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Amylase-treated rice flour oral rehydration solution with enhanced energy density. I. In vitro studies of viscosity, osmolality, and stability
Carolina Vettorazzi, Noel W. Solomons, Kenneth H. Brown, and Charles Shoemaker
Recent studies on the dietary management of dehydrated children with oral rehydration solution (ORS) showed that maximizing nutrient intake during treatment can curtail the nutritional deterioration of an episode of acute diarrhoea. The viscosity of a 10% or 15% (w/v) rice flour solution can be reduced to that of a drinkable solution by appropriate incubation and partial hydrolysis of rice starch with a commercial a -amylase. Cooking the solution for 5 minutes at 90°C inactivates the enzyme. When 0.32 ml of -amylase enzyme was added, the viscosity of a 10% rice-based oral rehydration solution (RB-ORS) measured at 0.3 revolution per minute declined from 50,539 to 636 centipoise (cp), and when 0.96 ml of enzyme was added to a 15% RB-ORS, the viscosity declined from 170,258 to 1,283 cp. The osmolality of enzyme-treated RB-ORS without electrolytes was 5 mOsm; that of the electrolyte mixture alone was 160 mOsm; that of 10% RB-ORS with electrolytes was 225 mOsm; and that of 15% RB-ORS with electrolytes was 295 mOsm. The 10% and 15% rice flour solutions incubated with a -amylase can be converted to easy-to-drink solutions that could potentially be used as ORS with greater nutritional value than current glucose-based solutions. Clinical trials are necessary to test the safety and efficacy of these enzyme-treated RB-ORS.
Each year millions of children in developing countries die from diarrhoeal diseases, principally as a result of fluid loss and secondary dehydration [1, 2]. Fluid replacement can prevent or reverse dehydration and reduce the risk of death. Several studies [1-4] showed that even though the standard glucose-based oral rehydration solution (GB-ORS) is effective in treating or preventing dehydration, it does not reduce stool output or shorten the duration of diarrhoea. Moreover, GB-ORS contributes little to the child's nutrition status.
The standard World Health Organization ORS contains 20 g/L glucose, enough to facilitate the transport of sodium into the intestinal mucosal cell, but the 80 kcal/L provided by this carbohydrate is not sufficient to maintain energy balance. Moreover, the energy content cannot be increased by adding more glucose, because the increased osmolality of this solution could dangerously increase diarrhoeal purging.
Some studies [1, 5-8] reported that patients with diarrhoea can be rehydrated with cereal-based ORS, using the glucose liberated from hydrolysed starch to promote electrolyte absorption. One of the cereals most frequently used, because of its widespread availability, is rice .
Rice starch is a large polymer that, when exposed to amylase, is hydrolysed to progressively smaller polymers and glucose . Rice protein, moreover, contains oligopeptides and amino acids such as glycine, lysine, leucine, and isoleucine, all of which facilitate the cotransport of sodium from the intestinal lumen. Using ORS with 50 g/L of rice in place of glucose, the stool volume was significantly reduced by 49% and the duration of diarrhoea by 30% . With rice powder at a concentration of 80 g/L, an ORS based on a large quantity of this cereal is much more efficiently absorbed than GB-ORS .
There is, however, a limitation to using high concentrations of rice flour solution; the maximum quantity of rice that can be added to a drinkable (low-viscosity) solution is approximately 80 g/L; beyond that level, the gruel becomes a very thick paste. The viscosity also depends on the variety of rice used to produce the rice flour. Some varieties will not yield a drinkable solution at 80 g/L.
Rice starch is composed of two polymers: a linear fraction, amylose, and a branched fraction, amylopectin. The granules are insoluble in cold water; on heating to gelatinization temperature, they swell [12,13]. When a cooked starch paste is cooled, it may take the form of a gel.
Viscosity is the flow property of a material, or the resistance of the material to flow under a mechanical stress [14, 15] expressed in units of centipoise (cp). Solutions with viscosity of less than 1,000 cp are freely flowing liquids. Between 1,000 and 3,000 cp they have a thick, soup-like consistency, and at higher viscosities the gruels become progressively thicker .
