Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-11-19
2003-07-01
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S014000, C435S004000, C435S967000, C435S003000
Reexamination Certificate
active
06586195
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates generally to an analytical method, and more particularly to a simple and rapid method for the detection of sugars in food and beverage items.
Diabetes is a major health issue in the United States. In fact, diabetes mellitus, the most prevalent form of diabetes, is the fourth most common reason for patient contact with a physician and is a major cause of premature disability and mortality. It is the leading cause of blindness among working-age people, of end-stage renal disease, and of non-traumatic limb amputations. It increases the risk of cardiac, cerebral, and peripheral vascular disease two- to seven-fold and is a major cause of neonatal morbidity and mortality.
Personal factors promoting diabetes are well known. Increased age, reduced physical activity, and especially obesity promote diabetic conditions. In particular, severe and prolonged obesity is understood to substantially enhance the risk of the disease. However, medical data now indicate that most of the debilitating complications of the disease can be prevented or delayed by prospective treatment of the above-mentioned risk factors. For most diabetic individuals, particularly those having Type II diabetes, diet and exercise are the key intervention required to restore metabolic control. In this regard, it is desirable for diabetic individuals, or those at risk of diabetes, to carefully monitor their diet as modest maintenance of a low sugar diet often leads to substantial reductions in diabetic symptoms. In addition, individuals suffering from Type I diabetes, an often more severe condition than Type II, must rely on exogenous sources of insulin due to an absent or reduced insulin secretion ability. Departures from a consistent diet require active adjustment of insulin dosages and increase the chances of dangerous, health-threatening mistakes in dosage delivery.
In light of the above, a simple and rapid method useful in determining the presence of sugars in food and beverage items is desirable for individuals who must closely monitor their carbohydrate intake. Specifically, a method efficient at providing a quick and clear indication of the presence of sugars in food and beverage items would be found useful and welcomed by a multitude of diabetic individuals. In addition, those with a general interest in weight loss, a popular pursuit in our current society, would also find such a method useful. Also, food manufacturers, distributors and retailers would have a great interest in a convenient method for monitoring the sugar content of their respective food products, especially those offered to the public as diet or low-calorie items.
A method useful for fulfilling the requirements highlighted above would ideally be in kit format allowing a user to conveniently carry out the method at any time without the need for complex manipulations and apparatus. In the case of test reagents useful in a kit format, it is generally desired that the reagents contain all necessary components in a pre-mixed state in order to reduce as much as possible the opportunity for errors arising from mixing of several components. Test reagents should also remain stable for as long as possible to avoid having to prepare new solutions continuously. Unfortunately, previously known methods did not fulfill these requirements. For example, enzymatic determination of glucose by use of the enzymes glucose oxidase and peroxidase requires the continual preparation of new enzymatic reagents as the enzyme components are unstable for long periods of time once diluted in a working test reagent. Obviously, this method is not convenient for those interested in a quick and convenient determination of sugar content in a snack or meal. Likewise, chemical indicators, particularly methylene blue, are subject to rapid photodegradation thereby making long term storage and use of such test reagents by consumers all but impossible.
SUMMARY OF THE INVENTION
Therefore, it is an object of the present invention to provide a simple, rapid and relatively low priced method of detecting sugars in a liquid sample. The method includes an initial step of selecting an indicator capable of producing a colorimetric change when contacted with a reducing sugar. Indicators suitable for use in the invention are selected from the phenazine class of compounds and include azocarmine B, indoine blue, methylene violet 3RAX, safranine O, phenosafronin, and Janus Green B. Once selected, the indicator is preferably dissolved or diluted in water to form an aqueous solution and the pH of the aqueous solution is adjusted to be alkaline by the addition of a base, such as NaOH, KOH, Sr(OH)
2
, Ba(OH)
2
, and NH
4
OH. Thusly prepared, the aqueous test reagent is particularly resistant to photodegradation and stable for long periods of time thereby eliminating the need to continually prepare fresh test reagent.
Subsequent to preparation, the method according to the present invention calls for the test reagent to be brought into contact with a sample. Reducing sugars, if present in the sample, cause the indicator in the test reagent to undergo a calorimetric change. Preferably, this calorimetric change is clearly visible to the naked eye of the user and provides definitive proof of the presence of a reducing sugar in the sample.
The method according to the invention may also include an additional step wherein the sample is pretreated with a glycosidase, such as invertase, prior to contacting the sample with the test reagent. This step allows nonreducing sugars, such as sucrose, to be converted into reducing forms capable of detection by the reducing sugar-specific indicator.
The invention also encompasses preparation of the test reagent by the addition of a material pre-absorbed with indicator to an aqueous solution thereby dissolving the indicator present in the pre-adsorbed material to form fresh test reagent. The indicator pre-adsorbed material offers the distinct advantage of maintaining the indicator in a dry, easily-stored form prior to carrying out the method.
Various other features, objects, and advantages of the invention will be made apparent to those skilled in the art from the following detailed description of the invention.
DETAILED DESCRIPTION OF THE INVENTION
In general, the invention disclosed herein may be used to detect the presence of sugars in samples. Appropriate samples may be liquid, solid or a combination of liquid and solid. An appropriate liquid may be a beverage item and an appropriate solid may be a food item.
Indicators useful with the present invention include compounds belonging generally to the phenazine class. Specifically, azocarmine B (1), indoine blue (2), methylene violet 3RAX (3), safranine O (4), phenosafronin (5), Janus Green B (6) and combinations thereof as shown below.
The preceding list is meant to be illustrative and not inclusive of all compounds from the phenazine class which are useful with the invention. However, not all phenazines are effective with the present invention. For instance, neutral red (7), shown above, fails to give a color change in the presence of reducing sugars. Compounds from other chemical classes, such as phenothiazine, phenoxazine, xanthine, acridine, anthraquinone, azo and sulfonephthalein classes, do not show the resistance to photodegradation exhibited by compounds within the phenazine class. Thus, compounds in the phenazine class are considered an improvement over non-phenazine compounds in regard to the present invention. Janus Green B is the preferred indicator for carrying out the invention.
The indicators useful in the invention are believed to undergo calorimetric changes visible to the naked eye in response to electron transfer reactions between the indicator and a reducing agent capable of donating electrons. The reducing agent is a reducing sugar in the case of the invention. As an example, blue-colored Janus Green B in alkaline media may be reversibly converted into a gray colored compound by an electron transfer from a reducing agent molecule to a Janus Green
Godfrey & Kahn S.C.
Leary Louise N.
R.E. Davis Chemical Corporation
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