk product
sample, ii. adding 1-10% of acid to the milk or milk product sample to
maintain pH at 4.0-4.5, iii. adding enzyme solution to the milk or milk product sample e
in step (iii), iv. incubating the solution in step (iii) for 5-30 mins at 60-65ºC, v. cooling the solution to room temperature vi. contacting the solution with glucose strips to detect change
in colour.
2. The method for the detection of maltodextrin and starch in milk and milk
product sample as claimed in claim 1 wherein the milk product sample is
prepared by diluting milk product to obtain total solid content of 10-
15%.
3. The method for the detection of maltodextrin and starch in milk and milk product sample as claimed in claim 2 wherein the milk product is milk concentrate, skimmed milk powder, whole milk powder, partially skimmed milk powder, khoya, paneer, curd, butter milk, chhana.
4. The method for the detection of maltodextrin and starch in milk and milk product sample as claimed in claim 1 wherein the acid is lactic acid, nitric acid, sulphuric acid, acetic acid or citric acid.
5. The method for the detection of maltodextrin and starch in milk and milk product sample as claimed in claim 1 wherein the enzyme is amyloglucosidase.
6. A method for the detection of maltodextrin and starch in milk and milk product sample as herein described with reference Jto the foregoing examples.