Our goal was to create a rice-based ORS (RB-ORS) that could be used not only in oral rehydration therapy, but also as nutritional therapy during diarrhoeal episodes. The objectives of this study were twofold: to determine the highest concentration of a rice flour solution that is still compatible with a drinkable solution based on its physical characteristics (viscosity) and appropriate osmolality, and to evaluate the use of an exogenous x-amylase enzyme to alter the viscosity of the solution. The aspects investigated were the amounts of enzyme required, the dilution of the enzyme, the temperature for inhibiting the breakdown of the rice starch, and the effects of adding electrolytes to the solution.
The first phase of the study examined the highest concentration at which a suspension of rice flour in water is no longer a free-flowing, drinkable liquid. For this process, solutions containing 5, 8, 9, 10, and 15 g flour/100 ml final volume were evaluated. The rice flour used was coded as 7011 and was produced by grinding and bolting clean quantities of California, milled, medium-grain rice (Pacific Rice, Inc., Woodland, California, USA).
Cooking the rice flour
Batches were made at different concentrations by cooking rice flour in proportions of 50, 80, 90, 100, and 150 g in 1 L water for five minutes at 90°C, and then replacing the evaporated water to achieve 1 L final volume. After cooking, samples were allowed to cool to an environmental temperature (25°C).
The viscosity was measured with a micro-viscometer (Brookfield Engineering Laboratories Inc., Stoughton, Massachusetts, USA). The solutions were measured first on the same day as prepared, and then again after 24 hours of refrigeration at 4°C. Measurements were always taken at room temperature. The purpose of measuring at 0 and 24 hours was to evaluate the stability of the solutions during storage. The viscosity was measured twice at eight different speeds: 0.3, 0.6, 1.5, 3, 6, 12, 30, and 60 rpm. Viscosity is measured in dynes-second/cm2 (or poise; 100 cp = 1 poise).
A fungal a -amylase (Clarase L-40,000; Solvay Enzymes, Inc., Elkhart, Indiana, USA) was selected for its sensitivity to heat inactivation at 90°C. Using an enzyme preparation, we wanted to determine the amount required to lower the viscosity of both 10 and 15 g/100 ml rice flour solutions (which are very thick pastes) to free-flowing, drinkable solutions. The solutions containing rice flour with amylase were prepared according to the cooking process described above with various amounts of enzyme added; the temperature of the solutions was maintained at 90°C for five to six minutes to inactivate the enzyme. After cooling to room temperature, viscosity was measured as described. This procedure allowed us to identify the right amount of enzyme to obtain solutions with appropriately low viscosity.
Osmolality, expressed as mOsm/L, was measured by freezing-point depression with an Osmette micro-osmometer (Precision Systems, Inc., Natick, Massachusetts, USA). The solution (50 µl) was placed in a microvial as required for the osmometer. Osmolality was measured on the day the solutions were made and on the day after. Additional NaCl 1.75 g, KCl 1.5 g, and NaHCO3 2.5 g were added to the RB-ORS containing enzyme to achieve final electrolyte concentrations of Na 60 mEq/L, K 20 mEq/L, and NaHCO3 20 mEq/L. Osmolality was measured with and without electrolytes.
Five percent, 10%, and 15% concentration solutions were prepared by cooking 50 g, 100 g, and 150 g of rice flour, respectively, in 1 L water for about 5 minutes at 90°C and replacing the evaporated water to restore the initial volume. For the 10% and 15% solutions, a dose of 0.32 ml (12,800 SKBU) or 0.96 ml (38,400 SKBU) of a -amylase, was added, respectively, together with the flour and water to be cooked for the established time and temperature. After the solution was cooked and the enzyme inactivated, the solutions were restored to 1 L and allowed to cool until they reached room temperature (25°C). The electrolytes were measured in an analytical scale (Fisher Scientific, Denver Instruments Co., Denver, Colorado, USA) and added to the solution.
The viscosities of the solutions at 5, 8, 9,10, and 15 g rice flour/100 ml concentrations, measured at 0.3 rpm, are shown in sable 1. The higher the concentration, the greater the viscosity of the solution. Above 8 g/100 ml. a gel is formed that cannot flow freely from a cup or a feeding bottle. Even at 5 g/ml, the rice solution has the consistency of a thick soup.
TABLE 1. Viscosity of the rice flour solutions at five concentrations
|Concentration of rice flour (w/v) (g/100 ml)||Time (h)|
Values are expressed as centipoise at 0.3 rpm.
When different amounts of enzyme were added to solutions with 10 and 15 g/100 ml concentrations, we found that 0.32 ml of the x-amylase enzyme (containing 12,800 SKBU) per litre of solution with 100 g rice flour was necessary to produce a drinkable solution with a viscosity of 105 cp at 60 rpm, and for 1 L of 150 g rice flour, 0.96 ml of the enzyme (38,400 SKBU) was necessary to produce a viscosity of 115 cp (see FIG. 1. Viscosity at 60 rpm of 10% and 15% concentrations of rice flour solution treated with amylase).
The thermal process (heating to above 90°C for 5 minutes) completely inactivated the enzyme (see FIG. 2. Stability of viscosity with time at 24 h of 10% rice flour solution after inactivation of the a -amylase enzyme and FIG. 3. Stability of viscosity with time at 24 h of 15% rice flour solution after inactivation of the a -amylase enzyme ). The lower the viscosity, the less was the tendency for any further spontaneous reduction with storage.
Table 2 shows the final composition of the solution when electrolytes were added, and sable 3 shows the osmolality measurements performed on the solutions with and without electrolytes. Both solutions had acceptably low osmolality, which is less than those of the human interstitial and intracellular spaces.
TABLE 2. Final electrolyte composition of solutions with the electrolytes added
|Source||Amount (g/L)||Concentration of chemicals (mmol)|
Several authors have commented on the importance of reducing the bulkiness of cereals, because their consistency can be the limiting factor in the intake of weaning foods mace from rice, maize, and other cereals [16-19]. Reducing the viscosity of the food should permit children to feed better, with higher energy intake per volume consumed, during the transition to household foods.
Our idea has been to use an a -amylase to modify the starch of rice, but with a specific application to oral rehydration. A controlled incubation with this hydrolytic enzyme could produce a solution with a low enough viscosity for easy drinking. Consequently, high concentrations of rice flour, treated with amylase, may not only improve the child's hydration state, but also prevent further deterioration of his or her nutrition status. Viscosity measured at 60 rpm is used most commonly for acceptability studies. The solution tested here reached a viscosity of 115 cp at 60 rpm, which is acceptable considering the desirable viscosity of less than 200 cp.
The rice-based solutions at 10 and 15 g/100 ml treated with exogenous enzyme are considered to have acceptable viscosity. Adding more enzyme will cause the solutions to form a precipitate. Even though a large amount of hydrolysis is produced, with a great deal of maltose formation, the osmolality is still within the desirable level, even after adding electrolytes to the solution. Our rice-based products never exceeded the osmolality of standard WHO ORS. In theory, this would make an ideal solution to cover all the purposes of a rehydration solution with a high energy content.
TABLE 3. Osmolality measurements (mOsm/L) performed with and without electrolytes and at different intervals from preparation
|Rice flour solution 5% (w/v)||5||-|
|Electrolytes in water||161||-|
|Rice flour 10%a (w/v) plus electrolytes||225||219|
|Rice flour 15%b (w/v)plus electrolytes||295||286|
One can reduce the viscosity of cereal-based solutions with exogenous a -amylase. Whether the effective osmolality in the colon due to incomplete removal of carbohydrate by the small intestine will exceed the absorptive capacity of the large bowel with the hypercaloric rice-based solutions is not known. The present observations are a prelude to clinical trials on the acceptability of and tolerance to 10% and 15% RB-ORS. The results of the application of this enzyme-treated ORS to rehydrate humane are presented in the companion papers in this series [20, 21].
We thank Ms. Jody Renner-Nants from the Laboratory of Food and Technology of the University of California at Davis, for her assistance; Dr. Pablo Yurrita of the Guatemalan Social Security Institute (IGSS); and Lic. Fabiola Micheo and Lic. Carmencita Arriola from the Central American Institute of Investigations and Industrial Technology (ICAITI), in Guatemala, who provided instruction about and access to the osmometer and viscometer. We also appreciate the help of Dr. Manolo Mazariegos who reviewed this manuscript.
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